# Patent

## Title
Methods for treating chronic pain

## Abstract

The invention provides a method of treating chronic pain by administering an effective amount of a low-molecular weight compound that inhibits the enzyme activity of autotaxin to a subject in need thereof, thereby treating chronic pain in the subject. The chronic pain preferably is fibromyalgia, chronic fatigue syndrome, or hypersensitivity colitis.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a divisional of copending U.S. patent application Ser. No. 13/057,888, filed on Mar. 24, 2011, which is the U.S. national phase of International Patent Application No. PCT/JP2009/064062, filed Aug. 7, 2009, which claims the benefit of Japanese Patent Application No. 2008-204762, filed on Aug. 7, 2008, and Japanese Patent Application No. 2009-112990, filed on May 7, 2009, which are incorporated by reference in their entireties herein.


INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 66,575 bytes ASCII (Text) file named “7169796SequenceListing.txt,” created Jun. 27, 2014.
TECHNICAL FIELD
The present invention relates to a therapeutic or prophylactic agent for generalized pain syndrome. Specifically, the present invention relates to the field of pharmaceuticals that help treat chronic pains.
BACKGROUND ART
There are two types of pains: acutely occurring pains as a self-warning to living organisms and chronically developing pains as an illness. While there is a demand for recovery from chronic pains by treatments, including drugs, a normegligible number of patients suffer intractable diseases for which no therapies have been established because the causal mechanism of onset remains unclear. Among them is generalized pain syndrome. Generalized pain syndrome is a disease of unknown cause that produces intense pain over a wide area of the body, and it is difficult to detect ecologically abnormal findings by examinations. Because generalized pain syndrome tends to often become chronic, it is positioned as an intractable chronic pain. Not only patients experience disturbances in their daily activities and labor, but also there are not a few cases wherein they have mental concerns, including the inability to determine which department to visit, the lack of an established diagnosis, even by extensive examinations, due to the low availability of specialist physicians, the absence of therapeutic drugs, being deemed idle, and the like. In fact, it is known that in addition to pains, generalized pain syndrome is often complicated by a sensation of fatigue, depression, anxiety and the like. Additionally, fibromyalgia, a form of generalized pain syndrome, occurs most prevalently among middle-aged to elderly women, and it has been found that physical traumas from surgery or accidents and stress-related mental factors are profoundly involved in the background of the onset.
Lysophosphatidic acid (LPA) is a lipid mediator produced at the time of tissue damage, known to act on 7-pass transmembrane receptors (LPA1, LPA2, LPA3) that couple with various G proteins (Gq/11/14, G12/13, Gi/o) to serve as a trophic factor for various cells, including nerve and glial cells.
The present inventor, through many years of research into the molecular mechanisms behind neuropathic pains, revealed that LPA is a substance that induces neuropathic pains, and reported that nerve injury-induced pain does not develop when a receptor of LPA is lacked using knockout mice prepared by knock out of LPA1, a kind of LPA receptor (Non-patent Documents 1 and 2).
The present inventor demonstrated that LPA is synthesized from lysophosphatidylcholine (LPC), which is a constituent of the cell membrane, by the LPA synthetase autotaxin (ATX), and is involved in irritation development (Non-patent Documents 3 and 4).
PRIOR ART DOCUMENTS
Non-Patent Documents


    Non-patent Document 1: Inoue M, et al Nat Med 10: p. 712-718, 2004
    Non-patent Document 2: Ueda H Pharmacol Ther 109: p. 57-77, 2006
    Non-patent Document 3: Inoue M, et al Neuroscience 152: p. 296-298, 2008
    Non-patent Document 4: Inoue M, et al Molecular Pain, 4: 6, 2008


SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
It is an object of the present invention to provide a therapeutic or prophylactic agent for generalized pain syndrome, for which no cause and therapies have been established.
Means of Solving the Problems
Taking note of stress as a cause of induction of generalized pain syndrome, the present inventor attempted to clarify the cause of generalized pain disease using animal models actually exposed to stress. The present inventor conducted further extensive investigations, found that LPA is profoundly involved not only in the peripheral nervous system, but also in the central nervous system in individuals affected by generalized pain syndrome, and has developed the present invention. Accordingly, the invention of this application is as follows:

    [1] A therapeutic or prophylactic agent for generalized pain syndrome comprising an LPA1 antagonist as an active ingredient.
    [2] The therapeutic or prophylactic agent described in [1] above, wherein the LPA1 antagonist is selected from the group consisting of antisense nucleic acids, siRNAs and antagonist antibodies against LPA1, soluble LPA1 and low-molecular compounds that bind to LPA1 to inhibit signaling.
    [3] The therapeutic or prophylactic agent described in [1] or [2] above, wherein the LPA1 antagonist is administered to the central nervous system.
    [4] The therapeutic or prophylactic agent described in any one of [1] to [3] above, wherein the generalized pain syndrome is fibromyalgia, chronic fatigue syndrome or hypersensitivity colitis.
    [5] A therapeutic or prophylactic agent for generalized pain syndrome comprising an autotaxin-inhibiting substance as an active ingredient.
    [6] The therapeutic or prophylactic agent described in [5] above, wherein the autotaxin-inhibiting substance is selected from the group consisting of antisense nucleic acids, siRNAs and inhibitory antibodies against autotaxin and low-molecular compounds that inhibit the enzyme activity of autotaxin.
    [7] The therapeutic or prophylactic agent described in [5] or [6] above, wherein the autotaxin-inhibiting substance is administered to the central nervous system.
    [8] The therapeutic or prophylactic agent described in any one of [5] to [7] above, wherein the generalized pain syndrome is fibromyalgia, chronic fatigue syndrome or hypersensitivity colitis.
    [9] A method of treating or preventing generalized pain syndrome comprising a step for administering an effective amount of an LPA1 antagonist to a subject in need thereof.
    [10] The therapeutic or prophylactic method described in [9] above, wherein the LPA1 antagonist is selected from the group consisting of antisense nucleic acids, siRNAs and antagonist antibodies against LPA1, soluble LPA1 and low-molecular compounds that bind to LPA1 to inhibit signaling.
    [11] The therapeutic or prophylactic method described in [9] or [10] above, wherein the LPA1 antagonist is administered to the central nervous system.
    [12] The therapeutic or prophylactic method described in any one of [9] to [11] above, wherein the generalized pain syndrome is fibromyalgia, chronic fatigue syndrome or hypersensitivity colitis.
    [13] A method of treating or preventing generalized pain syndrome comprising a step for administering an effective amount of an autotaxin-inhibiting substance to a subject in need thereof.
    [14] The therapeutic or prophylactic method described in [13] above, wherein the autotaxin-inhibiting substance is selected from the group consisting of antisense nucleic acids, siRNAs and inhibitory antibodies against autotaxin and low-molecular compounds that inhibit the enzyme activity of autotaxin.
    [15] The therapeutic or prophylactic method described in [13] or [14] above, wherein the autotaxin-inhibiting substance is administered to the central nervous system.
    [16] The therapeutic or prophylactic method described in any one of [13] to [15] above, wherein the generalized pain syndrome is fibromyalgia, chronic fatigue syndrome or hypersensitivity colitis.
    [17] A use of an LPA1 antagonist for producing the therapeutic or prophylactic agent for generalized pain syndrome described in any one of [1] to [4] above.
    [18] A use of an autotaxin-inhibiting substance for producing the therapeutic or prophylactic agent for generalized pain syndrome described in any one of [5] to [8] above.


