JP2007530069A6 - Ion channel - Google Patents

Abstract

  Disclosed is a transgenic non-human animal having a functionally disrupted endogenous TrpM8 gene. The TrpM8 gene comprises the nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence having at least 70% sequence identity thereto.

Description

  The present invention relates to newly identified nucleic acids, the polypeptides encoded by them, and their production and use. More particularly, the nucleic acids and polypeptides of the invention relate to an ion channel, hereinafter referred to as “TrpM8” or “TrpM8 ion channel”. The invention also relates to inhibiting or activating the action of such nucleic acids and polypeptides.

  The mammalian nervous system constantly evaluates internal and environmental temperatures to maintain homeostasis and avoid extreme heat. Transient receptor potential (TRP) channels form cation channels that are activated by a variety of factors including mechanical stimuli, changes in osmotic pressure, pH and temperature, and the exogenous stimulus capsaicin . These specialized sensory receptors are found in dorsal root ganglia (DRG).

  Several members of the transient receptor potential (TRP) family of ion channels have been implicated in heat-stimulated transducers. Among these members are TRPV1 and TRPV2 which are activated by heat and TRPM8 which is activated by cold.

  A cold menthol-sensitive receptor (CMRI) from rat was recently cloned [McKemy DD, Neuhausser WM, and Julius, D .: Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416: 5258, 2002]. This receptor is an excitable ion channel expressed by small diameter neurons of the trigeminal and dorsal root ganglia. This channel-type receptor is activated by low temperature (8-28 ° C.) and menthol as a chemical agonist of the thermoresponsive receptor, which induces the same sensation as cool. CNIR1 belongs to a member of the transient receptor potential (TRP) channel subfamily. CMRI is similar to VR1 and VRL1, which are other heat receptors that respond to noxious heat, and transports sensory information to the spinal cord and brain [Nagy L, Rang H .: Noxious heat activates all capsaicin-sensitive and a Neuroscience 88: 995-997, 1999] [Cesare P., McNaughton P .: A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin. Proc. Natl. Acad. sub-population of capsaicin insensitive dorsal root ganglion neurons. Sci. USA 93: 15435-15433% 1996].

  Recent electrophysiological studies of cultured dorsal root and trigeminal ganglion neurons have suggested a number of ion mechanisms underlying the detection of cryogenic temperatures in the periphery. Several candidate “cold receptors”, all of which are ion channel proteins, were suggested to be involved in this process. One candidate is TRPM8, a non-selective cation channel expressed in a subpopulation of sensory neurons. TRPM8 is activated by both temperature reduction and the cooling compound menthol. With combined fluorescent calcium imaging of cultured rat trigeminal neurons using single-cell RT-PCR, there is a distinct subpopulation of cold-responsive neurons and TRPM8 may contribute to cold transmission in one of them Proven to be high. TRPM8 is selectively expressed in a rapidly responsive and low threshold (less than 30 ° C.) cold sensitive neuron subset.

  The function of mouse TRPM8 was characterized as an ion channel opened by cold stimulation and menthol. In addition, its expression was limited to a subpopulation of pain and temperature sensitive DRG neurons [Peiet AM, Moqrich A., Hergarden AC, Reeve AJ, Andersson DA, Story GM, Earley TJ, Dragoni I., McIntyre P., Bevan S., Patapoutian A .: A TRP Channel that Senses Cold Stimuli and Menthol. Cell 108: 705-715, 2002].

  The present inventor has now found that mouse TrpM8 ion channel is also induced by noxious heat stimulation and can therefore affect pain. It also demonstrates that the TrpM8 ion channel is involved in stress and anxiety.

  According to a first aspect of the invention, a transgenic non-human animal having a functionally disrupted endogenous TrpM8 gene is provided. The TrpM8 gene comprises the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence having at least 70% sequence identity thereto.

  Preferably, the transgenic non-human animal has a deletion in the TrpM8 gene or a part thereof.

  Preferably, the animal is (a) preferably less sensitive to pain as measured by the tail flick test, (b) preferably measured by the open field test, when compared to the wild type animal. And (c) any one or combination of phenotypes of reduced plasma corticosterone levels.

  Furthermore, a transgenic non-human animal is provided, wherein at least part or all of the TrpM8 gene of the animal is replaced with a sequence of another animal, preferably another species, more preferably a human TrpM8 gene.

  Preferably, the transgenic non-human animal is a mouse.

  Preferably, said transgenic non-human animal comprises a functionally disrupted TrpM8 gene, preferably a deletion of the TrpM8 gene, said TrpM8 gene comprising at least 70 nucleic acid sequence as shown in SEQ ID NO: 4 or to them. It contains sequences with% sequence identity.

  According to a second aspect of the invention, there is provided a cell or tissue isolated from the non-human transgenic animal according to the first aspect of the invention.

  According to a third aspect of the present invention, there is provided a cell having a functionally disrupted endogenous TrpM8 gene, wherein the TrpM8 gene has the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or Includes sequences with at least 70% sequence identity to them.

  A fourth aspect of the present invention provides the use of a transgenic non-human animal, cell or tissue, or cell, respectively, as described above, as a model of pain or stress.

  According to a fifth aspect of the invention, there is provided the use of a transgenic non-human animal, cell or tissue, or cell, respectively as presented above, as a model for TrpM8-related disease.

  In a seventh aspect of the invention, a method of identifying an agonist or antagonist of a TrpM8 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto In non-human transgenic animals, as described respectively, isolated cells or tissues of the animals, or the use of cells.

  According to an eighth aspect of the invention, a method of identifying an agonist or antagonist of a TrpM8 polypeptide having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto Wherein the candidate compound is administered to an animal, preferably a wild-type animal or a transgenic non-human animal as presented above, as well as the following phenotype: (a) preferably measured in a tail flick test Measuring a change in any of: susceptibility to pain, (b) stress, preferably measured in an open field test, and (c) plasma corticosterone levels.

  Preferably, the method identifies an agonist of a TrpM8 polypeptide by identifying candidate compounds that are capable of exhibiting any increase in phenotype (a)-(c) in the animal.

  Alternatively or additionally, the method comprises identifying a TrpM8 polypeptide by identifying candidate compounds that are capable of exhibiting any of the phenotypes (a)-(c) or a decrease in such phenotype in the animal. Are identified.

  According to a ninth aspect of the invention, an agonist or antagonist of a TrpM8 polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto is identified. A method comprising exposing a candidate compound to a cell or tissue, preferably a wild type cell or tissue, or a cell or tissue or cell as described above, and the conductivity or intracellular of the cell or of the tissue Providing a method comprising measuring the change in calcium concentration r.

  Preferably, the method identifies an agonist of a TrpM8 polypeptide by identifying candidate compounds capable of increasing the conductivity or intracellular calcium concentration of the cell.

  Alternatively or additionally, the method identifies an antagonist of a TrpM8 polypeptide by identifying candidate compounds that can reduce the conductivity or intracellular calcium concentration of the cell.

  According to a tenth aspect of the present invention, there is provided a method for identifying a compound suitable for treating or alleviating pain or stress, preferably a TrpM8 related disease, comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, Or exposing a TrpM8 polypeptide comprising a sequence having at least 70% sequence identity to them to a candidate compound, and determining whether the candidate compound is an antagonist or antagonist of the TrpM8 polypeptide Provide a method.

  As an eleventh aspect of the invention, a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4 for identifying an agonist or antagonist for the treatment of pain or stress, preferably a TrpM8 related disease, or Use of TrpM8 polynucleotides comprising sequences having at least 70% sequence identity to them is provided.

  According to a twelfth aspect of the present invention, the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 for identifying pain or stress, preferably an agonist or antagonist for the treatment of TrpM8 related diseases, or to them Use of a TrpM8 polypeptide comprising a sequence having at least 70% sequence identity.

  According to a thirteenth aspect of the present invention, the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, or at least relative thereto, for use in a method of treatment of pain or stress in an individual, preferably a TrpM8 related disease An antagonist of a TrpM8 polypeptide having a sequence with 70% sequence identity is provided.

  According to a fourteenth aspect of the present invention, the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, or for the production of a pharmaceutical composition for the treatment of pain or stress in an individual, preferably a TrpM8 related disease There is provided the use of an antagonist of a TrpM8 polypeptide having a sequence having at least 70% sequence identity to.

  According to a fifteenth aspect of the present invention there is provided a method of treating an individual suffering from pain or stress, preferably suffering from a TrpM8-related disease, comprising the step of administering an antagonist of TrpM8 to the individual.

  According to a sixteenth aspect of the present invention, there is provided a method for diagnosing pain or stress in an individual, preferably a TrpM8-related disease, the method comprising detecting a change in TrpM8 expression, level or activity in an individual, or a cell or tissue thereof. A method comprising:

  Preferably, the TrpM8 related disease is pain, cancer, inflammation, inflammatory bowel disease, thermal hyperalgesia, visceral pain, migraine, postherpetic neuralgia, diabetic neuralgia, trigeminal neuralgia, postoperative pain, osteoarthritis, rheumatoid arthritis , Acute pain, chronic pain, skin pain, somatic pain, visceral pain, related pain including myocardial ischemia, phantom pain, neuropathic pain (neuralgia), pain due to injury and disease, headache, migraine, cancer pain Pain due to neurological disorders such as Parkinson's disease, pain due to spine and peripheral nerve surgery, brain tumor, traumatic brain injury (TBI), spinal cord injury, chronic pain syndrome, chronic fatigue syndrome; trigeminal neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia And neuralgia such as causalgia; pain due to lupus, sarcoidosis, arachnoiditis, arthritis, rheumatic diseases, menstrual pain, back pain, lower back pain, joint pain, abdominal pain, chest pain, labor pain, musculoskeletal Skin disease, diabetes, head trauma, and fibromyalgia; breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, and bone cancer; social anxiety, posttraumatic stress disorder, phobia, social phobia, specific Group consisting of phobia, panic disorder, obsessive compulsive disorder, acute stress disorder, separation anxiety disorder, generalized anxiety disorder, major depression, mood modulation, bipolar disorder, seasonal emotional disorder, postnatal depression, manic depression, bipolar depression More selected.

  According to a seventeenth aspect of the invention, a TrpM8 poly comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof having at least 70% sequence identity thereto A peptide is provided.

  According to an eighteenth aspect of the present invention there is provided a nucleic acid encoding such a polypeptide.

  Preferably, such nucleic acid comprises the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof having at least 70% sequence identity thereto .

The TRPM8 ion channel ion channel, in particular the TrpM8 ion channel, homologues, variants or derivatives thereof, and their use in the treatment and diagnosis of diseases including TrpM8 related diseases are described.

  TrpM8 is structurally related to other proteins in the ion channel family. This is shown by the results of sequencing the amplified cDNA product encoding human TrpM8. The cDNA sequence of SEQ ID NO: 1 comprises an open reading frame (SEQ ID NO: 2, nucleotide numbers 41 to 3352) encoding a polypeptide of 1104 amino acids shown in SEQ ID NO: 3. Human TrpM8 is known to be located on human (Homo sapiens) chromosome 2q37.

  In practicing the present invention, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the ability of one skilled in the art, are employed. Such techniques are explained in the literature. For example, J. Sambrook, EF Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, volumes 1-3, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons JM Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; MJ Gait (ed.), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; DMJ Lilley and JE Dahlberg, 1992 , Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA, Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Harlow (1999, Cold Spring) Harbor Laboratory Press, ISBN 0-87969-544-7) Antibodies: A Laboratory Manual, Harlow (ed), David Lane (ed) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson "Immunocytochemistry: Theory and Practice", CRC Press inc., Baca Raton, Florida, 1988, ISBN 0-8493-6078-1, John D. Pound (ed.); `` Immunochemical Protocols, Volume 80 '', `` Methods in Molecular Biology '' series, Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603-493-3, Handbook of Drug Screening, Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, edited by Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3; and The Merck Manual of Diagnosis and Therapy 17th edition, Beers, MH, and Berkow, R, edited by ISBN: 0911910107, John Wiley & Sons). Each of these general textbooks is incorporated herein by reference. Each of these general textbooks is incorporated herein by reference.

From the analysis of TrpM8 polypeptide (SEQ ID NO: 3) using the homology and similarity HMM structure prediction software pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml) with TRPM8 , TrpM8 Confirm that the peptide is an ion channel.

  A mouse homologue of the human TrpM8 ion channel was cloned, and its nucleic acid sequence and amino acid sequence are shown as SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The cDNA of the mouse TrpM8 ion channel of SEQ ID NO: 4 shows a high degree of identity with the sequence of the human TrpM8 ion channel (SEQ ID NO: 2), while the amino acid sequence of the mouse TrpM8 ion channel (SEQ ID NO: 5) is the human TrpM8 ion channel. It shows a high degree of identity and similarity with (SEQ ID NO: 3). Human and mouse TrpM8 ion channels are therefore members of a large ion channel family.

Expression profile of TRPM8 TrpM8 cDNA amplification by polymerase chain reaction (PCR) allows expression of TrpM8 in varying amounts in prostate (++++), liver (++++), muscle (+), testis (++), and ovary (+) Is detected.

  Using the TrpM8 cDNA of SEQ ID NO: 1, identity is found in the cDNA library by searching the human EST data source by BLASTN. This indicates that TrpM8 is expressed in these normal or abnormal tissues.

BE2744448.1 Human skin BE390627 Human uterus AW295430 Human prostate BG567490 Human liver BE791173 Human small cell lung cancer, MGC3
BE408880 Human placental choriocarcinoma BF244389 Human brain glioblastoma BG56597 Human liver BE207083 Human small cell lung cancer, MGC3
BE274448 human cutaneous melanin-deficient melanoma BE390627 human endometrial adenocarcinoma cell line

  Thus, TrpM8 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands can detect, diagnose, treat and otherwise detect diseases associated with excessive, under and abnormal expression of TrpM8 ion channels in these and other tissues. It is useful for the assay. Such diseases can include the TrpM8 related diseases presented below.

TRPM8 Ion Channel Related Diseases According to the methods and compositions described herein, TrpM8 ion channels are useful for the treatment and diagnosis of a range of diseases described in detail below. These diseases are referred to as TrpM8 related diseases for convenience.

  It is demonstrated herein that human TrpM8 maps to human chromosome 2q37. Thus, in certain embodiments, a TrpM8 ion channel that maps to this locus, chromosomal band, region, arm, or the same chromosome can be used to treat or diagnose a disease. For known diseases that are determined to be linked to the same locus, chromosomal band, region, arm, or chromosome as the chromosomal location of the TrpM8 ion channel (ie, human chromosome 2q37), the ion channel is upregulated. There is prostate cancer that was known to be done.

  Knockout mice lacking TrpM8 exhibit a range of phenotypes as demonstrated in the Examples.

  In particular, in Example 4 below, it is disclosed that TrpM8-deficient mice are less sensitive to painful analgesia, ie pain. TrpM8 and in particular modulators of TrpM8 activity including antagonists of TrpM8 can be used to treat or reduce diseases or syndromes characterized by pain. Specifically, TrpM8 activity or expression in an individual can be downregulated, for example, by administering an antagonist or blocker of TrpM8 for analgesia / pain reduction. Accordingly, the identification of TrpM8 antagonists for use as analgesics is disclosed.

  Thus, according to a preferred embodiment, pain and cancer using the methods and compositions described herein by any means as described herein, including TrpM8 ion channel and its modulators and antagonists. Can be diagnosed or treated or alleviated. In particular, pain includes neuropathic pain, inflammatory pain, inflammatory bowel disease, thermal hyperalgesia, visceral pain, migraine, postherpetic neuralgia, diabetic neuralgia, trigeminal neuralgia, postoperative pain, osteoarthritis, rheumatoid arthritis There is. Also included are acute pain, chronic pain, skin pain, somatic pain, visceral pain, related pain including myocardial ischemia, phantom pain, and neuropathic pain (neuralgia). The definition of pain includes pain due to injury and disease, headache, migraine, cancer pain, pain due to neurological disorders such as Parkinson's disease, pain due to spine and peripheral nerve surgery, brain tumor, traumatic brain injury (TBI), spinal cord Trauma, chronic pain syndrome, chronic fatigue syndrome; trigeminal neuralgia, glossopharyngeal neuralgia, neuralgia such as postherpetic neuralgia and causalgia; pain due to lupus, sarcoidosis, arachitis, arthritis, rheumatic diseases, menstrual pain, back pain, lower back Joint pain, abdominal pain, chest pain, labor pain, musculoskeletal and skin diseases, diabetes, head trauma, and fibromyalgia, but are not limited thereto. In particular, cancers include breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, and bone cancer.

  Example 5 describes an open field test, which shows that TrpM8 knockout mice are less susceptible to anxiety than wild-type equivalent animals. Furthermore, TrpM8 knockout mice are observed to have lower plasma corticosterone levels, which are indicators of stress and anxiety, than the corresponding wild type mice (Example 6). Thus, lack of TrpM8 activity correlates with reduced stress.

  Accordingly, disclosed is a method of reducing stress or anxiety, or both, in an individual, comprising reducing the level or activity of TrpM8 in the individual. As stated elsewhere, this can be achieved by down-regulating TrpM8 expression or by using antagonists to TrpM8.

  TrpM8 and in particular modulators of TrpM8 activity including antagonists of TrpM8 can be used to treat or reduce diseases or syndromes characterized by stress and anxiety. Such diseases include social anxiety, post-traumatic stress disorder, phobia, interpersonal phobia, specific phobia, panic disorder, obsessive compulsive disorder, acute stress disorder, isolated anxiety disorder, generalized anxiety disorder, major depression, If you have dysthymia, bipolar disorder, seasonal emotional disorder, postnatal depression, manic depression, or bipolar depression.

  In preferred embodiments, TrpM8-related diseases include diseases in which stress or anxiety is a symptom. In a highly preferred embodiment, the TrpM8 disease comprises the above list of anxiety and stress related diseases.

  As described above, the TrpM8 ion channel can be used to diagnose and / or treat any of these specific diseases using any of the methods and compositions described herein.

  In particular, nucleic acids, vectors containing TrpM8 ion channel nucleic acids, polypeptides (including homologues, variants or derivatives thereof), TrpM8 ion channel nucleic acids for the treatment or diagnosis of the specific diseases listed above And / or the use of pharmaceutical compositions, host cells, and transgenic animals comprising a polypeptide are specifically contemplated. Furthermore, a compound capable of interacting or binding to a TrpM8 ion channel, preferably an antagonist of TrpM8, preferably a compound capable of reducing the conductivity of the channel, in the diagnosis or treatment of the specific diseases mentioned above, TrpM8 Consider the use of antibodies against ion channels, as well as methods of making or identifying them. In particular, the use of any of these compounds, compositions, molecules, etc. in the manufacture of a vaccine for the treatment or prevention of a particular disease. Also disclosed are diagnostic kits for detecting specific diseases in an individual.

  Methods for linkage mapping to identify such or additional specific diseases that can be treated or diagnosed by use of TrpM8 ion channels are known in the art and are also described elsewhere herein.

Anxiety and stress Anxiety and stress and disorders in which they appear, including TrpM8-related diseases, are well known in the art. The outline is described below.