Effect of the Invention
According to a therapeutic or prophylactic agent for generalized pain disease of the present invention, it is possible to make a major contribution to the establishment of an effective and reliable therapeutic policy and prophylactic method for generalized pain disease, for which no therapies have been established to date because the cause remains unclear.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphic representation showing results of the mechanical stimulus-induced pain test of Example 3 performed on Vehicle-treated mice.
FIG. 2 is a graphic representation showing results of the mechanical stimulus-induced pain test of Example 3 performed on the AS-ODN-treated mice of Example 1.
FIG. 3 is a graphic representation showing results of the mechanical stimulus-induced pain test of Example 3 performed on the MS-ODN-treated mice of Example 1.
FIG. 4 is a graphic representation showing results of the mechanical stimulus-induced pain test of Example 3 performed on the AS-ODN-treated mice of Example 2.
FIG. 5 is a graphic representation showing results of the mechanical stimulus-induced pain test of Example 3 performed on the MS-ODN-treated mice of Example 2.
FIG. 6 is a graphic representation showing hypersensitive responses over days to mechanical stimulation in ICS. Control group: n=4, ICS stress group: n=6. ** indicates P<0.01 when comparing the control stress group and the ICS stress group.
FIG. 7 is a graphic representation showing results of measurements of corticosterone concentration for ICS/CCS stress model mice. ICS stress group: n=3, CCS group: n=6; ** indicates P<0.01 when comparing the CCS stress group and the ICS stress group.
FIG. 8 is a graphic representation showing changes over days in mechanical nociceptive responses to intracerebroventricular administration of ATX-AS to C57BL/6J. A: A plan for ATX-AS/MS administration is shown. Intracerebroventricular administration was performed, starting on 5 days before, including the starting day of ICS stress loading. B: Changes over days in mechanical nociceptive responses are shown. ACSF group: n=3, ACSF+ICS stress group: n=4, ATX-MS+ICS stress group: n=3, ATX-AS+ICS stress group: n=4. ## indicates P<0.01 when comparing the ATX-AS group and the ATX-MS group; * indicates P<0.05 when comparing the ATX-AS group and the ATX-MS group.
FIG. 9 is a graphic representation showing changes over days in mechanical nociceptive responses to intracerebroventricular administration of Bromoenol lactone (BEL) to C57BL/6J. A: A plan for BEL administration is shown. B: Changes over days in mechanical nociceptive responses are shown. ACSF group: n=4, BEL group: n=5, ACSF+ICS stress group: n=8, BEL+ICS stress group: n=8. ** indicates P<0.01 when comparing the ACSF group and the ACSF+ICS stress group; * indicates P<0.05 when comparing the ACSF group and the ACSF+ICS stress group.
FIG. 10 is a graphic representation showing changes over days in thermal nociceptive responses to intracerebroventricular administration of Bromoenol lactone (BEL) to C57BL/6J. ACSF group: n=2, BEL group: n=2, ACSF+ICS stress group: n=4, BEL+ICS stress group: n=4. ** indicates P<0.01 when comparing the ACSF group and the ACSF+ICS stress group.