  Anxiety and stress are also referred to as frustrated mood, tension, terrible nervousness, and concern. Stress can arise from any situation or idea that makes an individual feel frustrated, angry, or anxious. Because one person has a lot of stress, it cannot be said that another person has a lot of stress.

  Anxiety is a feeling of concern or fear. The source of this worry is not necessarily known or recognized, and the source can add to the suffering felt by the individual.

  Stress is a normal part of life. In small quantities, stress can be beneficial. Stress can motivate an individual so that the individual is more productive. However, too much stress or a strong response to stress is detrimental. Stress can cause certain body or heart illnesses, such as infection, heart disease, or depression, as well as general health deterioration in the individual. Often, persistent and unweakening stress leads to anxiety and unhealthy behavior such as overeating and alcohol or drug abuse.

  Emotional conditions such as grief or depression, and health conditions such as hyperthyroidism, hypoglycemia, or heart attacks can also cause stress.

  Anxiety is often accompanied by physical symptoms including spasm or tremor, muscle tension, headache, sweating, dry mouth, difficulty swallowing, and abdominal pain (especially in children this may be the only symptom of stress).

  Sometimes dizziness, fast irregular heartbeat, respiratory distress, diarrhea or frequent urination, fatigue; irritability including diarrhea, difficulty sleeping, and nightmares; anxiety of other symptoms such as poor concentration and sexual problems Accompanying.

  TrpM8 and its modulators can be used to treat or alleviate any of these symptoms.

  Anxiety disorders are a group of mental states with excessive anxiety. Anxiety disorders include generalized anxiety disorder, specific phobia, obsessive compulsive disorder, and interpersonal phobia. See also TrpM8 related diseases above.

  Certain drugs, both recreational and pharmaceutical, can lead to anxiety symptoms due to either side effects or withdrawal from the drug. Such drugs include caffeine, alcohol, nicotine, cold drugs, decongestants, asthma bronchodilators, tricyclic antidepressants, cocaine, amphetamines, diet drugs, ADHD drugs, and thyroid drugs. Disclosed is the use of TrpM8 and its modulators in combination with those drugs to reduce the stress and / or anxiety inducing effects of such drugs.

  Poor diets such as low levels of vitamin B12 can also contribute to stress or anxiety. Performance anxiety relates to specific situations such as taking a test or presenting in public. Post-traumatic stress disorder (PTSD) is a stress disorder that occurs after a traumatic event such as war, physical or sexual assault, or natural disaster.

  In very rare cases, an adrenal tumor (chromocytoma) can be the cause of anxiety. This occurs due to overproduction of hormones responsible for the feelings and symptoms of anxiety.

(Medline Plus, adapted from http://www.nlm.nih.gov/medlineplus/ency/article/003211.htm)
pain
Acute pain Acute pain is defined as short-term pain or pain with an easily identifiable cause. Acute pain is when the body warns of the presence of a wound or disease in tissue. Acute pain is rapid and sharp, often followed by tingling pain. Acute pain is concentrated in one area and then spreads somewhat.

Chronic pain Chronic pain is medically defined as pain lasting more than 6 months. This continuous or intermittent pain often loses its purpose over time. That is because chronic pain does not help the body prevent damage. Chronic pain is often more difficult to treat than acute pain. Special care is generally needed to treat any pain that becomes chronic. When opioids are used for long periods, drug resistance, chemical dependence, and even psychological addiction can occur. Drug resistance and chemical dependence are common among opioid users, but psychological addiction is rare.

  Depending on the source and the associated nociceptors (the nerves that detect pain), psychological pain experiences can be grouped into four types.

Skin pain Skin pain is caused by damage to the skin or superficial tissue. The skin nociceptor endings are just below the skin, resulting in short, clear, local pain due to the high concentration of nerve endings. Exemplary injuries that cause skin pain include paper cuts, minor (degree I) burns and lacerations.

Somatic pain Somatic pain originates from ligaments, tendons, bones, blood vessels, and even the nerve itself, and is detected by somatic nociceptors. Lack of pain receptors in these areas results in dull pain with poor localization that lasts longer than skin pain. Examples include ankle sprains and fractures.

Visceral pain Visceral pain originates from body organs, and visceral nociceptors are localized in body organs and lumens. The lack of nociceptors in these areas results in pain that usually tingles and lasts longer than somatic pain. Visceral pain is extremely difficult to define and various damages to visceral tissue indicate “related” pain. In associated pain, the sensation is localized in an area that has nothing to do with the site of injury. Myocardial ischemia (lack of blood flow to portions of myocardial tissue) may be the most well-known example of associated pain. The sensation can occur as a tingling pain in the upper chest as a sense of restraint, or in the left shoulder, arm, or even the hand.

Another type of pain phantom is a pain sensation from a foot that no longer has or a limb that no longer gives a physical signal, an experience reported in almost all limb amputees and limb paralysis patients. Neuropathic pain (neuralgia) can occur as a result of damage or disease to the nerve tissue itself. This may destroy the ability of sensory nerves to transmit the right information to the thalamus, so that the brain is painful even if there is no obvious or proven physiological cause for pain. Interpret the stimulus.

  Trigeminal neuralgia (“painful tic”) refers to pain caused by injury or wound to the trigeminal nerve. The trigeminal nerve has three branches. V1 gives sensation to the forehead and eye area, V2 gives sensation to the nose and face, and V3 gives sensation to the chin and the throat area. Each side of the face has a trigeminal nerve that gives a sense. Trilateral neuralgia unilateral pain may spread via the cheek, mouth, nose, and / or jaw muscles. Trigeminal neuralgia generally affects older people, but adolescents or those with multiple sclerosis may also experience trigeminal neuralgia.

  The primary symptom of trigeminal neuralgia is pain in either the forehead, cheeks, throat, or mandibular contour. In severe cases, all three regions or both left and right sides can be affected. The onset of pain is severe, convulsive, and brief, and is described as being similar to the pain you would feel as an electric shock. The pain can be triggered by common daily activities such as brushing, speaking, chewing, drinking, shaving or even kissing. The frequency of pain events increases with time, becoming increasingly destructive and depriving.

  Glossopharyngeal neuralgia is a clinical form characterized by an explosion of pain in the sensory distribution of the ninth cranial nerve. Seizures are identical to trigeminal neuralgia except for pain localization and pain stimulation. A typical pain is an electric shock-like puncture that repetitively pierces the tonsils on one side or the back of the tongue. In addition, the pain may dissipate or occur from the ear.

  The sensory stimulus that induces the pain is swallowing, during which a patient sits still, bends his head forward and leaves saliva dripping from the mouth. Cardiac arrest, syncope (fall), and epileptic seizures have been associated with seizures of glossopharyngeal neuralgia. The cause of glossopharyngeal neuralgia is not known in most cases. However, some cases are attributed to tumors, compression of the ninth nerve by vertebral arteries, and vascular malformations.

  Postherpetic neuralgia refers to chronic pain that persists after herpes zoster virus infection. Shingles, also known as herpes zoster, is a recurrent infection of varicella-zoster (varicella) virus infection. This virus lies in the nerve until the patient's immunity is weakened. Acute lesions of herpes zoster cause pain, which usually heals. However, in some patients pain continues chronically. This is postherpetic neuralgia.

  The symptoms of shingles include deep, continuous piercing pain that is 65% in the chest and 20% in the face. If the face is affected, the virus shows a prevalence to the trigeminal optic nerve branch (the upper edge of the face above the eyebrows). This pain resolves spontaneously, usually in 2 to 4 weeks. However, a small number of patients may have persistent pain. The pain is present in the area of the previous rash and is exacerbated by gently stroking the affected skin and reduced by applying pressure to the area. Clothing friction is often very painful. This persistent pain is called postherpetic neuralgia. The incidence of postherpetic neuralgia is high in cases of shingles that affect the face.

  Causalgia is a rare syndrome following partial peripheral nerve injury. Causalgia is characterized by a triple of burning pain, autonomic dysfunction, and nutritional changes. Severe cases are called severe causalgia. Mild causalgia presents a less severe form similar to reflex sympathetic dystrophy (RSD). RSD has major muscle and joint symptoms and osteoporosis is common on X-rays.

  Causalgia is caused by peripheral nerve damage, usually brachial plexus damage. Nephrectomy results in increased pain as a result of hypersensitivity and increased norepinephrine release results in sympathetic findings. Symptoms include pain, usually burning pain, which is prominent on the hands or feet. The majority of onset is within 24 hours of injury. The median nerve, ulnar nerve, and sciatic nerve are usually affected. Almost any sensory stimulus exacerbates this pain. That is, either vascular changes, either blood increase due to vasodilation (warm, pink) or blood decrease due to vasoconstriction (cold, mottled blue), nutritional changes, ie dry / scaled skin, joint stiffness, tapered There are changes in perspiration, fingers, ungrooved nails, either long / stiff hair or hair loss.

TrpM8 ion channel polypeptide As used herein, the term “TrpM8 ion channel polypeptide” means a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof. . Preferably, the polypeptide comprises or is a homologue, variant or derivative of the sequence shown in SEQ ID NO: 3.

  The term “polypeptide” refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds, or to two or more amino acids linked to each other by modified peptide bonds, ie any peptide isostere. Also refers to things. “Polypeptide” refers to both short and long chains, the former usually referred to as peptides, oligopeptides or oligomers, and the latter typically referred to as proteins. Polypeptides may also contain amino acids other than the 20 amino acids encoded by the gene.

  “Polypeptides” include amino acid sequences modified by natural processes such as post-translational processing as well as amino acid sequences modified by chemical modification techniques well known in the art. Such modifications are described in detail in basic textbooks and in more detailed research books, as well as numerous research papers. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. A given polypeptide may also contain many types of modifications.

  Polypeptides may be branched as a result of ubiquitination, and polypeptides may be cyclic, in which case they may be branched or unbranched There is also. Cyclic polypeptides, branched polypeptides, and cyclic branched polypeptides can occur in natural post-translational processes or can be produced synthetically. Modifications include acetylation, acylation, ADP ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond, Cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cystine formation, pyroglutamic acid formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation , Transfer RNA-mediated amino acid addition to proteins, such as myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination It is. For example, `` Proteins-Structure and Molecular Properties '', 2nd edition, TE Creighton, WH Freeman and Company, New York, 1993, and Wold, F., `` Posttranslational Protein Modifications: Perspectives and Prospects '', `` Posttranslational Covalent '' 1-12 in `` Modification of Proteins '', BC Johnson, Academic Press, New York, 1983; Seifter et al., `` Analysis for protein modifications and nonprotein cofactors '', Meth Enzymol (1990), 182: 626-646, And Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci (1992), 663: 48-62.

  As used herein, the term “variant”, “homolog”, “derivative”, or “fragment” includes any substitution of one (or more) amino acids from or to a sequence. Substitution, mutation, modification, replacement, deletion, or addition is also included. Unless otherwise noted by context, references to “TrpM8” and “TrpM8 ion channel” include references to such TrpM8 variants, homologues, derivatives, and fragments.

  Preferably, when referring to TrpM8, the resulting amino acid sequence has ion channel activity, more preferably at least the same activity as the TrpM8 ion channel shown in SEQ ID NO: 3 or SEQ ID NO: 5. In particular, the term “homologue” encompasses identity with respect to structure and / or function as long as the resulting amino acid sequence has ion channel activity. With respect to sequence identity (ie similarity) this is preferably at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. This is more preferably at least 95% sequence identity, more preferably at least 98% sequence identity. These terms also encompass polypeptides derived from amino acids that are allelic changes of the TrpM8 ion channel nucleic acid sequence.

  When referring to “channel activity” or “biological activity” of an ion channel, such as a TrpM8 ion channel, these terms shall refer to the metabolic or physiological function of the TrpM8 ion channel, which includes similar Included are those activities that are active or improved or that have reduced attendant undesirable side effects. Also included are antigenic and immunogenic activities of the TrpM8 ion channel. Examples of ion channel activity and methods for assaying and quantifying these activities are known in the art and are described in detail elsewhere herein.

  As used herein, a “deletion” is defined as a change in either the nucleotide sequence or amino acid sequence, wherein one or more nucleotides or amino acid residues are absent, respectively. . As used herein, “insertion” or “addition” is a change in nucleotide sequence or amino acid sequence, each of one or more nucleotides or amino acid residues compared to a naturally occurring substance. It is a change that brings about an addition. As used herein, “substitution” occurs by replacing one or more nucleotides or amino acids with different nucleotides or amino acids, respectively.

  The TrpM8 polypeptides described herein may have amino acid residue deletions, insertions or substitutions that cause silent changes and result in functionally equivalent amino acid sequences. Careful amino acid substitutions can be introduced based on residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic similarity. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and uncharged polar head groups with similar hydrophilicity. Amino acids possessed include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column, preferably amino acids in the same row in the third column may be substituted for each other:

  A TrpM8 polypeptide may comprise a heterologous amino acid sequence, typically at its N-terminus or C-terminus, preferably at its N-terminus. Heterologous sequences include sequences (such as leader sequences) that affect intracellular or extracellular protein targeting. Heterologous sequences also include sequences that enhance the immunogenicity of the polypeptide and / or facilitate identification, extraction and / or purification of the polypeptide. Another particularly preferred heterologous sequence is a polyamino acid sequence (such as polyhistidine) which is preferably present at the N-terminus. Particularly preferred are polyhistidine sequences of at least 10 amino acids, preferably at least 17 amino acids, but less than 50 amino acids.

  The TrpM8 ion channel polypeptide may be in the form of a “mature” protein or may be part of a larger protein such as a fusion protein. It is advantageous to include an additional amino acid sequence including a secretory or leader sequence, a pro sequence, a sequence assisting purification (such as multiple histidine residues), or an additional sequence for stability during recombinant production There is also.

  The TrpM8 polypeptide is advantageously produced using known methods by recombinant techniques. However, they can also be made by synthetic techniques using techniques well known to those skilled in the art, such as solid phase synthesis. The polypeptides described herein can also be made as fusion proteins, eg, to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain) and β-galactosidase. It is also convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of the fusion protein sequence (eg, a thrombin cleavage site). Preferably, the fusion protein does not interfere with the function of the protein sequence of interest.

  The TrpM8 polypeptide can exist in a substantially isolated form. This term means artificially changing from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide, nucleic acid or polypeptide naturally occurring in a living organism is not “isolated”, but the same polynucleotide, nucleic acid or polypeptide separated from a coexisting material in the natural state is the term Are “isolated” as used herein.

  However, it is understood that the TrpM8 ion channel protein may be mixed with a carrier or diluent that does not interfere with the intended purpose of the protein and still be considered substantially isolated. The TrpM8 polypeptide may also be in a substantially purified form, in which case 90% or more, eg 95%, 98% or 99% of the protein in the preparation is in the preparation where the TrpM8 polypeptide is present. In general.

  The inventor further describes peptides comprising a portion of a TrpM8 polypeptide. Thus, it includes fragments of TrpM8 ion channel and homologues, variants or derivatives thereof. Peptides can be 2 to 200 amino acids long, preferably 4 to 40 amino acids long. Peptides can be derived from TrpM8 polypeptides as described herein, for example, by digestion with a suitable enzyme (such as trypsin). Alternatively, peptides, fragments, etc. can be produced by recombinant techniques or synthetic techniques.

  The term “peptide” includes various synthetic peptide changes known in the art, such as retro-inverso D peptides. The peptide may be an antigenic determinant and / or a T cell epitope. The peptide may be immunogenic in vivo. Preferably, the peptide is capable of inducing neutralizing antibodies in vivo.

  By aligning TrpM8 ion channel sequences from different species, regions of amino acid sequences conserved between different species (homologous regions) and regions that differ between different species (non-homologous regions ( heterologous region)) can be determined.

  Accordingly, the TrpM8 polypeptide may include a sequence corresponding to at least a part of the homologous region. A homologous region shows high homology between at least two species. For example, a homologous region may exhibit at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identity at the amino acid level using the tests described above. Peptides containing sequences corresponding to homologous regions can be used in therapeutic methods as described in more detail below. Alternatively, the TrpM8 ion channel peptide may include a sequence corresponding to at least part of the heterologous region. A heterologous region shows low homology between at least two species.

TrpM8 ion channel polynucleotides and nucleic acids Further disclosed are TrpM8 polynucleotides, TrpM8 nucleotides and TrpM8 nucleic acids, methods for their production, uses, etc., as described in further detail herein.

  The terms “TrpM8 polynucleotide”, “TrpM8 nucleotide” and “TrpM8 nucleic acid” are used interchangeably and include a nucleic acid / nucleic acid comprising the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or It is intended to mean a homologue, variant or derivative. Preferably, the polynucleotide / nucleic acid comprises or is a homologue, variant or derivative of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, most preferably SEQ ID NO: 2.

  These terms are also intended to encompass nucleic acids sequences that can encode polypeptides and / or peptides, ie, TrpM8 polypeptides. Accordingly, TrpM8 ion channel polynucleotides and nucleic acids comprise a nucleotide sequence that can encode a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof. Preferably, the TrpM8 ion channel polynucleotide and nucleic acid comprise a nucleotide sequence capable of encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, or a homologue, variant or derivative thereof.

  In general, “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleoside, which can be unmodified RNA or DNA, or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single stranded and double stranded DNA, DNA that is a mixture of single stranded and double stranded regions, single stranded RNA and double stranded RNA, single stranded RNA, which is a mixture of regions and double stranded regions, and hybrid molecules comprising DNA and RNA, these hybrid molecules may be single stranded, but more typically double stranded Or a mixture of single and double stranded regions. In addition, “polynucleotide” also refers to triple-stranded regions comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA having a backbone modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications have been made to DNA and RNA, and thus “polynucleotides” are not only the chemical forms of DNA and RNA characteristic of viruses and cells, but also the chemically modified forms, enzymes normally found in nature Also included are modified or metabolically modified polynucleotides. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.

  One skilled in the art will appreciate that as a result of the degeneracy of the genetic code, multiple nucleotide sequences can encode the same polypeptide.

  As used herein, the term “nucleotide sequence” refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences, and variants, homologues, fragments, and derivatives thereof (such as portions thereof). Nucleotide sequences may be DNA, RNA, genome-derived, chemically synthesized or recombinant-derived, representing either double-stranded, single-stranded, sense strand or antisense strand However, it may represent a combination thereof. The term nucleotide sequence may mean that prepared by use of recombinant DNA techniques (eg, recombinant DNA).

  The term “nucleotide sequence” preferably means DNA.

  As used herein, the term “variant”, “homolog”, “derivative”, or “fragment” includes 1 (or from a sequence of a TrpM8 nucleotide sequence or from a sequence of a TrpM8 nucleotide sequence. Any substitution, mutation, modification, substitution removal, deletion, or addition of nucleic acid (s) is included. Unless otherwise noted by context, references to “TrpM8” and “TrpM8 ion channel” include references to such TrpM8 variants, homologues, derivatives, and fragments.

  Preferably, the resulting nucleotide sequence encodes a polypeptide having ion channel activity, preferably a polypeptide having at least the same activity as the ion channel shown in SEQ ID NO: 3 or SEQ ID NO: 5. Preferably, the term “homologue” is intended to encompass identity in terms of structure and / or function as long as the resulting nucleotide sequence encodes a polypeptide having ion channel activity. With respect to sequence identity (ie similarity) this is preferably at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. This is more preferably at least 95% sequence identity, more preferably at least 98% sequence identity. These terms also encompass allelic variations of the above sequences.

Calculation of Sequence Homology Sequence identity for any of the sequences presented herein is determined by a simple “eyeball” comparison (ie, strict comparison) of any one or more sequences with another sequence, It can be ascertained whether a sequence has, for example, at least 70% sequence identity to the sequence (s).

  Relative sequence identity can also be determined by a commercially available computer program that can calculate% identity between two or more sequences, for example using any suitable algorithm for determining identity using default parameters. Can do. A typical example of such a computer program is CLUSTAL. Other computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).

  % Homology can be calculated over contiguous sequences, i.e., aligning one sequence with another sequence and directly comparing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. This is referred to as “no gap” alignment. Typically, such ungapped alignments are only performed on a relatively small number of residues.

  This is a very simple and consistent method, but for example, in other identical pairs of sequences, one insertion or deletion occurred after the amino acid residue subjected to the alignment, thus performing an overall alignment. In some cases, this does not take into account that the% homology may result in a significant decrease. As a result, most sequence comparison methods are designed to obtain optimal alignments that take into account possible insertions and deletions so as not to unduly penalize the overall homology score. This is done by inserting “gaps” in the sequence alignment to maximize local homology.

  However, these more complex methods give a “gap penalty” for each gap that occurs in the alignment, and for the same number of identical amino acids, sequence alignments with as few gaps as possible (the higher association between the two comparison sequences). Reflect a higher score than sequences with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap score system. Create an optimal alignment with fewer gaps as well as higher gap penalties. Most alignment programs can change the gap penalty. However, it is preferred to use the default values when using such sequence comparison software. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

  Therefore, the calculation of the maximum homology requires that the optimum alignment is first performed in consideration of the gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A .; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparison include, but are not limited to, the BLAST package (Ausubel et al., 1999, supra, Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the comparison tool GENEWORKS package software. Both BLAST and FASTA are available by offline and online search (Ausubel et al., 1999, supra, pages 7-58 to 7-60).

  Although the final% homology can be determined in terms of identity, the alignment process itself is typically not based on an “all or nothing” pairwise comparison. Instead, a scaled similarity score matrix is commonly used, which assigns a score for each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix, which is the default matrix for the program's BLAST package software. The GCG Wisconsin program typically uses either public default values or custom symbol comparison tables if supplied. It is preferred to use public default values for the GCG package, or in the case of other software, a default matrix such as BLOSUM62.

  It is advantageous to use the BLAST algorithm with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporated herein by reference. Search parameters can be defined and can be advantageously set against defined default parameters.

  When assessed by BLAST, “substantial identity” advantageously corresponds to sequences that match EXPECT values of at least about 7, preferably at least about 9, and most preferably 10 or more. The threshold of EXPECT in the BLAST search is normally 10.

  BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm used by the programs blastp, blastn, blastx, tblastn, and tblastx, these programs are Karlin and Altschul (Karlin and Altschul 1990, Proc. Natl. Acad). Sci. USA 87: 2264-68; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-7; see http://www.ncbi.nih.gov/BLAST/blast_help.html) The significance is attributed to these findings using slight improvements to the statistical method. The BLAST program is tailored for sequence similarity searches, for example, to identify homologies to query sequences. For a discussion of basic issues in sequence database similarity searches, see Altschul et al (1994) Nature Genetics 6: 119-129.

  Five BLAST programs are available from http://www.ncbi.nlm.nih.gov that perform the following tasks: compare amino acid query sequences against blastp-protein sequence database; blastn-nucleotide sequence database Compare 6 nucleotide conceptual translation products of nucleotide query sequence (both strands) against blastx-protein sequence database; compare protein query sequence against tblastn-nucleotide sequence database Dynamically translate and compare all 6 reading frames (both strands); compare 6 frame translation of nucleotide query sequence against 6 frame translation of tblastx-nucleotide sequence database.

BLAST uses the following search parameters:
HISTOGRAM—Displays a histogram of scores for each search. The default is yes (see parameter H in the BLAST Manual).

DESCRIPTIONS-Limits the number of short descriptions of reported matching sequences to the specified number. The default limit is a description of 100 (see parameter V in the manual page).

EXPECT-Statistical significance threshold for reported matches against database sequences. The default value is 10, which is expected to result in 10 matches simply by chance according to the probabilistic model of Karlin and Altschul (1990). If the statistical significance given to a match is greater than the EXPECT threshold, the match will not be reported. The lower the EXPECT threshold, the more stringent and the less likely matches are reported. Decimal values are allowed (see parameter E in the BLAST Manual).

CUTOFF—Cutoff score that reports high score segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for database sequences only if the statistical significance given to them is at least as high as that given to a single HSP with a score equal to the CUTOFF value. The higher the CUTOFF value, the higher the strictness and the less chance of a match being reported (see parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.

ALIGNMENTS—Limits the database sequence to a specific number that reports high-scoring segment pairs (HSPs). The default limit is 50. If there are more database sequences than this would occur to meet the statistical significance threshold for the report, only the match given the greatest statistical significance is reported (see parameter B in the BLAST Manual) )

Specify alternative score matrices for MATRIX-BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). Valid alternative choices include PAM40, PAM120, PAM250 and IDENTITY. An alternative score matrix is not available for BLASTN and returns to an error response by specifying a MATRIX command in BLASTN.

Limit the STRAND-TBLASTN search to only the top or bottom strand of the database sequence; or limit the BLASTN, BLASTX or TBLASTX search to only the reading frame in the top or bottom strand of the query sequence.

FILTER-Wootton & Federhen (1993) Computers and Chemistry 17: 149-163, a segment of a query sequence with low compositional complexity as assessed by the SEG program, or Claverie & States (1993) Computers and Chemistry 17: 191-201 Eliminates segment masks consisting of short-period internal repeats as evaluated by the XNU program or in the case of BLASTN by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov) To do. Filtering can eliminate statistically significant but biologically insignificant reports from the blast output (eg hits against common acidic, basic or proline rich regions) and specific to database sequences Leave more biologically important regions of the query sequence available for matches.

  Low complexity sequences discovered by the filter program use the letter “N” in the nucleotide sequence (eg “NNNNNNNNNNNNN”) and the letter “X” in the protein sequence (eg “XXXXXXXXX”) Replaced.

  Filtering applies only to query sequences (or their translation products), not database sequences. The default filtering is DUST for BLASTN and SEG for other programs.

  When applied to sequences in SWISS-PROT, it is not normal that nothing is masked by SEG, XNU, or both, so filtering should not always be expected to be effective. Further, in some cases, the entire sequence may be masked, which may be suspicious of any statistical significance of matches reported against unfiltered query sequences.

In addition to the NCBI-gi-accession and / or locus name, the NCBI gi identifier is indicated in the output.

  Most preferably, the sequence comparison is performed using a simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST. In some embodiments, no gap penalty is used when determining sequence identity.

Hybridization Further disclosed are nucleotide sequences that are capable of hybridizing to the sequences presented herein, or any fragment or derivative thereof, or any of the above complementary sequences.

  Hybridization means “a process in which a single strand of nucleic acid binds to a complementary strand by base pairing” (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY), and Dieffenbach CW and GS Dveksler (1995). , PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY).

  Hybridization conditions are determined by the melting temperature (Tm) of the nucleic acid binding complex as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA). Based on this, the predetermined “stringency” described below is provided.

  Nucleotide sequences that are selectively hybridizable to the nucleotide sequences presented herein or their complementary sequences are generally at least 20, preferably at least 25, or relative to the corresponding nucleotide sequences presented herein. Over at least 70, preferably at least 75%, more preferably at least 85% or 90%, even more preferably at least 95% or 98% over a region of 30, eg at least 40, 60 or 100 or more contiguous nucleotides Have homology. Preferred nucleotide sequences comprise a region homologous to SEQ ID NO: 1, 2 or 4 and are at least 70%, 80% or 90%, more preferably at least 95% homologous to one of these sequences Have

The term “selectively hybridizable” is used under conditions where the nucleotide sequence used as a probe is found to hybridize with the probe at a level where the target nucleotide sequence is significantly above background. Means. Background hybridization can occur due to other nucleotide sequences such as those present in the cDNA or genomic DNA library being screened. In this event, the background is between the probe and the non-specific DNA member of the library at a strength less than 10 times, preferably less than 100 times compared to the specific interaction observed with the target DNA. It shows the level of signal produced by the interaction. The strength of the interaction can be measured, for example, by radioactively labeling the probe (eg, using ³² P).

  Also encompassed are nucleotide sequences that can hybridize to the nucleotide sequences presented herein under moderate to maximal stringency conditions. Hybridization conditions are determined by the melting temperature (Tm) of the nucleic acid binding complex as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA). Based on this, the predetermined “stringency” described below is provided.

  Typically, maximum stringency is approximately Tm-5 ° C (5 ° C below the Tm of the probe), high stringency is about 5 ° C to 10 ° C below Tm, and moderate stringency is At temperatures about 10-20 ° C. below Tm, low stringency occurs at temperatures about 20-25 ° C. below Tm. As can be appreciated by one skilled in the art, maximal stringency hybridization can be used to identify or detect identical nucleotide sequences, and moderate (or low) stringency hybridization can be similar or It can be used to identify or detect related nucleotide sequences.

In a preferred embodiment, stringent conditions (e.g., 65 ° C. and 0.1 × SSC {1 × SSC = 0.15M NaCl, 0.015M citric acid _Na 3 pH 7.0}) of the TrpM8 ion channel nucleotide sequences 1 Nucleotide sequences capable of hybridizing to the above are disclosed. When the nucleotide sequence is double stranded, both strands of the double strand are included individually or in combination. It should be understood that if the nucleotide sequence is single stranded, it may also be a complementary sequence of the nucleotide sequence.

Disclosed are nucleotide sequences that are hybridizable to sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. Similarly, the present disclosure encompasses nucleotide sequences that are complementary to sequences that are capable of hybridizing to the above sequences. These types of nucleotide sequences are examples of variant nucleotide sequences. In this regard, the term “variant” encompasses sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences presented herein. Preferably, however, the term "variant" stringent conditions (e.g., 65 ° C. and 0.1 × SSC {1 × SSC = 0.15M NaCl, 0.015M citric acid _Na 3 pH 7.0}) in It includes sequences that are complementary to sequences that can hybridize to the nucleotide sequences presented herein.

Cloning of TrpM8 Ion Channels and Homologues The present disclosure encompasses nucleotide sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. If the sequence is complementary to the fragment, the sequence can be used as a probe to identify and clone similar ion channel sequences in other organisms, and the like.

  This makes it possible to clone TrpM8 ion channels, their homologues derived from humans and other species (mouse, pig, sheep, etc.) and other structurally or functionally related genes. A polynucleotide having the same or sufficient identity as the nucleotide sequence contained in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or a fragment thereof is a full-length or partial cDNA encoding a TrpM8 ion channel from a suitable library and It can be used as a hybridization probe for cDNA and genomic DNA to isolate genomic clones. Such probes also include cDNA of other genes having sequence similarity, preferably high sequence similarity to the TrpM8 ion channel gene, including genes encoding homologs and orthologs from species other than humans, and Can be used to isolate genomic clones. Hybridization screening, cloning and sequencing techniques are known to those skilled in the art and are described, for example, in Sambrook et al (supra).

  Typically, a nucleotide sequence suitable for use as a probe has 70% identity, preferably 80% identity, more preferably 90% identity, even more preferably to the subject sequence. It has 95% identity. A probe generally can comprise at least 15 nucleotides. Such probes preferably have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes are in the range of 150-500 nucleotides, more particularly about 300 nucleotides.

  In one embodiment, to obtain a polynucleotide encoding a TrpM8 polypeptide (including homologs and orthologs derived from non-human species), SEQ ID NO: 1, SEQ ID NO: 2, sequence under stringent hybridization conditions Screening a suitable library with a labeled probe having number 4 or a fragment thereof, isolating full-length or partial cDNA and genomic clones containing such polynucleotide sequences. Such hybridization techniques are well known to those skilled in the art. Stringent hybridization conditions are as described above, or alternatively 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% Conditions with overnight incubation at 42 ° C. in a solution containing dextran sulfate and 20 μg / ml denatured sheared salmon sperm DNA, followed by washing of the filter at 0.1 ° SSC at 65 ° C.

Functional assay of TrpM8 ion channel The cloned putative TrpM8 ion channel polynucleotide can be verified by sequence analysis or functional assay. In particular, the conductivity of Xenopus oocytes transfected as described above can be detected as a means of guaging and quantifying TrpM8 activity, useful in the screening assays described below. Such a conductivity assay is referred to as a “TrpM8 functional assay (conductivity)” for simplicity.

A putative TrpM8 ion channel or homologue can be assayed for activity in a “TrpM8 functional assay (conductivity)” as follows. Capped RNA transcripts from linearized plasmid templates encoding TrpM8 ion channel cDNA are synthesized in vitro using RNA polymerase according to standard procedures. The in vitro transcript is suspended in water to a final concentration of 0.2 mg / ml. The leaf ovary is removed from the adult female toad, stage V ovulated oocytes are obtained, and the RNA transcript (10 ng / oocyte) is injected with a 50 nl bolus using a microinjection device. Two electrode voltage clamps are used to measure the current from individual Xenopus oocytes in response to agonist exposure. Recording is performed at room temperature in a solution of 96 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 0.1 mM CaCl ₂ , 5 mM Hepes, pH 7.4 with 200 mM mannitol. OSM is 210 mOsm. The Xenopus system can also be used to screen known ligands and tissue / cell extracts for ligand activation as described in further detail below.

  Other functional assays include whole cell electrophysiology, fluorescence resonance energy transfer (FRET) analysis and FLIPR analysis.

TrpM8 Ion Channel Expression Assay Determining the expression profile of TrpM8 (wild type or specific mutants) is useful for designing therapeutic agents useful for treating TrpM8 ion channel related diseases. Thus, methods known in the art will be used to determine the organ, tissue, and cell type (also developmental stage) in which TrpM8 is expressed. For example, traditional Northern or “electronic” Northern can be performed. Reverse transcriptase PCR (RT-PCR) can also be used to assay expression of the TrpM8 gene or mutant. More sensitive methods for determining the expression profile of TrpM8 include RNAse protection assays known in the art.

  Northern analysis is a laboratory technique used to detect the presence of gene transcripts and involves the hybridization of labeled nucleotide sequences to membranes bound with RNA from specific cell types or tissues ( Sambrook, supra, chapter 7 and Ausubel, FM et al., Supra, chapters 4 and 16). Similar computer techniques utilizing BLAST ("electronic Northern") can be used to search for identical or related molecules in nucleotide databases such as the GenBank database or the LIFESEQ database (Incyte Pharmaceuticals). The advantage of this type of analysis is that they are faster than multiple membrane-based hybridizations. In addition, computer searches can determine whether a particular match is classified as accurate or homologous by changing its sensitivity.

  The polynucleotides and polypeptides described herein, including the probes described above, can be used to discover treatments and diagnoses for animal and human diseases, as described in more detail elsewhere herein. Can be used as a reagent and substance.

Expression of TrpM8 Ion Channel Polypeptide Further disclosed is a method for producing a TrpM8 polypeptide. The method generally involves culturing a host cell comprising a nucleic acid encoding a TrpM8 ion channel polypeptide, or a homologue, variant or derivative thereof, under suitable conditions (ie, conditions under which the TrpM8 ion channel polypeptide is expressed). Including that.

  In order to express a biologically active TrpM8 ion channel, a nucleotide sequence encoding the TrpM8 ion channel, or a homologue, variant or derivative thereof, is converted into an appropriate expression vector, ie, an inserted coding sequence. It is inserted into a vector containing the elements necessary for transcription and translation.

  Construction of an expression vector containing a sequence encoding a TrpM8 ion channel and appropriate transcriptional and translational control elements is performed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989; “Molecular Cloning, A Laboratory Manual”, chapters 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, NY), and Ausubel, (1995 and periodic supplements; “Current Protocols in Molecular Biology”, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY).

  A variety of expression vector / host systems that contain and express sequences encoding the TrpM8 ion channel are available. These include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines infected with viral expression vectors (eg, baculovirus) Plant cell lines transformed with viral expression vectors (eg cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or bacterial expression vectors (eg Ti or pBR322 plasmid); or animal cell lines However, it is not limited to these. The nature of the host cell used is not a problem.

  “Control elements” or “regulatory sequences” are untranslated regions of a vector (ie, enhancers, promoters, and 5 ′ and 3 ′ untranslated regions) that interact with host cell proteins. Transcription and translation are performed. The strength and specificity of such elements can vary. Depending on the vector system and host utilized, a variety of suitable transcription and translation elements can be used, including constitutive and inducible promoters. For example, when cloning into bacterial systems, inducible promoters such as BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) Or PSPORT1 plasmid (GIBCO / BRL) hybrid lacZ promoter, and the like may be used. it can. In insect cells, the baculovirus polyhedrin promoter can be used. Promoters or enhancers derived from plant cell genomes (eg, heat shock, RUBISCO, and storage protein genes), or those derived from plant viruses (eg, viral promoters or leader sequences) can be cloned into vectors. . In mammalian cell systems, promoters from mammalian genes or mammalian viruses are preferred. If it is necessary to create a cell line containing multiple copies of a sequence encoding the TrpM8 ion channel, an SV40-based or EBV-based vector with an appropriate selectable marker can be used.

  In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the TrpM8 ion channel. For example, if a large amount of TrpM8 ion channel is required to induce antibodies, vectors that direct high level expression of easily purified fusion proteins can be used. Without limitation, such vectors include BLUESCRIPT (Stratagene), pIN vector (Van Heeke, G. and SM Schuster (1989), J. Biol. Chem. 264: 5503-5509), Multifunctional E. coli cloning and expression vectors, such as and the like, in BLUESCRIPT, the sequence encoding the TrpM8 ion channel, the amino terminal Met of β-galactosidase, followed by the 7 residue sequence It can be ligated into the vector so that it is in-frame, thereby producing a hybrid protein. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. . Proteins produced in such systems can be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released freely from the GST moiety. .

  In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For a review, see Ausubel (supra) and Grant et al. (1987; Methods Enzymol. 153: 516-544).

  When using plant expression vectors, the expression of the sequence encoding the TrpM8 ion channel can be driven by any of a number of promoters. For example, viral promoters such as CaMV 35S and 19S promoters can be used alone or in combination with TMV omega leader sequences (Takamatsu, N. (1987), EMBO J. 6: 307-311). . Alternatively, plant promoters such as the RUBISCO small subunit promoter or heat shock promoter can be used (Coruzzi, G. et al. (1984), EMBO J. 3: 1671-1680; Broglie, R. et al. ( 1984), Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into these plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (eg, Hobbs, S. or Murry, LE, McGraw Hill, New York, McGraw Hill Yearbook of Science and Technology (1992)). NY; see pages 191-196).

  Insect systems can also be used to express the TrpM8 ion channel. For example, in one such system, autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. Use. The sequence encoding the TrpM8 ion channel can be cloned into a non-essential region, such as the polyhedrin gene in this virus, and placed under the control of the polyhedrin promoter. Successful insertion of the TrpM8 ion channel will render the polyhedrin gene inactive and produce a recombinant virus lacking the coat protein. This recombinant virus is then described, for example, in S. It can be used to infect Frugiperda cells or Trichopulcia larvae, in which the TrpM8 ion channel can be expressed (Engelhard, EK et al. (1994) Proc. Nat. Acad. Sci. 91: 3224 ~ 3227).

  In mammalian host cells, a number of viral-based expression systems are available. When using an adenovirus as an expression vector, the sequence encoding the TrpM8 ion channel can be ligated to an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. By using insertion into the E1 or E3 region, which is a non-essential region of the viral genome, a viable virus capable of expressing the TrpM8 ion channel in infected host cells can be obtained (Logan, J. and T. Shenk ( 1984), Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to enhance expression in mammalian host cells.

  Thus, for example, the TrpM8 ion channel can be expressed in either human embryonic kidney 293 (HEK293) cells or adherent CHO cells. To achieve maximum expression, typically all 5 'and 3' untranslated regions (UTRs) are removed from the TrpM8 cDNA prior to insertion into a pCDN or pCDNA3 vector. Cells are transfected with individual cDNAs by lipofection and selected in the presence of 400 mg / ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 cells or CHO cells transfected with the vector alone are used as negative controls. In order to isolate cell lines that stably express individual ion channels, approximately 24 clones are typically selected and analyzed by Northern blot analysis. TrpM8 ion channel mRNA is generally detectable in about 50% of the G418 resistant clones analyzed.

  Human artificial chromosomes (HACs) can also be utilized to deliver larger DNA fragments than can be contained and expressed in a plasmid. For therapeutic purposes, approximately 6 kb to 10 Mb HACs are constructed and delivered by conventional delivery methods (liposomes, polycation amino polymers, or vesicles).

  Specific initiation signals can also be used to achieve more efficient translation of the sequence encoding the TrpM8 ion channel. Such signals include the ATG start codon and adjacent sequences. If the sequence encoding the TrpM8 ion channel and its initiation codon and upstream sequence are inserted into an appropriate expression vector, no additional transcriptional or translational control signals will be required in addition to them. . However, if only the coding sequence or fragment thereof is inserted, an exogenous translational control signal including the ATG start codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert sequence. Exogenous translation elements and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by including enhancers appropriate for the particular cell line used, such as those described in the literature (Scharf, D. et al. (1994), Results Probl. Cell Differ. 20: 125-162).

  In addition, a host cell strain can be selected for its ability to modulate the expression of the inserted sequences, or to process the expressed protein in the desired fashion. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves “prepro” type proteins can also be utilized to facilitate correct insertion, folding, and / or function. A variety of host cells (eg, CHO, HeLa, MDCK, HEK293, and WI38) with specific cellular mechanisms and unique mechanisms for post-translational activity are obtained from ATCC (American Type Culture Collection, Bethesda, Md.). And can be selected to ensure correct modification and processing of the foreign protein.

  Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines capable of stably expressing the TrpM8 ion channel use expression vectors that will contain the viral origin of replication and / or endogenous expression elements and the selectable marker gene on the same or separate vectors. Can be transformed. Cells can be grown in concentrated media for about 1 to 2 days after introducing the vector and before switching to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows for the growth and recovery of cells that express the introduced sequence well. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Any of a number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase gene (Wigler, M. et al. (1977), Cell 11: 223-32) and the adenine phosphoribosyltransferase gene (Lowy, I. et al. (1980)). Year), Cell 22: 817-23), which can be used in tk ⁻ cells or apr ⁻ cells, respectively. Antimetabolites, antibiotics, or herbicide resistance can also be used as a basis for selection. For example, dhfr confers resistance to methotrexate (Wigler, M. et al. (1980), Proc. Natl. Acad. Sci. 77: 3567-70); npt confers resistance to aminoglucoside neomycin and G-418. (Colbere-Garapin, F. et al. (1981), J. Mol. Biol. 150: 1-14); and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry , Supra). Other selectable genes, such as trpB or hisD, have already been described, trpB allows the use of indole by the cell instead of tryptophan, and hisD allows the use of histinol by the cell instead of histidine. (Hartman, SC and RC Mulligan (1988), Proc. Natl. Acad. Sci. 85: 8047-51). Recently, the use of visible markers has become preferred, and such markers include anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression resulting from a particular vector system (Rhodes, CA et al. (1995). ), Methods Mol. Biol. 55: 121-131).

  The presence / absence of marker gene expression suggests that the gene of interest is also present, but the presence and expression of the gene may need to be confirmed. For example, when a sequence encoding a TrpM8 ion channel is inserted into a marker gene sequence, transformed cells containing a sequence encoding a TrpM8 ion channel can be identified by the absence of marker gene function.

  Alternatively, the marker gene can be placed in tandem with a sequence encoding the TrpM8 ion channel and under the control of a single promoter. Expression of a marker gene in response to induction or selection usually also indicates expression of a tandem gene.

  Alternatively, host cells containing a nucleic acid sequence encoding a TrpM8 ion channel and expressing a TrpM8 ion channel can be identified by various techniques known to those skilled in the art. These techniques include DNA-DNA or DNA-RNA hybridization, and protein bioassay techniques, including membrane, solution, or chip-based techniques for detecting and / or quantifying nucleic acid or protein sequences. Or include, but are not limited to, immunoassay techniques.

  The presence of a polynucleotide sequence encoding a TrpM8 ion channel can be detected by DNA-DNA or DNA-RNA hybridization, or amplification using a probe or fragment of the polynucleotide encoding the TrpM8 ion channel. Nucleic acid amplification-based assays involve the use of oligonucleotides or oligomers based on sequences encoding TrpM8 ion channels to detect transformants containing DNA or RNA encoding TrpM8 ion channels.

  Various protocols are known in the art for detecting and measuring the expression of TrpM8 ion channels using polyclonal or monoclonal antibodies specific for proteins. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). Although a two-site monoclonal based immunoassay utilizing a monoclonal antibody that reacts with two non-interfering epitopes on the TrpM8 ion channel is preferred, a competitive binding assay can also be utilized. These and other assays have been described in detail in the art, for example, Hampton, R. et al. (1990; “Serological Methods, a Laboratory Manual”, Section IV, APS Press, St Paul, Minn.) And Maddox, DE et al. (1983; J. Exp. Med. 158: 1211-1216).

  A variety of labels and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for generating labeled hybridization probes or PCR probes for detection of sequences related to polynucleotides encoding TrpM8 ion channels include oligo-labeling, nick translation, end-labeling, or labeled nucleotides. The PCR amplification used is included. Alternatively, the sequence encoding the TrpM8 ion channel or any fragment thereof can be cloned into a vector to generate mRNA probes. Such vectors are known in the art and are commercially available, and RNA probes can be obtained in vitro by adding an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Can be used to synthesize. These techniques involve a variety of commercially available kits such as those sold by Pharmacia & Upjohn (Kalamazoo, Mich.), GE Healthcare (UK), and US Biochemical Corp. (Cleveland, Ohio). Can be implemented. Suitable reporter molecules, or labels that can be used to facilitate detection, include radionuclides, enzymes, fluorescence, chemiluminescence, or chromogens, as well as substrates, cofactors, inhibitors, magnetic particles , And the like.

  Host cells transformed with a nucleotide sequence encoding a TrpM8 subunit can be cultured under conditions suitable for protein expression and protein recovery from cell culture. Depending on the sequence and / or vector used, the protein produced by the transformed cell may be localized in the cell membrane, secreted, or contained within the cell. As will be appreciated by those skilled in the art, an expression vector containing a polynucleotide encoding a TrpM8 subunit contains a signal sequence that directs secretion of the TrpM8 subunit through the cell membrane of prokaryotic or eukaryotic cells. Can be designed as Other constructs can be used that link the sequence encoding the TrpM8 subunit to a nucleotide sequence encoding a polypeptide domain that facilitates purification of the soluble protein. Such domains that facilitate purification allow for metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metal, purification on immobilized immunoglobulin Protein A domains and domains used in the FLAGS extension / affinity purification system (Immunex Corp., Seattle, Wash.) Are included but are not limited to these. To facilitate purification, cleaving between the purification domain and the sequence encoding the TrpM8 subunit, such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) Various linker sequences can also be included. One such expression vector provides for the expression of a fusion protein containing the TrpM8 subunit and a nucleic acid encoding a 6 histidine residue preceding the thioredoxin or enterokinase cleavage site. The histidine residue facilitates purification by immobilized metal ion affinity chromatography (IMIAC; described in Porath, J., et al. (1992), Prot. Exp. Purif. 3: 263-281). The kinase cleavage site provides a means to purify the TrpM8 subunit from the fusion protein. A discussion on vectors containing fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12: 441-453).

  TrpM8 subunit fragments can be generated not only by recombinant production, but also by direct peptide synthesis using solid phase methods (Merrifield J. (1963), J. Am. Chem. Soc. 85: 2149-2154). Protein synthesis can be performed by manual techniques or by automation. Synthesis by automation can be realized using, for example, Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Various fragments of the TrpM8 subunit can be synthesized separately and then combined to generate a full-length molecule.

Biosensor TrpM8 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands are useful as (and for the production of) biosensors.

  According to Aizawa (1988), Anal. Chem. Symp. 17: 683, a biosensor is a characteristic of a receptor for molecular recognition, such as a selective layer, and an immobilized antibody or ion channel (such as TrpM8). A combination is defined as a transducer that conveys measured values. One group of such biosensors detects changes that occur in the optical properties of the surface layer due to the interaction between the receptor and the surrounding medium. Among such techniques, mention is made in particular of ellipsometry and surface plasmon resonance. A biosensor incorporating TrpM8 can be used to detect the presence or level of TrpM8 ligand. The construction of such biosensors is well known in the art.

  Therefore, cell lines expressing TrpM8 subunits should be used as reporter systems for the detection of ligands (such as ATP) through receptor-stimulated phosphate [3H] inositol or other second messenger formation. (Watt et al., 1998, J Biol Chem May 29; 273 (22): 14053-8). Receptor-ligand biosensors are also described in Hoffman et al., 2000, Proc Natl Acad Sci USA Oct 10; 97 (21): 11215-20. Optical and other biosensors including TrpM8 are also described, for example, in Figler et al, 1997, Biochemistry Dec 23; 36 (51): 16288-99 and Sarrio et al., 2000, Mol Cell Biol 2000 Jul; 20 (14): As described by 5164-74, it can be used to detect the level or presence of interactions with G proteins and other proteins. A sensor unit for a biosensor is described, for example, in US Pat. No. 5,492,840.

Screening assays TrpM8 polypeptides, including homologues, variants and derivatives, bind to TrpM8 ion channels and activate TrpM8 (agonists) or inhibit activation, whether natural or recombinant It can be used in screening methods for (antagonist or blocker) compounds. Thus, such polypeptides can also be used to assess the binding of small molecule substrates and ligands, eg, in cells, cell-free preparations, chemical libraries and natural product mixtures. These substrates and ligands may be natural substrates and ligands, or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

  TrpM8 ion channel polypeptides contribute to many biological functions, including many pathologies. Therefore, it is desirable to find compounds and drugs that stimulate the TrpM8 ion channel on the one hand but can inhibit the function of the TrpM8 ion channel on the other hand. In general, agonists and antagonists are used for therapeutic and prophylactic purposes for conditions such as anxiety, stress, depression, cancer or pain.

  Rational design of candidate compounds that are likely to interact with the TrpM8 ion channel protein can be based on structural studies of the molecular shape of the polypeptide. One means for identifying sites that interact with other specific proteins is physical structure determination, such as X-ray crystallography or two-dimensional NMR techniques. These can give an indication of which amino acid residues form the molecular contact region. For a detailed description of protein structure determination, see, for example, Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

  An alternative to rational design utilizes screening techniques that generally involve generating appropriate cells that express the TrpM8 ion channel polypeptide on their surface. Such cells include cells from animals, yeast, Drosophila or E. coli. Cells expressing TrpM8 (or cell membrane containing the expressed protein) are then contacted with a test compound to observe binding or stimulation or inhibition of a functional response. For example, Xenopus oocytes are injected with TrpM8 mRNA or polypeptide and the voltage induced method is used to measure the current induced by exposure to the test compound as detailed herein.

  Instead of individually testing each candidate compound with the TrpM8 ion channel, it may be advantageous to create and screen a library or bank of candidate ligands. Thus, for example, a bank of over 200 putative ligands has been constructed for screening. Banks include transmitters, hormones and chemokines known to act through ion channels; natural compounds considered to be putative agonists of ion channels; Biologically active peptides; as well as compounds that activate ion channels with unknown natural ligands that are not found in nature.

  Use this bank to refer to functional assays (ie, calcium, microphysiometer, FLIPR assay, whole cell electrophysiology, oocyte electrophysiology, etc. as detailed herein) ) As well as binding assays can be used to screen TrpM8 ion channels for known ligands. However, many mammalian receptors still remain free of cognate activating ligands (agonists) or deactivating ligands (antagonists). Therefore, active ligands for these receptors may not have been identified to date and are included in the ligand bank. Thus, TrpM8 may also be functionally screened against tissue extracts to identify natural ligands (using functional screens such as calcium, microphysiometer, oocyte electrophysiology, etc.). Extracts that produce a positive functional response can be sequentially fractionated until the activating ligand is isolated and identified, with fractions assayed as described herein.

  Ion channels expressed in HEK293 cells have been shown to be functionally associated with PLC activation and calcium mobilization and / or cAMP stimulation or inhibition. Accordingly, one screening technique involves the use of cells that express TrpM8 (eg, transfected Xenopus oocytes, CHO or HEK293 cells) in a system that measures extracellular pH or intracellular calcium changes caused by channel activity. . In this technique, the compound can be contacted with a cell that expresses a TrpM8 polypeptide. Second messenger responses, such as signal transduction, pH changes or preferably changes in calcium levels, are then measured to determine if potential compounds activate or inhibit the channel.

  Preferably, the response is a change in calcium levels and such an assay is referred to as a “TrpM8 functional assay (calcium concentration)” for convenience.

In such experiments, it is observed whether basal calcium levels in transfected HEK293 cells or vector control cells are normal, i.e. in the range of 100 nM to 200 nM. HEK293 cells expressing homomeric or heteromeric TrpM8 ion channels, or homomeric or heteromeric recombinant TrpM8 ion channels loaded with fura2 in one day and over 150 selected ligands or tissue / cell extracts are induced by an agonist Assess calcium mobilization. Similarly, HEK293 cells expressing TrpM8 ion channels or recombinant TrpM8 ion channels are evaluated for increased or decreased Ca ²⁺ flux. Agonists that exhibit calcium transients are tested in vector control cells to see if the response is unique to transfected cells expressing ion channels.

  In another method, ion channel inhibitors are screened by measuring inhibition or stimulation of the TrpM8 ion channel. In such a method, a TrpM8 subunit is transfected into a eukaryotic cell so that it forms a homomeric channel alone or together with other Trp channel subunits, on its cell surface. Ion channels are expressed. The cells are then exposed to candidate antagonists in the presence of the TrpM8 ion channel. Cells are tested using whole cell electrophysiology to measure changes in current conductance or kinetics.

  Another method for detecting agonists or antagonists of TrpM8 is a yeast-based technique as described in US Pat. No. 5,482,835, which is hereby incorporated by reference.

  In a preferred embodiment, the screening utilizes detection of a change in conductivity to screen for agonists and antagonists of TrpM8. Specifically, a method is disclosed in which an antagonist of TrpM8 reduces the conductivity of suitably transfected cells. Preferably, the conductivity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an antagonist of TrpM8. Preferably, the conductivity is reduced in the presence of an antagonist of TrpM8 by 1pS, 2pS, 3pS, 4pS, 5pS, 10pS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more.

  Further disclosed is a method in which an agonist of TrpM8 increases the conductivity of suitably transfected cells. Preferably, the conductivity is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an agonist of TrpM8. Preferably, the conductivity is increased in the presence of an agonist of TrpM8 by 1pS, 2pS, 3pS, 4pS, 5pS, 10pS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more.

  In a further preferred embodiment, the screening utilizes detection of changes in intracellular calcium concentration to screen for agonists and antagonists of TrpM8. Preferably, the screening utilizes the functional assay described above in “TrpM8 Functional Assay (Calcium Concentration)”.

  Specifically, a method is disclosed in which an antagonist of TrpM8 reduces the calcium concentration of suitably transfected cells. Preferably, the calcium concentration is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an antagonist of TrpM8. Preferably, the calcium concentration is reduced by 1 pS, 2 pS, 3 pS, 4 pS, 5 pS, 10 pS, 15 pS, 25 pS, 35 pS, 45 pS, 60 pS, 70 pS or more in the presence of an antagonist of TrpM8.

  Further disclosed is a method in which an agonist of TrpM8 increases the calcium concentration of suitably transfected cells. Preferably, the conductivity is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an agonist of TrpM8. Preferably, the calcium concentration is increased by 1 pS, 2 pS, 3 pS, 4 pS, 5 pS, 10 pS, 15 pS, 25 pS, 35 pS, 45 pS, 60 pS, 70 pS or more in the presence of an agonist of TrpM8.

  If the candidate compound is a protein, particularly an antibody or peptide, a library of candidate compounds can be screened using phage display methods. Phage display is one of the molecular screening protocols using recombinant bacteriophage. In this technique, a bacteriophage is transformed with a gene that encodes one compound from a library of candidate compounds such that each phage or phagemid expresses a particular candidate compound. The transformed bacteriophage (preferably linked to a solid support) expresses the appropriate candidate compound and displays it on the phage coat. Specific candidate compounds that can bind to a TrpM8 polypeptide or peptide are enriched by selection techniques based on affinity interactions. The candidate drug that responded well is then characterized. Phage display has advantages over standard affinity ligand screening techniques. The phage surface displays candidate drugs in a three-dimensional structure that is more similar to the natural structure. This makes the affinity binding for screening purposes more specific and high.

Another method of screening a compound library utilizes eukaryotic or prokaryotic host cells that are stably transformed with recombinant DNA molecules that express the compound library. Such cells, whether viable or fixed, can be used in standard binding partner assays. Parce et al. (1989) Science 246: 243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87; 4007, which also describes sensitive methods for detecting cellular responses. See -4011. A competitive assay is particularly useful, in which cells expressing a compound library are contacted with or incubated with a labeled antibody (such as a ¹²⁵ I-antibody) known to bind to a TrpM8 polypeptide, and the binding composition A test sample (candidate compound, etc.) having a binding affinity for a product is measured. Subsequently, bound and free labeled binding partners for the polypeptide are separated and the extent of binding is assessed. The amount of test sample bound is inversely proportional to the amount of labeled antibody bound to the polypeptide.

  Any one of a number of techniques is used to separate bound binding partner from free binding partner and assess the extent of binding. This separation step typically includes procedures such as attachment to a filter and subsequent washing, attachment to plastic and subsequent washing, or centrifugation of the cell membrane.

  Another approach uses solubilized, unpurified or solubilized purified polypeptides or peptides, such as those extracted from transformed eukaryotic or prokaryotic host cells. This allows for “molecular” binding assays with the advantages of increased specificity, automatability, and drug test high throughput.

  Another approach for candidate compound screening includes, for example, high throughput screening methods for novel compounds having suitable binding affinity for TrpM8 polypeptides, including, in particular, International Patent Application No. WO 84/03564 (Commonwealth Serum Labs., (Published September 13, 1984). Initially, a number of different small peptide test compounds are synthesized on a solid phase substrate, such as plastic pins or some other suitable surface. See Fodor et al. (1991). Subsequently, all pins are reacted with solubilized TrpM8 polypeptide and washed. The next step involves detection of bound polypeptide. Then, a compound that specifically interacts with the polypeptide is identified.

  Ligand binding assays provide a direct method for elucidating drug pharmacology and are adaptable to a high-throughput format. The purified ligand can be radiolabeled to high specific activity (50-2000 Ci / mmol) for binding studies. Subsequently, a determination is made that the radiolabeling process does not weaken the activity of the ligand for that target. Assay conditions for buffers, ions, pH and other variables (such as nucleotides) are optimized to establish a functional signal-to-noise ratio for the membrane and whole cell receptor or ion channel source. For these assays, specific binding is defined as total associated radioactivity minus the radioactivity measured in the presence of excess unlabeled competing ligand. Where possible, more than one competing ligand is used to define the remaining non-specific binding.

  This assay is intended to detect the binding of candidate compounds to detect adherence to cells with receptors or ion channels by means of a label directly or indirectly associated with the candidate compound or in an assay that competes with a labeled competitor. Are simply tested. In addition, these assays can test whether a candidate compound provides a signal resulting from target activation, using a detection system appropriate for cells having the target on its surface. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the candidate compound is observed.

  In addition, the assay comprises mixing a candidate compound with a solution containing a TrpM8 polypeptide to form a mixture, measuring the TrpM8 ion channel activity in the mixture, and comparing the TrpM8 ion channel activity of the mixture to a standard. It may simply include the step of performing.

  Alternatively, an assay for detecting the effect of added compounds on TrpM8 subunit mRNA and protein production in cells can be set up using TrpM8 subunit cDNA, protein and antibodies to the protein. For example, an ELISA can be constructed to measure the secretion level or cell binding level of TrpM8 subunit protein using monoclonal and polyclonal antibodies by standard methods known in the art, which is preferably Can be used to discover agents (also referred to as antagonists or agonists, respectively) that can inhibit or enhance the production of TrpM8 subunits from cells or tissues that have been engineered. Standard methods for performing screening assays are well known in the art.

  Examples of potential TrpM8 ion channel antagonists and blockers include antibodies or, in some cases, nucleotides and their analogs (including purines and purine analogs), oligonucleotides or ligands for TrpM8 ion channels. A protein (eg, a fragment of a ligand) or a small molecule that binds to an ion channel but does not cause a response and interferes with the activity of the channel.

  Accordingly, compounds that can specifically bind to TrpM8 polypeptides and / or peptides are also provided.

  The term “compound” refers to chemical compounds (natural or synthetic), such as biopolymers (eg, nucleic acids, proteins, non-peptides or organic molecules), or bacteria, plants, fungi or animal (especially mammalian) cells or tissues, etc. It means an extract made from a biomaterial, or even an inorganic element or molecule. Preferably the compound is an antibody.

  Substances necessary for carrying out such screening may be packaged in a screening kit. Such screening kits are useful for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for TrpM8 polypeptides, or compounds that reduce or increase the production of TrpM8 ion channel polypeptides. The screening kit comprises (a) a TrpM8 polypeptide, (b) a recombinant cell that expresses a TrpM8 polypeptide, (c) a cell membrane that expresses a TrpM8 polypeptide, or (d) an antibody against the TrpM8 polypeptide. The screening kit may optionally include instructions for use.

In addition, transgenic animals that are capable of expressing a natural or recombinant TrpM8 ion channel, or a homologue, variant or derivative thereof, at a normal level, a high level or a low level compared to a normal expression level. Disclose animals. Preferably, such transgenic animals are non-human mammals such as pigs, sheep or rodents. Most preferably, the transgenic animal is a mouse or a rat.

  Also disclosed are transgenic animals in which all or part of the native TrpM8 gene is replaced with a TrpM8 sequence from another organism. Preferably the organism is another species, most preferably a human. In a highly preferred embodiment, a mouse is disclosed in which the substantially full-length TrpM8 gene has been replaced with a human TrpM8 gene. Such transgenic animals, as well as animals in which TrpM8 is wild-type, can be used to screen for agonists and / or antagonists of TrpM8.

  For example, such assays assay for exposing a wild-type or transgenic animal, or part thereof (preferably cells, tissues or organs of the transgenic animal) to a candidate substance, a TrpM8-related phenotype such as pain or stress. Includes steps. It is also possible to perform a cell-based screen using cells from the relevant animal to assay for effects on conductivity or intracellular calcium concentration.

  Further disclosed are transgenic animals comprising a functionally disrupted TrpM8 gene, wherein one or more functions of TrpM8 disclosed herein are partially or completely abolished. For example, transgenic animals (TrpM8 knockout) that do not express a functional TrpM8 ion channel as a result of one or more loss-of-function mutations of the TrpM8 gene, eg, deletion.

  Also included are partial loss-of-function mutants, such as incomplete knockouts, such as having deletions in selected portions of the TrpM8 gene. Such animals can be created by selectively substituting or deleting relevant portions of the TrpM8 sequence, eg, functionally important protein domains.

  Such full or partial loss of function mutants are useful as models for TrpM8 related diseases, particularly pain or stress related diseases. Animals that exhibit partial loss of function can be exposed to candidate substances to identify substances that enhance the phenotype, ie, increase the observed hypoalgesia or reduced stress level phenotype (in the case of TrpM8). Other parameters such as reduced conductivity or reduced intracellular calcium levels can also be detected using the methods described herein.

  Partial and complete knockouts can also be used to identify selective agonists and / or antagonists of TrpM8. For example, agonists and / or antagonists can be administered to wild type and TrpM8 deficient animals (knockouts). It will be shown that selective agonists or antagonists of TrpM8 affect wild-type animals but not TrpM8-deficient animals. Specifically, a specific assay is designed such that a potential agent (candidate ligand or compound) is evaluated to determine whether it produces a physiological response in the absence of the TrpM8 ion channel. This can be accomplished by administering an agent to the transgenic animal as described above, followed by assaying the animal for a specific response. Similar cell-based methods of assaying effects on conductivity or intracellular calcium concentration may be performed using cells from the relevant animal. Such animals can also be used to test for the efficacy of agents identified by the screening described herein.

  In another embodiment, transgenic animals with a partial loss of function phenotype are used for screening. In such embodiments, the screening comprises assaying for partial or complete recovery or reversion to the wild type phenotype. Cell-based screening can also be performed in which cells from the relevant animal are used to assay effects on conductivity or intracellular calcium concentration. Candidate compounds found to be able to do so can be considered TrpM8 agonists or analogs. Such agonists can be used, for example, to ameliorate or increase sensitivity to a stimulus, such as pain, or to increase stress levels in an individual.

  In preferred embodiments, transgenic TrpM8 animals, particularly TrpM8 knockouts (complete loss of function), preferably exhibit the phenotypes shown in the examples as measured by the tests described herein. Thus, TrpM8 animals, particularly TrpM8 knockouts, preferably exhibit any one or more of the following: reduced grip strength, tendency to drink more than wild type mice, low sensitivity to pain (painlessness), reduced stress, Reduced plasma corticosterone levels.

  In a highly preferred embodiment, the transgenic TrpM8 animals, particularly TrpM8 knockouts are at least 10%, preferably at least 20%, more preferably at least 30%, 40%, 50%, compared to the corresponding wild type mice. 60%, 70%, 80%, 90% or 100% higher or lower (optional) measurement parameters are indicated. Thus, for example, TrpM8 knockout has a pain threshold of 1 second, 2 seconds, 5 seconds, 10 seconds, 30 seconds or more, or 5 compared to wild-type mice in response to the tail flick test shown in the Examples. %, 10%, 20%, 50% or more. When measured in the open field analysis described in the Examples, TrpM8-deficient mice have a 3, 4, 5, 6, 7, 8, 9, 9 10, 11, 12, 13, 14, 15, 20 seconds or more, or the distance traveled in the central region is 15, 20, 25, 30, 50 cm or more longer.

  It is clear that the phenotypes disclosed herein for TrpM8-deficient transgenic animals can be usefully used in screens using wild type animals to detect compounds that cause effects similar to loss of function of TrpM8. In other words, exposing wild-type animals to candidate compounds and observing changes such as associated TrpM8 phenotypes such as hypoalgesia, pain insensitivity, decreased stress levels, decreased corticosterone levels, and modulators of TrpM8 function In particular, antagonists can be identified. Cell phenotypes, such as reduced conductivity or reduced intracellular calcium levels, can also be detected using the methods described herein.

  The compounds identified by such screening can be used as antagonists of TrpM8, for example as analgesics or stress relievers, especially for the treatment or alleviation of TrpM8 related diseases.

  The screening described above may involve observation of any suitable parameters, such as behavioral responses, physiological responses or biochemical responses. Preferred responses include physiological responses and may include one or more of the following: changes in disease resistance, changes in inflammatory response, changes in tumor susceptibility, changes in blood pressure, neovascularization, changes in eating and drinking behavior, weight changes, Changes in bone density, changes in body temperature, insulin secretion, gonadotropin secretion, nasal and bronchial secretion, vasoconstriction, memory loss, anxiety, changes in anxiety, decreased reflex or hyperreflex, pain or stress response.

  Biochemical parameters can also be used, such as changes in conductivity or intracellular calcium concentration. Preferably, conductivity is measured using a “TrpM8 functional assay (conductivity)”, and intracellular calcium concentration is measured using a “TrpM8 functional assay (calcium concentration)”. This is particularly useful in cell-based screening.

  In preferred embodiments, the conductivity of cells exposed to a TrpM8 agonist (eg, wild type cells or partially loss of function cells) is at least 10%, preferably at least 20%, more preferably at least + 30%, more preferably at least 40. %, More preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%. In a preferred embodiment, this is measured using the “TrpM8 Functional Assay (Conductivity)” described herein.

  In preferred embodiments, the intracellular calcium concentration of cells exposed to a TrpM8 agonist (eg, wild type cells or partially lost function cells) is at least 10%, preferably at least 20%, more preferably at least + 30%, more preferably Increase by at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%. In a preferred embodiment, this is measured using the “TrpM8 Functional Assay (Intracellular Calcium Concentration)” described herein.

  In preferred embodiments, corticosterone levels in wild-type or TrpM8 partial knockout animals exposed to a TrpM8 agonist are at least 10%, preferably at least 20%, more preferably at least + 30%, more preferably at least 40%, more Preferably it is increased by at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%.

  In a preferred embodiment, the antagonist of TrpM8 is such that a wild type or partially dysfunctional animal exposed to such antagonist has at least a partial phenotype that is at least partially identical to the TrpM8 partial or full loss of function mutant. It is as shown. That is, preferred antagonists are those that cause hypoalgesia, or decreased stress, or decreased serum corticosterone levels, or decreased conductivity, or decreased intracellular calcium levels, or any combination thereof. Preferably, the related phenotype is expressed to the same extent as the TrpM8 knockout animal.

  In a preferred embodiment, the conductivity of wild type cells or partially lost cells exposed to a TrpM8 antagonist is + 80%, preferably + 70%, more preferably + 60%, more preferably + 50% of the conductivity of TrpM8 deficient cells. , More preferably + 40%, more preferably + 30%, more preferably + 20%, more preferably + 10%, more preferably + 5%. In a preferred embodiment, this is measured using the “TrpM8 Functional Assay (Conductivity)” described herein.

  In a preferred embodiment, the intracellular calcium concentration of wild type cells or partially lost function cells exposed to a TrpM8 antagonist is + 80%, preferably + 70%, more preferably + 60%, more than the intracellular calcium concentration of TrpM8 deficient cells. It is preferably in the range of + 50%, more preferably + 40%, more preferably + 30%, more preferably + 20%, more preferably + 10%, more preferably + 5%. In a preferred embodiment, this is measured using the “TrpM8 Functional Assay (Intracellular Calcium Concentration)” described herein.

  In a preferred embodiment, the corticosterone level of wild-type or partial TrpM8 knockout animals exposed to a TrpM8 antagonist is + 80%, preferably + 70%, more preferably + 60%, of corticosterone levels in TrpM8-deficient transgenic animals, More preferably, it is in the range of + 50%, more preferably + 40%, more preferably + 30%, more preferably + 20%, more preferably + 10%, more preferably + 5%.

  Tissue obtained from TrpM8 knockout animals can be used in binding assays to determine whether a potential agent (candidate ligand or compound) binds to TrpM8. Such an assay is known to bind to a first ion channel preparation from a transgenic animal engineered to be deficient in TrpM8 ion channel production and any TrpM8 ligand or compound identified. By obtaining a second ion channel preparation from a different source. In general, the first ion channel preparation and the second ion channel preparation will be similar in all respects except for the source from which they were obtained. For example, when brain tissue obtained from a transgenic animal (such as those described above and the following) is used in the assay, the corresponding brain tissue obtained from a normal (wild-type) animal is used as the second ion channel. Used as a source of preparation. Each ion channel preparation is incubated with a ligand known to bind to the TrpM8 ion channel, both incubated alone and in the presence of a candidate ligand or compound. Candidate ligands or compounds can preferably be tested at several different concentrations.

  The extent to which binding by the known ligand is displaced by the test compound is measured in both the first ion channel preparation and the second ion channel preparation. Tissue obtained from the transgenic animal can be used directly in the assay or the tissue can be processed to isolate membranes or membrane proteins, which are themselves used in the assay. A preferred transgenic animal is a mouse. The ligand may be labeled using any means compatible with the binding assay. These will include, but are not limited to, radioactive labels, enzyme labels, fluorescent labels, or chemiluminescent labels (as well as other labeling techniques described in more detail above).

  In addition, TrpM8 ion channel antagonists have been identified as candidate compounds, etc. that exhibit any phenotypic characteristics associated with reduced or abolished expression of TrpM8 function in wild-type animals expressing functional TrpM8. It can be identified by administering to animals.

  Detailed methods for producing transgenic non-human animals are described in detail below. In order to create a transgenic mammal, a transgenic gene construct can be introduced into the germline of the animal. For example, one or several copies of the construct can be integrated into the genome of a mammalian embryo by standard transgenic techniques.

  In an exemplary embodiment, a transgenic non-human animal is created by introducing a transgene into the germ line of the non-human animal. Target embryo cells at various developmental stages can be used to introduce the transgene. Various methods are used depending on the stage of development of the target embryo cell. For any animal, the particular lineage of that animal is selected according to overall health, good embryo yield, good pronuclear visibility in the embryo, and good self-propagating fitness. In addition, haplotypes are an important factor.

  Introduction of the transgene into the embryo can be performed by any means known in the art, such as, for example, microinjection, electroporation, or lipofection. For example, a TrpM8 transgene can be introduced into a mammal by microinjection of the construct into the pronucleus of a mammalian fertilized egg, thereby making one or more copies in a developing mammalian cell. Maintenance of the structure is caused. After introducing the transgene construct into the fertilized egg, the egg can be incubated in vitro for various lengths of time, reimplanted into a surrogate host, or both. In vitro incubation to maturity can also be performed. One common method is to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into a surrogate host.

  Embryonic offspring engineered by the transgenic technique can be tested for the presence of the construct by Southern blot analysis of tissue fragments. A permanent transgenic mammalian system that retains the added construct as a transgene when one or more copies of the cloned exogenous construct is stably integrated into the genome of such a transgenic embryo Can be established.

  Mammalian littermates that have been modified by transgenic techniques can be assayed after delivery to see if the construct has been incorporated into the progeny genome. This assay can be performed by hybridizing a DNA sequence encoding the desired recombinant protein product, or a probe corresponding to a segment thereof, to chromosomal material from the offspring. Offspring mammals found to contain at least one copy of the construct in the genome are raised to maturity.

  For purposes herein, a zygote is essentially the formation of a diploid cell that can occur in a complete organism. Usually, a zygote will consist of an egg containing one nucleus formed by natural or artificial fusion of two haploid nuclei derived from one or more gametes. Thus, their gamete nuclei must be compatible in nature, ie, produce live zygotes that can undergo differentiation and development into functional organisms. Usually, an euploid zygote is preferable. If an aneuploid zygote is obtained, the number of chromosomes should not differ by more than 2 compared to the euploid number of the organism from which any gamete was derived.

  In addition to similar biological requirements, physical requirements also determine the amount (eg, volume) of exogenous genetic material that can be added to, or form part of, the zygote nucleus. If genetic material is not removed, the amount of exogenous genetic material that can be added is limited to the amount that would be absorbed without causing physical destruction. Usually, the volume of exogenous genetic material inserted will not exceed about 10 picoliters. The physical effect of the addition should not be so great as to physically destroy the survival of the zygote. Biological restrictions on the number and diversity of DNA sequences are such that the genetic material of the resulting zygote, including exogenous genetic material, can initiate and maintain the differentiation and development of the zygote into a functional organism. Since it must be biologically possible, it will vary depending on the particular conjugate and the function of the exogenous genetic material, and will be readily apparent to those skilled in the art.

  The number of copies of the transgene construct added to the zygote will depend on the total amount of exogenous genetic material added and will be an amount that allows for the realization of genetic transformation. Theoretically, only one copy is required, but many copies are usually used, for example 1000-20000 copies of the transgene construct, to ensure that there is one copy of the functional one. In order to enhance the phenotypic expression of exogenous DNA sequences, it will often be advantageous to have multiple functional copies of each exogenous DNA sequence inserted.

  Any technique that allows the addition of exogenous genetic material to the nuclear genetic material can be used as long as it is not disruptive to the cell, nuclear membrane, or other existing cell or genetic structure. Exogenous genetic material is selectively inserted into nuclear genetic material by microinjection. Microinjection of cells and cell structures is known and used in the art.

  Retransplantation is performed using standard methods. Usually, the surrogate host is anesthetized and the embryo is inserted into the fallopian tube. The number of embryos transferred into a particular host will vary from species to species, but will usually be comparable to the number of offspring that the species naturally occurs.

  Screening for surrogate host transgenic progeny can be done for the presence and / or expression of the transgene by any suitable method. Screening is often performed by Southern blot analysis or Northern blot analysis using a probe complementary to at least a portion of the transgene. Western blot analysis using antibodies against the protein encoded by the transgene can be used as an alternative or additional method of screening for the presence of the transgene product. Usually, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, a tissue or cell in which the transgene is thought to be expressed at the highest level is examined for the presence and expression of the transgene using Southern analysis or PCR, but any tissue or cell type is analyzed by this analysis. You may use for.

  Alternative or additional methods of assessing the presence of the transgene include suitable biochemical assays, such as enzyme assays and / or immunological assays, histological staining of specific markers and enzyme activity, flow cytometric analysis, and The same is included without limitation. Blood analysis can also be useful in detecting the presence of transgene products in the blood and assessing the effects of the transgene on the levels of various types of blood cells and other blood components.

  The progeny of the transgenic animal can be obtained by mating the transgenic animal with a suitable partner or by in vitro fertilization of an egg and / or sperm obtained from the transgenic animal. When mated with a partner, the partner may or may not be transgenic and / or knocked out, and if transgenic, includes the same or different transgenes or both. May be. Alternatively, the partner may be a parent line. When in vitro fertilization is used, the fertilized embryo is transferred to a foster parent host, incubated in vitro, or both. With either method, the offspring can be assessed for the presence of the transgene using the methods described above or other suitable methods.

  Transgenic animals produced according to the methods described herein will contain exogenous genetic material. As described above, in certain embodiments, the exogenous genetic material is a DNA sequence that results in the production of a TrpM8 ion channel. Further, in such embodiments, the sequence adds a transcriptional control element, such as a promoter, preferably a promoter that allows expression of the transgene product in a particular cell type.

  Retroviral infection can be used to introduce a transgene into a non-human animal. Developing non-human embryos can be cultured in vitro up to the blastocyst stage. During this time, the blastomeres can be targeted for retroviral infection (Jaenich, R. (1976), PNAS 73: 1260-1264). Enzymatic treatment to remove the zona pellucida achieves efficient infection of the blastomeres ("Manipulating the Mouse Embryo", edited by Hogan (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the gene is a replication defective retrovirus with a transgene (Jahner et al. (1985), PNAS 82: 6927-6931; Van der Putten et al. (1985), PNAS 82. : 6148-6152) Transfection can be performed easily and efficiently by culturing blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987), EMBO J. 6: 383-388. Alternatively, infection can be performed at a later stage, and the virus or virus-producing cells can be injected into the cleavage space (Jahner et al. (1982) Nature 298: 623-628) Transgene import Since most occur in only a subpopulation of cells that form a transgenic non-human animal, the majority of the primary will be a mosaic of transgenes. Although they may contain retroviral insertions, these will usually be segregated in the offspring.In addition, transgenes may be introduced into the germline by intrauterine retroviral infection of pregnant embryos. It is possible (Jahner et al. (1982), supra).

  A third type of target cell for introducing the transgene is an embryonic stem cell (ES). ES cells are obtained from unimplanted embryos cultured in vitro and fused with the embryo (Evans et al. (1981) Nature 292: 154-156; Bradley. Et al. (1984) Nature 309: 255- 258; Gossler. Et al. (1986), PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322: 445-448). Transgenes can be efficiently introduced into ES cells by DNA transfection or retrovirus-mediated transduction. Thereafter, such transformed ES cells can be combined with blastocysts obtained from non-human animals. ES cells then colonize the embryo and join the resulting chimeric animal germline. For a review, see Jaenisch, R. (1988), Science 240: 1468-1474.

  Also provided is a non-human transgenic animal having a modified TrpM8 gene, preferably a gene as described above, as a model of TrpM8 ion channel function. Genetic modifications include the introduction of exogenous genes with nucleotide sequences by deletion or other loss-of-function mutations, targeting or random mutations, the introduction of exogenous genes from another species, or combinations thereof It is. The transgenic animal may be homozygous for the modification or heterozygous. Animals and cells derived therefrom are useful for screening for biologically active factors that may modulate TrpM8 ion channel function. Screening methods are used in particular to determine the specificity and action of possible treatments for pain and cancer, specifically prostate cancer. Animals are useful as models for studying the role of TrpM8 ion channels in normal tissues and organs (such as brain, heart, spleen and liver) and their effects on function.

  Another embodiment is a transgenic animal that has a functionally disrupted endogenous TrpM8 gene, but at the same time retains and expresses in its genome a transgene encoding a heterologous TrpM8 protein (ie, TrpM8 from another species). About. Preferably, the animal is a mouse and the heterologous TrpM8 is human TrpM8. Animals reconstituted with human TrpM8 or cell lines derived from such animals can be used to identify agents that inhibit human TrpM8 in vivo and in vitro. For example, a stimulus that induces signal transduction through human TrpM8 in the presence and absence of the agent to be tested can be applied to the animal or cell line and the response in the animal or cell line can be measured. An agent that inhibits human TrpM8 in vivo or in vitro can be identified based on a decrease in response in the presence of the agent as compared to a response in the absence of the agent.

  Also provided is a TrpM8 deficient transgenic non-human animal (“TrpM8 subunit knockout”). Such animals are preferably those that do not express or underexpress TrpM8 ion channel activity as a result of disruption or deletion of the endogenous TrpM8 ion channel genomic sequence. Preferably, such animals do not express ion channel activity. More preferably, the animal does not express the activity of the TrpM8 ion channel shown in SEQ ID NO: 3 or SEQ ID NO: 5. The TrpM8 ion channel knockout can be made by various means known in the art, as described in further detail below.

  The present disclosure also relates to a nucleic acid construct for functionally disrupting the TrpM8 gene in a host cell. The nucleic acid construct comprises a first homologous region having a nucleotide sequence having substantial identity with the first TrpM8 gene sequence, the first homologous region located upstream of a) a non-homologous replacement portion and b) a non-homologous replacement portion. Region; c) a second homologous region located downstream of the non-homologous replacement portion, the second homologous region having a nucleotide sequence having substantial identity to the second TrpM8 gene sequence (the second TrpM8 gene sequence is Located downstream of the first TrpM8 gene sequence in the native endogenous TrpM8 gene). In addition, the first and second homologous regions are long enough for homologous recombination between the nucleic acid construct and the host cell's endogenous TrpM8 gene to occur when these nucleic acid molecules are introduced into the host cell. That's it. In a preferred embodiment, the heterologous replacement moiety comprises an expression reporter, which preferably includes lacZ and a positive selection expression cassette, which preferably includes a neomycin phospho operatively linked to a regulatory element. A transferase gene is included.

  Preferably, the first and second TrpM8 gene sequences are derived from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or homologues, variants or derivatives thereof.

  Another aspect relates to a recombinant vector incorporating the nucleic acid construct described above. Yet another aspect relates to a host cell into which a nucleic acid construct has been introduced, whereby homologous recombination occurs between the nucleic acid construct and the host cell's endogenous TrpM8 gene, and the endogenous TrpM8 gene is functionally disrupted. . The host cell can be a mammalian cell that normally expresses TrpM8, derived from the liver, brain, spleen or heart, or a pluripotent cell (such as a mouse embryonic stem cell). Transgenic non-human animals having cells derived from said embryonic stem cells by introduction of a nucleic acid construct and homologously recombined with an endogenous TrpM8 gene, and thus having a TrpM8 gene disruption in their genome Create. Subsequently, animals having a TrpM8 gene disruption in their germline are selected and mated to create animals having the TrpM8 gene disruption in all somatic and germ cells. Such mice can then be bred for homozygosity for the TrpM8 gene disruption.

For the purposes of this specification, the term “antibody” includes polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by Fab expression libraries, unless specified to the contrary. It is not limited to. Such fragments include whole antibody fragments, Fv, F (ab ′), and F (ab ′) ₂ fragments, which retain binding activity to the target substance, as well as antibody antigen binding. Single chain antibodies (scFv) containing sites, fusion proteins, and other synthetic proteins are included. The antibodies and their fragments may be humanized antibodies, such as those described in EP-A-239400. In addition, antibodies (or fragments thereof) having fully human variable regions, such as those described in US Pat. Nos. 5,545,807 and 6,075,181 may be used. Neutralizing antibodies, ie those that inhibit the biological activity of the subject amino acid sequence, are particularly preferred for diagnostic and therapeutic purposes.

  Antibodies can be produced by standard techniques such as immunization or use of phage display libraries.

  The polypeptide or peptide can be used to develop antibodies by known techniques. Such antibodies can be those that can specifically bind to TrpM8 ion channel protein, or homologues, fragments, and the like.

  Where polyclonal antibodies are desired, selected mammals (eg, mice, rabbits, goats, horses, etc.) can be immunized with an immunogenic composition comprising the relevant polypeptide or peptide. Depending on the host species, various adjuvants can be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, mussel hemocyanin, dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are purified to subject amino acid sequences and administered to immunocompromised individuals to stimulate systemic defense responses A potentially useful human adjuvant available in some cases.

  Serum obtained from the immunized animal is collected and processed according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from a TrpM8 polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order to be able to make such antibodies, the inventor also provides the amino acid sequence of TrpM8 or a fragment thereof conjugated with another amino acid sequence for use as an immunogen in animals or humans as a hapten.

  One skilled in the art can readily produce monoclonal antibodies raised against a TrpM8 polypeptide or epitope obtainable from a peptide. General methods for producing monoclonal antibodies by hybridomas are well known. Immortal antibody-producing cell lines are generated by cell fusion and by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. be able to. A series of monoclonal antibodies generated against orbit epitopes can be screened for various properties, ie isotype and epitope affinity.

  Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules by continuous cell culture systems. These include the hybridoma technique, trioma technique, human B-cell hybridoma technique (Kosbor et al. (1983), Immunol Today 4: 72; Cote, originally described by Koehler and Milstein (1975, Nature 256: 495-497). (1983), Proc Natl Acad Sci 80: 2026-2030), and EBV hybridoma technology (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, pp. 77-96, Alan R. Liss, Inc., 1985). Is included, but is not limited thereto.

  In addition, using the techniques developed to splice mouse antibody genes into human antibody genes to create “chimeric antibodies”, ie, to obtain molecules with the appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454). Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946,779) can be adapted to produce a specific single chain antibody of interest.

  Antibodies raised against epitopes obtainable from TrpM8 polypeptides or peptides are particularly useful for diagnosis, both monoclonal and polyclonal antibodies, and neutralizing antibodies are useful for passive immunotherapy. In particular, monoclonal antibodies can be used to make anti-idiotype antibodies. An anti-idiotype antibody is an immunoglobulin that retains an “internal image” of the substance and / or drug for which protection is desired. Techniques for making anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful for therapy.

  Antibodies induce in vivo production in lymphocyte populations as described by Orlandoi et al. (1989, Proc Natl Acad Sci 86: 3833-3837) and Winter G and Milstein C (1991; Nature 349: 293-299) Or by screening a recombinant immunoglobulin library or panel to obtain a substance that binds with high specificity.

Antibody fragments that contain specific binding sites for the polypeptide or peptide can also be generated. For example, such fragments include F (ab ′) ₂ fragments that can be generated by pepsin digestion of antibody molecules and Fabs that can be generated by reducing disulfide bridges of F (ab ′) ₂ fragments. Fragment, but not limited to. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al. (1989) Science 256: 1275-1281). .

  Techniques for making single chain antibodies (US Pat. No. 4,946,778) can also be adapted to make single chain antibodies to TrpM8 polypeptides. Also, other organisms, including transgenic mice or other mammals, can be used to express humanized antibodies.

  Using the antibodies described above, clones expressing the polypeptide can be isolated or identified, or the polypeptide can be purified by affinity chromatography.

  In addition, antibodies against TrpM8 ion channel polypeptides can be used to treat pain and cancer, particularly neuropathic pain and prostate cancer.

Diagnostic Assays This specification relates to the use of TrpM8 ion channel polynucleotides and polypeptides (as well as homologues, variants, and derivatives thereof) for use as diagnostic reagents in diagnosis or for use in genetic analysis. For such assays, nucleic acids complementary to or hybridizable to TrpM8 ion channel nucleic acids (including homologues, variants, and derivatives) are useful, and antibodies to TrpM8 polypeptides are also useful. is there.

  Detection of a mutated form of the TrpM8 ion channel gene associated with dysfunction adds to or defines the diagnosis of a disease or susceptibility to a disease resulting from underexpression, overexpression, or altered expression of a TrpM8 ion channel Would provide a diagnostic tool that could Individuals with mutations in the TrpM8 ion channel gene (including regulatory sequences) can be detected at the DNA level by a variety of techniques.

  For example, DNA can be isolated from a patient to determine the TrpM8 DNA polymorphism pattern. The identified pattern is compared to a control patient known to suffer from a disease associated with TrpM8 overexpression, underexpression, or expression abnormality. Thereafter, patients exhibiting a genetic polymorphism pattern associated with a TrpM8-related disease can be identified. Genetic analysis of the TrpM8 ion channel gene can be performed by any technique known in the art. For example, individuals can be screened, such as by RFLP or SNP analysis, by DNA sequencing of the TrpM8 allele. Identifying a patient with a genetic predisposition to a disease associated with TrpM8 over-expression, under-expression, or expression abnormality by detecting the presence of a DNA polymorphism of the TrpM8 gene sequence or any sequence that controls its expression Can do.

  Patients so identified should be treated to prevent the onset of TrpM8-related disease, or more aggressively treated early in TrpM8-related disease to prevent subsequent onset or progression of the disease Can do. TrpM8 related diseases include stress, anxiety, depression, pain and cancer, particularly neuropathic pain and prostate cancer.

  Further disclosed is a kit for identifying a patient's genetic polymorphism pattern associated with a TrpM8-related disease. The kit includes means for collecting a DNA sample and means for determining a genetic polymorphism pattern, which are then compared to a control sample to determine the patient's susceptibility to a TrpM8 related disease. Also provided is a diagnostic kit for a TrpM8-related disease comprising a TrpM8 polypeptide and / or an antibody (or fragment thereof) against such a polypeptide.

Nucleic acids for diagnosis can be obtained from a subject's cells, such as blood, urine, saliva, tissue biopsy samples, or autopsy material. In a preferred embodiment, DNA is collected from blood cells obtained from a patient's finger puncture and the blood is collected on blotter paper. In a further preferred embodiment, blood is collected in AmpliCard ^™ (University of Sheffield, Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF).

  The DNA may be used for direct detection or may be amplified enzymatically using PCR or other amplification techniques prior to analysis. Oligonucleotide DNA primers targeting a specific polymorphic DNA region of the gene of interest can be prepared such that PCR reaction amplification of the target sequence is realized. RNA or cDNA can be used as a template as well. The DNA sequence amplified from the template DNA can then be analyzed using restriction enzymes to determine whether the genetic polymorphism is present in the amplified sequence, thereby determining the genetics of the patient. Provide a polymorphic profile. The length of the restriction fragment can be identified by gel analysis. Alternatively, or in combination with this, techniques such as SNP (single nucleotide polymorphism) analysis can also be used.

  Deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled TrpM8 ion channel nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched double-stranded molecules by differences in RNAse digestion or melting temperatures. DNA sequence differences can also be detected by changes in mobility in electrophoresis of DNA fragments in gels in the presence or absence of denaturing agents, or by direct DNA sequencing. See, for example, Myers et al., Science (1985), 230: 1242. Sequence changes at specific positions can also be revealed by nuclease protection assays such as RNAse protection and S1 protection, or by chemical cleavage methods. See Cotton et al., Proc Natl Acad Sci USA (1985), 85: 4397-4401. In another embodiment, an array of oligonucleotide probes comprising TrpM8 ion channel nucleotide sequences or fragments thereof can be constructed, for example, for efficient screening of genetic mutations. Array technology methods are well known and have general applicability and can be used to address various questions in molecular genetics, including gene expression, gene linkage, and genetic variability (eg, (See Science, 274, 610-613 (1996)).

  Single-stranded conformational polymorphism (SSCP method) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989), Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton (1993), Mutat. Res 285: 125-144; and Hayashi (1992), Genet. Anal. Tech. Appl. 9: 73-79 reference). Single-stranded DNA fragments of sample and control TrpM8 nucleic acids can be denatured and renatured. The secondary structure of a single-stranded nucleic acid varies depending on the sequence, and the resulting change in electrophoretic mobility makes it possible to detect even a single base change. The DNA fragment may be labeled or detected with a labeled probe. The sensitivity of the assay can be enhanced by using RNA (rather than DNA), and the secondary structure of the RNA is more sensitive to sequence changes. In a preferred embodiment, such methods use heteroduplex analysis to separate double stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7: Five).

  The diagnostic assay provides a method of diagnosing or determining susceptibility (susceptibility) to psoriasis, pain and cancer, particularly neuropathic pain and prostate cancer, through detection of mutations in the TrpM8 gene by the described methods.

  The presence of TrpM8 polypeptide and nucleic acid can be detected in the sample. Accordingly, the above infections and diseases can be diagnosed by a method comprising determining an abnormal decrease or increase in the level of TrpM8 polypeptide or TrpM8 ion channel mRNA from a sample derived from a subject. A sample is a cell or tissue from an organism suffering from or suspected of having a disease associated with other abnormalities, including increased or decreased TrpM8 expression, or a spatial or temporal change in the level or pattern of expression It may contain a sample. As a method of diagnosing a disease, one can usually compare the level or pattern of TrpM8 expression in an organism suffering from or suspected of such a disease with the level or pattern of expression in a normal organism. .

  Accordingly, in general, a method for detecting the presence of a nucleic acid comprising a TrpM8 nucleic acid in a sample comprising contacting the sample with at least one nucleic acid probe specific for said nucleic acid, and monitoring the sample for the presence of said nucleic acid Disclosed is a method comprising the steps of: For example, the nucleic acid probe may specifically bind to a TrpM8 subunit nucleic acid or a portion thereof, the binding between the two can be detected, and the presence of the complex itself can be detected. Furthermore, the present invention provides a method for detecting the presence of a TrpM8 polypeptide, comprising contacting a cell sample with an antibody capable of binding to the polypeptide, and monitoring the sample for the presence of the polypeptide. Includes methods. This may conveniently be achieved by monitoring the presence of a complex formed between the antibody and the polypeptide or by monitoring the binding between the polypeptide and the antibody. Methods for detecting binding between two substances are known in the art, and include FRET (fluorescence resonance energy transfer), surface plasmon resonance, and the like.

  Decreasing or increasing expression can be achieved using any method known in the art for quantifying polynucleotides, such as, for example, PCR, RT-PCR, RNAse protection, Northern blots, and other hybridization methods. It can be measured at the RNA level. Assay techniques that can be used to measure levels of a protein, such as TrpM8, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

  Further disclosed are kits for diagnosing diseases (including infections), such as pain and cancer, particularly neuropathic pain and prostate cancer, or susceptibility (susceptibility to) disease. The diagnostic kit comprises a TrpM8 polynucleotide or fragment thereof; a complementary nucleotide sequence; a TrpM8 polypeptide or fragment thereof, or an antibody to a TrpM8 polypeptide.

Chromosome assays The nucleotide sequences described herein are also valuable for chromosome identification. Sequences can be specifically targeted and hybridized to specific locations on individual human chromosomes. As mentioned above, the human TrpM8 ion channel has been found to map to the human (Homo sapiens) chromosome 2q37.

  Mapping of related sequences to the chromosome is the first important step in correlating these sequences with disease-related genes. Once the sequence has been mapped to the correct chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data is found, for example, in V. McKusick, Mendelian heritance in Man (available online by Johns Hopkins University Welch Medical Library). Subsequently, the relationship between the gene mapped to the same chromosomal region and the disease is identified by linkage analysis (simultaneous inheritance of physically adjacent genes).

  Differences in cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals and not in normal individuals, the mutation may be a causative factor of the disease.

Prophylactic and therapeutic methods Further provided are methods of treating both abnormal conditions associated with excessive TrpM8 ion channel activity and abnormal conditions associated with insufficient amounts thereof.

  If the TrpM8 ion channel activity is excessive, several approaches are available. One approach is to block the binding of a ligand to the TrpM8 ion channel with an inhibitor compound (antagonist) as described herein above, together with a pharmaceutically acceptable carrier, or to inhibit a second signal, Thereby administering to the subject in an amount effective to inhibit activation by alleviating the abnormal condition.

  In another approach, a soluble form of a TrpM8 polypeptide can be administered that is still able to compete with the endogenous TrpM8 ion channel and bind to the ligand. An exemplary embodiment of such a competitor comprises a fragment of a TrpM8 polypeptide.

  In yet another approach, expression blocking techniques can be used to inhibit expression of a gene encoding an endogenous TrpM8 ion channel. Known such techniques include the use of antisense sequences that are generated internally or administered separately. See, for example, O'Connor, J Neurochem (1991), 56: 560, CRC Press, Boca Raton, Fla. (1988) in “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression”. Alternatively, an oligonucleotide that forms a triple helix with the gene can be provided. See, for example, Lee et al., Nucleic Acids Res (1979), 6: 3073; Conney., Et al, Science (1988), 241: 456; Dervan et al., Science (1991), 251: 1360. These oligomers can be administered per se or the appropriate oligomers can be expressed in vivo.

  Several approaches are also available for the treatment of abnormal conditions related to the underexpression of the TrpM8 ion channel and its activity. One approach involves administering to a subject a therapeutically effective amount of a compound that activates the TrpM8 ion channel, ie, an agonist as described above, together with a pharmaceutically acceptable carrier, thereby alleviating the abnormal condition. including. Alternatively, gene therapy can be utilized to affect the endogenous production of TrpM8 ion channels by appropriate cells in the subject. For example, a TrpM8 polynucleotide can be constructed to be expressed in a replication defective retroviral vector, as discussed above. The retroviral expression construct can then be isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding the TrpM8 polypeptide, thereby containing the gene of interest. Infectious viral particles are produced by the packaging cells. These production cells can be administered to a subject for manipulation of the cells in vivo and expression of the polypeptide in vivo. For an overview of gene therapy, see “Human Molecular Genetics”, T Strachan and AP Read, BIOS Scientific Publishers Ltd (1996), Chapter 20, “Gene Therapy and other Molecular Genetic-based Therapeutic Approaches” (and (References cited in).

Formulation and Administration Peptides such as soluble TrpM8 ion channel polypeptides, agonist peptides and antagonist peptides, or small molecules can be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration and is well within the skill of the art. The present disclosure further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the aforementioned composition.

  TrpM8 polypeptides and other compounds can be used alone or in combination with other compounds such as therapeutic compounds.

  Preferred forms for systemic administration of these pharmaceutical compositions include injection, usually by intravenous injection. Other routes of injection can also be used, such as subcutaneous, intramuscular, or intraperitoneal. Alternative methods of systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other surfactants. In addition, oral administration may be possible if properly formulated in enteric preparations or capsules. Administration of these compounds can also be performed topically and / or locally in the form of ointments, pastes, gels, and the like.

  The required dosage range will depend on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending physician. However, suitable doses are in the range of 0.1-100 μg / kg per subject body weight. However, given the variety of available compounds and the differing efficiencies of the various routes of administration, the required dose is expected to be in a wide range. For example, oral administration would be expected to require a higher dose than administration by intravenous injection. These dose level variations can be adjusted using empirical standard routines for optimization, as is well understood in the art.

  In the manner of treatment often referred to as “gene therapy” as described above, the polypeptide used for treatment can also be produced endogenously in the body of the subject. Thus, for example, cells obtained from a subject can be engineered with a polynucleotide, such as DNA or RNA, to encode the polypeptide ex vivo, eg, by use of a retroviral plasmid vector. Those cells are then introduced into the subject's body.

A pharmaceutical composition further comprising a TrpM8 polypeptide, a polynucleotide, peptide, vector or antibody thereof, and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof) In accordance with the invention, pharmaceutical compositions comprising administering in a therapeutically effective amount are also disclosed.

  This pharmaceutical composition may be used for humans or animals in human and veterinary medicine and is usually any one of pharmaceutically acceptable diluents, carriers, or excipients. Will include one or more. Carriers or diluents that are acceptable for use in therapy are well known in the pharmaceutical art and are described, for example, in “Remington's Pharmaceutical Sciences”, Mack Publishing Co. (A. R. Gennaro, 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can include any suitable binder, lubricant, suspending agent, coating agent, solubilizer as, or in addition to, a carrier, excipient or diluent.

  Preservatives, stabilizers, dyes, and even perfumes can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

  There will be different composition / formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical compositions described herein can be formulated to be delivered using a minipump, or by mucosal route, for example as a nasal spray or aerosol for inhalation, or as an ingestible solution. Alternatively, the composition can be formulated in an injectable form and delivered parenterally, for example, by intravenous, intramuscular, or subcutaneous routes. Alternatively, the formulation can be designed to be delivered by both routes.

  For mucosal delivery of a drug through the gastrointestinal mucosa, the drug must be able to remain stable while temporarily passing through the gastrointestinal tract, for example, it is resistant to proteolytic degradation and has an acidic pH Should be stable and resistant to bile surfactant effects.

  If desired, the pharmaceutical composition is by inhalation, in the form of a suppository or suppository, topically in the form of a lotion, solution, cream, ointment, or garment, by the use of a skin patch. Orally in the form of tablets containing excipients such as starch or lactose, alone or in admixture with excipients, in capsules or suppositories, or containing flavoring or coloring agents Can be administered in the form of elixirs, solutions, or suspensions, or they can be injected parenterally, eg, intravenously, intramuscularly, or subcutaneously. For parenteral administration, the composition will best be used in the form of a sterile aqueous solution. In this case, the sterile aqueous solution can contain sufficient salts and monosaccharides to make other substances, eg, isotonic solutions with blood. For buccal or sublingual administration, the compositions can be administered in the form of tablets or lozenges that can be formulated by conventional methods.

Another embodiment of a vaccine is a method of inducing an immunological response in a mammal, which protects the animal from anxiety, stress, depression, pain and cancer, particularly prostate cancer It relates to a method comprising inoculating with sufficient TrpM8 ion channel polypeptide or fragment thereof to generate an antibody and / or T cell immune response.

  Another embodiment is a method of inducing an immunological response in a mammal, wherein the TrpM8 polypeptide is delivered via a vector that directs the expression of the TrpM8 polynucleotide in vivo to protect the animal from disease. Inducing an immunological response to produce the antibody.

  A further embodiment is an immunological / vaccine formulation (composition) that, when introduced into a mammalian host, induces an immunological response to the TrpM8 polypeptide in the mammal, wherein the TrpM8 polypeptide Or an immunological / vaccine formulation (composition) comprising the TrpM8 gene. The vaccine formulation may further comprise a suitable carrier.

  Since TrpM8 polypeptide can be degraded in the stomach, it is preferably administered parenterally (for example, subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostatic agents and solutes that render the formulation isotonic with the blood of the recipient, and suspending agents or Aqueous and non-aqueous sterile suspensions that may contain thickening agents are included. The formulations can be stored in unit-dose containers or multi-dose containers, such as sealed ampoules and vials, and in a lyophilized state requiring only the addition of a sterile liquid carrier just prior to use. Vaccine formulations may also include adjuvant systems that increase the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

  A vaccine can be prepared from one or more TrpM8 polypeptides or peptides.

  The preparation of vaccines comprising an immunogenic polypeptide or peptide (s) as active ingredient (s) is known to those skilled in the art. Typically, such vaccines can be prepared for injection as liquid solvents or suspensions, and are prepared in solid form suitable for solution or suspension in liquid prior to injection. It is also possible to do. The preparation may also be emulsified or a protein encapsulated in liposomes. The immunogenic active ingredient may be mixed with excipients that are pharmaceutically acceptable and miscible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof.

  In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants that enhance the effectiveness of the vaccine. Examples of effective adjuvants include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L- Alanyl-D-isoglutamine (CGP11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero) -3-Hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MTP-PE), and 3 components extracted from bacteria in 2% squalene / Tween 80 emulsion, monoforphoryl lipid A, trehalose dimycolate ) And cell wall skeleton (MPL + TDM + CWS) Including RIBI, and the like.

  Further examples of adjuvants and other drugs include aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsion, oil-in-water emulsion, muramyl dipeptide, bacterial endotoxin , Lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotide, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposome, levamisole, DEAE-dextran, block Copolymers or other synthetic adjuvants may be mentioned. Such adjuvants are commercially available from various sources, such as Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ) or Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Michigan). .

  Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human application.

The ratio of immunogen to adjuvant can vary widely as long as both are present in effective amounts. For example, aluminum hydroxide may be present in an amount of about 0.5% (Al ₂ O ₃ basis) of the vaccine mixture. Conveniently, the vaccine is formulated to contain the immunogen in a final concentration range of 0.2-200 μg / ml, preferably 5-50 μg / ml, most preferably 15 μg / ml.

  After formulation, the vaccine can be placed in a sterile container, which is then sealed and stored at low temperature, eg, 4 ° C., or lyophilized. Freeze drying allows long-term storage in a stabilized form.

  Vaccines are conventionally administered parenterally by injection, for example subcutaneously or intramuscularly. Other formulations suitable for other routes of administration include suppositories and, in some cases, oral formulations. For suppositories, conventional binders and carriers include, for example, polyalkylene glycols or triglycerides, and such suppositories are effective in the range of 0.5% to 10%, preferably 1% to 2%. It can be formed from a mixture containing the components. Oral formulations include commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solvents, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% -95%, preferably 25% -70% active ingredient. Including. If the vaccine composition is lyophilized, the lyophilized material can be reconstituted, eg, into a suspension, prior to administration. Reconstitution is preferably performed in a buffer.

  Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate, or hydroxypropylmethylcellulose.

  TrpM8 polypeptides can be formulated into vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of peptides), such as those formed with inorganic salts such as hydrochloric acid or phosphoric acid, acetic acid, oxalic acid, tartaric acid and maleic acid, etc. And those formed together with the organic salt. Salts formed with free carboxyl groups are also from inorganic bases such as sodium, potassium, ammonia, calcium or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine and procaine. It may be induced.

The actual dosage that is most suitable for the individual subject to be administered will usually be determined by a physician. And it will vary depending on the age, weight and response of the particular patient. The following doses are examples of the average case. Of course, in individual cases, higher or lower dose ranges may be beneficial.

  The pharmaceutical and vaccine compositions described herein can be administered directly by injection. This composition can be formulated for parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, or transdermal administration. Usually, each protein can be administered at a dose of 0.01 to 30 mg / kg body weight, preferably 0.1 to 10 mg / kg, more preferably 0.1 to 1 mg / kg body weight.

  The term “administered” includes delivery by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Non-viral delivery mechanisms include lipid-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic surface amphiphile (CFA), and combinations thereof. Routes for such delivery mechanisms include, but are not limited to, mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.

  The term “administered” includes delivery by a mucosal route, for example as a nasal spray or aerosol for inhalation, or as an ingestible solution, for example, in an injectable form by intravenous, intramuscular or subcutaneous route. Including but not limited to the parenteral route to perform.

  The term “co-administered” means that the site and time at which each additional substance, eg, TrpM8 polypeptide, and adjuvant is administered, is such that the necessary immune system modulation is achieved. Thus, the polypeptide and adjuvant can be administered at the same time and at the same site, but it may be advantageous to administer the polypeptide at a different time and at different sites than the adjuvant. is there. The polypeptide and adjuvant can even be delivered in the same delivery vehicle, and the polypeptide and antigen can be combined and / or separated and / or genetically combined and / or genetic. Can be separated.

  The TrpM8 polypeptide, polynucleotide, peptide, nucleotide, antibody thereof, and optionally an adjuvant can be administered separately to the host subject or can be co-administered as a single dose or in multiple doses.

  Vaccine and pharmaceutical compositions can be administered by many different routes such as injection (including parenteral, subcutaneous, and intramuscular injection), intranasal, mucosal, oral, intravaginal, urethral, or ocular administration. it can.

  The vaccines and pharmaceutical compositions described herein can be administered parenterally by conventional methods, for example, by subcutaneous or intramuscular injection. Other formulations suitable for other methods of administration include suppositories and, optionally, oral formulations. Suppositories may contain traditional binders and carriers, such as polyalkylene glycols or triglycerides, and such suppositories range from 0.5% to 10%, in some cases 1% to 2%. It can be formed from a mixture containing active ingredients in the range. Oral formulations usually include commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solvents, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70% To do. Where the vaccine composition is lyophilized, the lyophilized material can be reconstituted, eg, as a suspension, prior to administration. Reconstitution is preferably performed in a buffer.

Further aspects Further aspects and embodiments of the invention are described in the following numbered sections. The present invention should be understood to encompass these embodiments.

1st item: TrpM8 polypeptide containing the amino acid sequence shown by sequence number 3 or sequence number 5, or its homologue, variant, or derivative.

Second item: a nucleic acid encoding the polypeptide according to the first item.

Third item: The nucleic acid according to the second item, comprising the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.

Fourth item: A polypeptide comprising a fragment of the polypeptide according to the first item.

Fifth item: including one or more regions having homology between SEQ ID NO: 3 and SEQ ID NO: 5 or including one or more regions different between SEQ ID NO: 3 and SEQ ID NO: 5. The polypeptide according to item 3.

Sixth item: a nucleic acid encoding the polypeptide according to the fourth or fifth item.

Item 7: A vector comprising the nucleic acid according to Item 2, 3 or 6.

Item 8: A host cell comprising the nucleic acid according to Item 2, 3 or 6, or the vector according to Item 7.

Item 9: A transgenic non-human animal comprising the nucleic acid according to item 2, 3, or 6, or the vector according to item 7.

Item 10: The transgenic non-human animal according to item 9, which is a mouse.

Item 11: Use of the polypeptide according to Item 1, 4 or 5 in a method for identifying a compound capable of specifically interacting with an ion channel.

Item 12: Use of the transgenic non-human animal according to item 9 or 10 in a method for identifying a compound capable of specifically interacting with an ion channel.

Item 13: A method for identifying an antagonist of TrpM8, comprising contacting a cell expressing a TrpM8 receptor with a candidate compound, and the level of cyclic AMP (cAMP) in the cell decreased as a result of the contact Determining whether or not.

Item 14: A method for identifying a compound capable of reducing an endogenous level of cyclic AMP in a cell, comprising contacting a cell expressing TrpM8 with a candidate compound, and cyclic AMP (cAMP) in the cell Determining whether the level has decreased as a result of the contact.

Item 15: A method for identifying a compound capable of binding to a TrpM8 polypeptide, comprising the steps of contacting a TrpM8 polypeptide with a candidate compound, and determining whether the candidate compound binds to a TrpM8 ion channel polypeptide A method comprising the steps of:

Item 16: A compound identified by the method according to any one of Items 11 to 15.

Item 17: A compound capable of specifically binding to the polypeptide according to Item 1, 4, or 5.

Item 18: Use of the polypeptide according to Item 1, 4 or 5 or a part thereof, or the nucleic acid according to Item 2, 3 or 6 in the antibody production method.

Item 19: It can specifically bind to the polypeptide according to item 1, 4, or 5 or a part thereof, or the polypeptide encoded by the nucleotide according to item 2, 3, or 6 or a part thereof antibody.

Item 20: A pharmaceutical composition comprising any one or more of the following and a pharmaceutically acceptable carrier or diluent: the polypeptide according to item 1, 4 or 5, or a part thereof, 2, 3 or 6 The nucleic acid according to item or a part thereof, the vector according to item 7, the cell according to item 8, the compound according to item 16 or 17, and the antibody according to item 19.

Item 21: Vaccine composition comprising any one or more of the following: The polypeptide according to item 1, 4, or 5, or a part thereof, the nucleic acid according to item 2, 3, or 6, or a part thereof, The vector according to item 7, the cell according to item 8, the compound according to item 16 or 17, and the antibody according to item 19.

Item 22: Diagnostic kit for disease or disease susceptibility, comprising any one or more of the following: Polypeptide according to item 1, 4, or 5, or a part thereof, item 2, 3, or 6 Or a part thereof, a vector according to item 7, a cell according to item 8, a compound according to item 16 or 17, and an antibody according to item 19.

Item 23: A method for treating a patient suffering from a disease associated with increased activity of TrpM8, comprising administering to the patient an antagonist of a TrpM8 ion channel.

Item 24: A method for treating a patient suffering from a disease associated with reduced activity of TrpM8, comprising administering to the patient an agonist of a TrpM8 ion channel.

Item 25: The method according to Item 23 or 24, wherein the TrpM8 ion channel comprises a polypeptide having the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5.

Item 26: A method for treating and / or preventing a disease in a patient, comprising the step of administering any one or more of the following to the patient: The polypeptide according to Item 1, 4, or 5, or one of the polypeptides Part, the nucleic acid according to item 2, 3 or 6 or a part thereof, the vector according to item 7, the cell according to item 8, the compound according to item 16 or 17, the compound according to item 19 The antibody, the pharmaceutical composition according to item 20, and the vaccine according to item 20.

Item 27: The polypeptide according to item 1, 4 or 5 or a part thereof, the nucleic acid according to item 2, 3 or 6, or a part thereof for use in a method for treating or preventing a disease, A drug comprising the vector according to item 7, the cell according to item 8, the compound according to item 16 or 17, and / or the antibody according to item 19.

Item 28: The polypeptide according to item 1, 4 or 5 or a part thereof, the nucleic acid according to item 2, 3 or 6 for the manufacture of a pharmaceutical composition for treating or preventing a disease, Use of a part thereof, the vector according to item 7, the cell according to item 8, the compound according to item 16 or 17, and the antibody according to item 19.

Item 29: A non-human transgenic animal comprising a modified TrpM8 gene.

Item 30: The modification is a deletion of TrpM8, a mutation in TrpM8 that results in a loss of function, introduction of an exogenous gene having a nucleotide sequence that includes targeting or random mutation to TrpM8, an exogenous origin from another species to TrpM8 30. The non-human transgenic animal according to item 29, which is selected from the group consisting of introduction of a sex gene and any combination of the above.

Item 31: A non-human transgenic animal having a functionally disrupted endogenous TrpM8 gene, the genome comprising and expressing a transgene encoding a heterologous TrpM8 protein.

Item 32: A nucleic acid construct for functionally disrupting the TrpM8 gene in a host cell, wherein (a) a non-homologous replacement portion, (b) a first homologous region located upstream of the non-homologous replacement portion, A first homologous region having a nucleotide sequence with substantial identity to the first TrpM8 gene sequence, and (c) a second homologous region located downstream of the non-homologous replacement portion, substantially identical to the second TrpM8 gene sequence A nucleic acid construct comprising a second homologous region having a nucleotide sequence having a unique identity, wherein the second TrpM8 gene sequence is located downstream of the first TrpM8 gene sequence in the native endogenous TrpM8 gene.

Item 33: A method for producing a TrpM8 polypeptide, comprising culturing the host cell according to item 8 under conditions under which a nucleic acid encoding the TrpM8 polypeptide is expressed.

Item 34: A method for detecting the presence of the nucleic acid according to item 2, 3 or 6 in a sample, comprising contacting the sample with at least one nucleic acid probe specific for the nucleic acid, and the nucleic acid A method comprising monitoring a sample for the presence of.

Item 35: A method for detecting the presence of the polypeptide according to item 1, 4 or 5 in a sample, comprising contacting the sample with the antibody according to item 19, and the presence of the polypeptide A method comprising monitoring a sample for.

Item 36: A method for diagnosing a disease or syndrome caused by or associated with an increase, decrease or abnormality in TrpM8 expression, wherein (a) the expression level of TrpM8 in an animal suffering from or suspected of suffering from such disease Or detecting the expression pattern, and (b) comparing the expression level or expression pattern with that of a normal animal.

Item 37: The disease is social anxiety, post-traumatic stress disorder, phobia, interpersonal phobia, specific phobia, panic disorder, obsessive compulsive disorder, acute stress disorder, separation anxiety disorder, generalized anxiety disorder, major depression, 40. A kit, method, medicament or use according to any of items 22-28, or a method according to item 36, which is mood modulation, bipolar disorder, seasonal affective disorder, or postnatal depression.

Transgenic TrpM8 Knockout Mouse: Construction of TrpM8 Gene Targeting Vector The TrpM8 gene was identified by bioinformatics using a homology search of the genomic database. Genomic contigs with an 87 kb gap were constructed from various databases. Since this contig has sufficient flanking sequence information, it was possible to design a homology arm for cloning into a targeting vector.

  The mouse TrpM8 gene has 23 coding exons. The targeting method is designed to remove part of the 15th coding exon. The 4.0 kb 5 'homology arm and the 1.6 kb 3' homology arm adjacent to the region to be removed are amplified by PCR and the fragment is cloned into a targeting vector. The 5 'end of each oligonucleotide primer used to amplify the arm is compatible with the cloning site of the vector polylinker and recognizes different restriction enzymes that do not exist in the arm itself and are not frequently cleaved. Synthesize to contain sites. In the case of TrpM8, the primers are designed as described in the primer table below, which have AgeI / NotI as the 5 ′ arm cloning site and AscI / FseI as the 3 ′ arm cloning site (of the targeting vector used). The structure is shown in Figure X, including the associated restriction sites).

In addition to the arm primer pair (5′armF / 5′armR) and (3′armF / 3′armR), a primer specific for the TrpM8 locus is further designed for the following purposes. The 5 ′ and 3 ′ probe primer pairs (5′prF / 5′prR, and 3′prF / 3′prR) are outside of each arm and beyond the repeat genomic DNA that is beyond them. This is to amplify two short fragments of 150-300 bp and also to allow Southern analysis of target loci in isolated putative target clones. Mouse genotyping primer pairs (hetF and hetR) allow discrimination between wild type, heterozygous and homozygous mice when used in multiplex PCR with vector specific primers, in this case Asc403 To do. Finally, the target screening primer (3′scr) is annealed downstream of the end of the 3 ′ arm region and used as a pair with a primer specific to the 3 ′ end of the vector (TK5IBLMNL) (in this case Asc403). If so, it produces a 1.7 kb amplimer specific to the target event. This amplimer can only be obtained from template DNA derived from cells that have undergone the desired genomic change, allowing the identification of correctly targeted cells from background clones containing randomly integrated vector copies To. The location of these primers used in the targeting strategy and the genomic structure of the TrpM8 locus are shown.

  The position of the homology arm is selected to functionally disrupt the TrpM8 gene. In the prepared targeting vector, the expression-promoted neomycin phosphotransferase (neo) gene is arranged in the same direction as the TrpM8 gene, and an endogenous gene expression reporter (frame) upstream of the selection cassette constituted by this neo gene. The TrpM8 region to be deleted is replaced with a non-homologous sequence constituted by an independent lacZ gene).

  Once the 5 'and 3' homology arms have been cloned into the targeting vector TK5IBLMNL (see FIG. 5), large scale, high purity DNA preparations are prepared using standard molecular biology techniques. 20 μg of freshly prepared endotoxin-free DNA is digested with another low-frequency cleavage restriction enzyme, SwaI. This is present at a site specific to the vector backbone between the ampicillin resistance gene and the bacterial origin of replication. The linearized DNA is then precipitated and resuspended in 100 μl phosphate buffered saline and ready for electroporation.

  24 hours after electroporation, the transfected cells are cultured for 9 days in medium containing 200 μg / ml neomycin. Clones were picked into 96-well plates, replicated and propagated, and then PCR (primer 3 described above) to identify clones undergoing homologous recombination between the endogenous TrpM8 gene and the targeting construct. 'using scr and Asc146). Positive clones can be identified at a rate of 1% to 5%. High quality DNA for Southern blots using 5 'and 3' external probes, prepared as described above, all using standard procedures to freeze replicas and confirm targeting events These clones are propagated (Russ et al., 2000, Nature 2000 Mar 2; 404 (6773): 95-99). When Southern blots of DNA digested with diagnostic restriction enzymes are hybridized with an external probe, the presence of mutant and unchanged wild-type bands confirms homologously targeted ES cell clones Is done. For example, wild type genomic DNA digested with BstEII produces an 8.0 kb band when hybridized with any external probe, and by digesting genomic DNA containing the target allele in the same way, an additional approximately 13 kb Knockout specific bands can occur.

Transgenic TrpM8 Knockout Mice: Generation of TrpM8 Ion Channel Deficient Mice Female and male C57BL / 6 mice are mated and blastocysts are isolated on day 3.5 of gestation. Cells from selected clones are injected with 10-12 cells per blastocyst and 7-8 blastocysts are implanted into pseudopregnant F1 female uterus. Litters of chimeric offspring mice containing several high levels (up to 100%) of Agoutios are born (Agouti skin color indicates the distribution of cells that are the descendants of the targeted clone). These male chimeras are mated with female MF1 and 129 mice, and germline transmission is determined by agouti skin color and by respective genotyping by PCR.

  PCR genotyping of the lysed tail clip is performed using primers hetF and hetR and a third, vector specific primer (Asc306). This multiplex PCR allows amplification from the wild type locus (if present) from primers hetF and hetR, giving a 230 bp band. This amplification may not be from the targeted allele because the hetF site is deleted in knockout mice. However, the Asc306 primer will amplify a 338 bp band from the targeted locus in conjunction with a hetR primer that anneals to the region immediately in the 3 'arm. Therefore, this multiplex PCR reveals the litter genotype as follows. The wild type sample will show a single band of 230 bp; the heterozygous DNA sample will give two bands of 230 bp and 338 bp, and the homozygous sample will show only a 338 bp band specific for the target.

Biological data: Gene expression pattern
1) RT-PCR
Gene expression in the prostate, liver and testis is shown using RT-PCR (Figure 2).

2) List of LacZ stained structures
Xgal staining of the tissue excised by LacZ staining is performed as follows.

Representative tissue sections are made from large organs. All organelles and tubes are dissected and infused with fixative and stain. The tissue is washed thoroughly with PBS (phosphate buffered saline) to remove blood or gastrointestinal contents. Tissue fixative put 30-45 minutes in (2% formaldehyde, 0.2% glutaraldehyde, PBS containing 0.02% NP40 MgCl _2, sodium deoxycholate 0.23 mM). After 3 washes with PBS for 5 minutes, Xgal staining solution (4 mM ferrocyanated K, 4 mM ferricyanide K, 2 mM MgCl ₂ , 1 mg / ml X-gal in PBS) at 30 ° C. for 18 hours. Put the organization inside. The tissue is washed 3 times with PBS, post-fixed in 4% formaldehyde for 24 hours, washed again with PBS and stored in 70% ethanol.

  By using LacZ staining, it can be seen that TrpM8 is expressed in DRG and especially in neurons.

Biological Data: Tail Flick Test A tail flick analgesia test is performed using a tail flick analgesia meter. This instrument makes it easy to use methods for accurately and reproducibly measuring pain sensitivity in rodents (D'Amour, FE and DL Smith, 1941, Expt. Clin. Pharmacol., 16 : 179-184). This device comprises a lamp controlled by a shutter as a heat source. The lamp is placed under the animal, creating a less confined environment. The tail flick is detected by an automatic detection circuit that does not require the user to manipulate the animal. The animal is restrained in a ventilated tube and its tail is placed in the sensor groove at the top of the device.

  The activation of a powerful ray on the tail by the opening of the shutter makes it uncomfortable at several points, where the animal moves its tail to a place where there is no ray. In automated mode, the photodetector detects tail movement, stops the clock, and closes the shutter. Record the total time elapsed between the shutter opening and the animal reaction.

  The response of the mutant transgenic animals is compared to wild type mice of matched age and gender. Single animals are subjected to testing at different heat settings and increased to tail temperatures not exceeding 55 ° C.

  This test is used as an indicator of the response of knockout mice to tissue damaging pain.

  The mutant is observed to be less sensitive to heat-induced pain and exhibit hypoalgesia. The mutant is observed to have a longer duration of moving its tail from the heat source compared to age- and gender-matched wild-type mice (FIG. 3). This therefore indicates that TrpM8 is involved in pain sensing.

Biological data: Open field test Knockout mice and wild type control mice are tested in the open field test. Those skilled in the art are familiar with such tests and understand how to perform them. Briefly, a mouse is placed in the center of a windproof glass box having a transparent side, and the movement of the mouse at a certain time is recorded as a video. Analyze the distance moved and the position of the mouse at a certain time. Control animals generally spend the most time traveling around the perimeter of their box area. Changes from this normal pattern, especially the time spent in the central area of the box area, are recorded and this increase can mean that the animal is less anxious.

  The results showed that the knockout mice migrated as much as the wild type control (FIG. 4A). However, the analysis result of this data showed that the knockout mouse generally traveled more in the central region of the open field box region than the surrounding region (FIG. 4B). This was reflected in the distance that the knockout animal compared to the wild type was very large and moved in the central region (FIG. 4C).

Biological data: Physiology: Plasma corticosterone Corticosterone is a hormone released in response to stress and anxiety. The lower the corticosterone level, the lower the level of anxiety and stress that the animal may be experiencing.

  Plasma corticosterone was analyzed using an ELISA assay in TrpM8 − / − mice and age-matched + / + mice. Corticosterone levels were found to be lower in mutant animals than in wild type (FIG. 5). This indicates that TrpM8 mice are less anxious and less stressed than their wild type controls.

  Each patent application and patent mentioned in this specification, including those in each patent application and patent procedure, as well as each document cited or referenced in each of the above patent applications and patents ("patent application citations") ), And any manufacturer's instructions and catalogs of any products cited or referenced in any of the patent applications and patents and patent application citations are hereby incorporated herein by reference. In addition, any documentation by the manufacturer of all documents cited in the text, and all documents cited or referenced in documents cited in the text, and any products cited or referenced in the text. And catalogs are hereby incorporated herein by reference.

  It will be apparent to those skilled in the art that various modifications and variations of the described methods and system of the invention do not depart from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, the claims of the invention should not be unduly limited to such specific embodiments and are within the scope of the invention. It should be understood that many changes and additions can be made in. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. Furthermore, the features of the independent claims can be used to make various combinations of the features of the dependent claims without departing from the scope of the invention.

It is a figure which shows a knockout vector. It is a figure which shows the result of the gene expression by RT-PCR test. It is a graph which shows the result of the tail flick test regarding a knockout mouse (mutant) compared with a wild type mouse (wt). FIG. 6 is a graph showing the results of an open field test for knockout mice (mutants, − / −) compared to wild type mice (wt, + / +). FIG. 4A shows the action time in the central region. FIG. 4B shows the distance moved in the central region. FIG. 4C shows the total distance traveled. FIG. 5 is a graph showing the results of an assay of plasma corticosterone levels for female knockout mice (mutants, white bars) compared to female wild type mice (black bars) at 3 months of age.

SEQ ID NO: 1 shows the cDNA sequence of human TrpM8. SEQ ID NO: 2 shows the open reading frame obtained from SEQ ID NO: 1. SEQ ID NO: 3 shows the amino acid sequence of human TrpM8. SEQ ID NO: 4 shows the open reading frame of mouse TrpM8 cDNA. SEQ ID NO: 5 shows the amino acid sequence of mouse TrpM8. SEQ ID NOs: 6-18 show the genotyping primers used to construct the knockout plasmid. SEQ ID NO: 19 shows the sequence of a knockout plasmid.

Claims (28)

  A transgenic non-human animal having a functionally disrupted endogenous TrpM8 gene, wherein the TrpM8 gene is a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or at least 70% relative thereto A transgenic non-human animal comprising a sequence having the following sequence identity:   The transgenic non-human animal according to claim 1, which has a deletion in the TrpM8 gene or a part thereof. When compared to wild-type animals,
(A) reduced susceptibility to pain, preferably measured by tail flick test,
3. The phenotype of any one or combination of (b) reduced stress, preferably measured in an open field test, and (c) reduced plasma corticosterone levels. Transgenic non-human animals.   A transgenic non-human animal, wherein at least part or all of the TrpM8 gene of the animal is replaced with a sequence of another animal, preferably another species, more preferably a human TrpM8 gene .   The transgenic non-human animal according to any one of claims 1 to 4, which is a mouse.   A functionally disrupted TrpM8 gene, preferably comprising a deletion in the TrpM8 gene, wherein the TrpM8 gene comprises the nucleic acid sequence shown in SEQ ID NO: 4 or a sequence having at least 70% sequence identity thereto The transgenic non-human animal according to claim 5.   A cell or tissue isolated from the non-human transgenic animal according to any one of claims 1 to 6.   A cell having a functionally disrupted endogenous TrpM8 gene, wherein the TrpM8 gene is a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or at least 70% sequence identity thereto A cell comprising a sequence having   Use of the transgenic non-human animal according to any one of claims 1 to 6, the cell or tissue according to claim 7 or the cell according to claim 8 as a model of pain or stress.   Use of the transgenic non-human animal according to any one of claims 1 to 6, the cell or tissue according to claim 7, or the cell according to claim 8 as a model of a TrpM8-related disease.   7. A method of identifying an agonist or antagonist of a TrpM8 polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto. Use of the non-human transgenic animals according to claim 8, their isolated cells or tissues according to claim 7, or the cells according to claim 8.   A method of identifying an agonist or antagonist of a TrpM8 polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto, comprising an animal, preferably wild A candidate compound to a non-human animal or a transgenic non-human animal according to any of claims 1 to 6, and the following phenotype: (a) sensitivity to pain, preferably measured in a tail flick test Measuring the change in any of stress, and (c) plasma corticosterone levels, preferably as measured in an open field test.   13. A method of identifying an agonist of a TrpM8 polypeptide according to claim 12, comprising identifying candidate compounds capable of exhibiting any increase in the phenotypes (a)-(c) in the animal.   13. A TrpM8 polypeptide antagonist according to claim 12, comprising identifying a candidate compound capable of exhibiting any of the phenotypes (a) to (c) or such a decrease in phenotype in the animal. How to identify.   A method for identifying an agonist or antagonist of a TrpM8 polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto, comprising a cell or tissue, preferably Exposing a candidate compound to a wild type cell or tissue, or a cell or tissue according to claim 7, or a cell according to claim 8, and the conductivity or intracellular calcium of the cell or of the tissue Measuring the concentration change.   16. A method of identifying an agonist of a TrpM8 polypeptide according to claim 15, comprising identifying a candidate compound capable of increasing the cell conductivity or intracellular calcium concentration.   16. The method of identifying an antagonist of a TrpM8 polypeptide according to claim 15, comprising identifying a candidate compound capable of reducing the cellular conductivity or intracellular calcium concentration.   A method for identifying a compound suitable for treating or alleviating pain or stress, preferably a TrpM8-related disease, comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or at least 70% sequence thereto Exposing a TrpM8 polypeptide comprising a sequence having identity to a candidate compound and determining whether the candidate compound is an antagonist or antagonist of the TrpM8 polypeptide.   A nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or at least relative thereto, for identifying an agonist or antagonist of a TrpM8 polynucleotide for the treatment of pain or stress, preferably a TrpM8 related disease Use of a TrpM8 polynucleotide comprising a sequence having 70% sequence identity.   Amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence of at least 70% relative thereto for identifying an agonist or antagonist of a TrpM8 polypeptide for the treatment of pain or stress, preferably a TrpM8 related disease Use of a TrpM8 polypeptide comprising a sequence with identity.   Having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence having at least 70% sequence identity thereto, for use in a method of treating pain or stress in an individual, preferably a TrpM8-related disease An antagonist of a TrpM8 polypeptide.   Amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, or at least 70% sequence identity thereto for the manufacture of a pharmaceutical composition for the treatment of pain or stress in an individual, preferably a TrpM8 related disease Use of an antagonist of a TrpM8 polypeptide having a sequence having   A method of treating an individual suffering from pain or stress, preferably a TrpM8-related disease, comprising administering to the individual an antagonist of TrpM8.   A method of diagnosing pain or stress in an individual, preferably a TrpM8-related disease, comprising detecting a change in the expression, level or activity of TrpM8 in the individual or its cells or tissues.   TrpM8 related diseases include pain, cancer, inflammation, inflammatory bowel disease, hyperalgesia, visceral pain, migraine, postherpetic neuralgia, diabetic neuralgia, trigeminal neuralgia, postoperative pain, osteoarthritis, rheumatoid arthritis, acute pain, Chronic pain, skin pain, somatic pain, visceral pain, related pain including myocardial ischemia, phantom pain, neuropathic pain (neuralgia), pain due to injury and disease, headache, migraine, cancer pain, Parkinson's disease Pain due to neuropathy, pain from spinal and peripheral nerve surgery, brain tumors, traumatic brain injury (TBI), spinal cord trauma, chronic pain syndrome, chronic fatigue syndrome; such as trigeminal neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia and causalgia Pain due to lupus, sarcoidosis, arachnoiditis, arthritis, rheumatic diseases, menstrual pain, back pain, lower back pain, joint pain, abdominal pain, chest pain, labor pain, musculoskeletal and skin diseases, Urine disease, head injury, and fibromyalgia; breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, and bone cancer; social anxiety, post-traumatic stress disorder, phobia, interpersonal phobia, specific phobia, Selected from the group consisting of panic disorder, obsessive compulsive disorder, acute stress disorder, separation anxiety disorder, generalized anxiety disorder, major depression, mood modulation, bipolar disorder, seasonal emotional disorder, postnatal depression, manic depression, bipolar depression 23. Use according to claim 10, 19, 20 or 22, method according to claim 18, 23 or 24, or antagonist according to claim 21.   A TrpM8 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or a homologue, variant, or derivative thereof having at least 70% sequence identity thereto.   27. A nucleic acid encoding the polypeptide of claim 26.   The nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof having at least 70% sequence identity thereto The nucleic acid described.

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