MODE FOR EMBODYING THE INVENTION
Detailed Description of the Invention
A therapeutic or prophylactic agent of the present invention comprises an LPA1 antagonist as an active ingredient.
In the present invention, LPA1 (lysophosphatidic acid receptor 1) refers to a kind of 7-pass transmembrane receptor, a ligand thereto being LPA (lysophosphatidic acid), which couples with G protein, and is also referred to as EDG2 (endothelial differentiation lysophosphatidic acid G protein coupled receptor 2). While LPA1, LPA2 and LPA3 have been identified as lysophosphatidic acid receptors so far, LPA1 is targeted in relation to the diseases to which the present invention is directed. The gene that encodes LPA1 may have a base sequence derived from any animal. For example, for developing a therapeutic drug for humans, the human LPA1 gene is preferred. When using mice, which are readily utilizable laboratory animals, the analytical results from the mice are well expected to reflect pathologic conditions in other mammals, including humans; therefore, it is also preferable to use the mouse LPA1 gene. Herein, the human LPA1 gene is based on the base sequence disclosed in Genbank Accession No. NM—001401 (SEQ ID NO:1). The mouse LPA1 gene is based on the base sequence disclosed in Genbank Accession No. NM—010336 (SEQ ID NO:3). LPA1 homologues derived from other animals can be identified using HomoloGene (www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, a particular human base sequence is applied to BLAST (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, www.ncbi.nlm.nih.gov/BLAST/), and the accession number of the identical (exhibiting the highest Score, an E-value of 0 and 100% Identity) sequence is acquired. The accession number is input to UniGene (www.ncbi.nlm.nih.gov/UniGene/), and the thus-obtained UniGene Cluster ID (a number shown by Hs.) is input to HomoloGene. From the resulting list of gene homologue correlations between the genes of other biological species and the human gene, genes of other biological species can be selected as genes corresponding to the human gene shown by the particular base sequence (homologues).
Likewise, in the present invention, the human LPA1 protein is based on the amino acid sequence disclosed in Genbank Accession No. NM—001401 (SEQ ID NO:2). The mouse LPA1 protein is based on the amino acid sequence disclosed in Genbank Accession No. NM—010336 (SEQ ID NO:4).
In the present invention, an LPA1 antagonist generically refers to substances acting to antagonize the receptor LPA1, possessing the action reverse to that of LPA, which is a natural agonist of LPA1. LPA1 antagonists include not only substances that bind to LPA1 to inhibit the signaling from the receptor, but also substances that inhibit the binding of LPA1 and LPA, for example, substances that capture LPA (for example, soluble LPA1, which captures LPA in the blood, and the like) and the like. Furthermore, LPA1 antagonists also include substances that inhibit factors downstream of the signaling from the LPA1 receptor. Such substances include Botulinus toxin C3, which inhibits RhoA activity, Y-27632, which inhibits Rho kinase activity, and the like.
LPA1 antagonists include antagonists that have been shown to possess antagonistic activity to date, or all substances whose antagonistic activity can be confirmed using the model of stress-induced generalized pain described below. Specifically, ones selected from the group consisting of an antisense nucleic acid, siRNA, viral vector-recombined shRNA, microRNA and antagonist antibody against LPA1, soluble LPA1 and a low-molecular compound that binds to LPA1 to inhibit signaling can be mentioned as examples. These may be natural substances or artificially synthesized substances.
The aforementioned antisense nucleic acid against LPA1 refers to, for example, a single-stranded nucleic acid complementary to at least a part of base sequence selected from among the base sequence of SEQ ID NO:1 or 3 or the base sequence that encodes the amino acid sequence of SEQ ID NO:2 or 4. The nucleic acid may be a naturally derived or artificial nucleic acid, or may be one based on DNA or RNA. The length of the antisense nucleic acid is normally about 15 bases to the same as the full length of the mRNA, preferably about 15 to about 30 bases long. The complementarity of the antisense nucleic acid does not always need to be 100%, and may be of a degree that allows complementary binding to the DNA or RNA of LPA1 in a living organism.
The aforementioned siRNA refers to a double-stranded RNA synthesized artificially to cause the degradation of the mRNA transcribed from the LPA1 gene (RNA interference) on the basis of the base sequence thereof, or a vector capable of supplying the double-stranded RNA in a living organism, in order to suppress the expression of the LPA1 gene. Using the siRNA or siRNA expression vector of the present invention, it is possible to have an action to suppress generalized pains by reducing the expression of LPA1 to inhibit the signaling from LPA1. To construct an siRNA or siRNA expression vector, a publicly known method can be used (Ui-Tei K, et al., Nucleic Acids Res. 2004; 32: 936-948; Miyagishi M, and Taira K, Nature biotechnology 2002; 20: 497-500). The length of the siRNA is preferably 19 to 27 bp, more preferably 21 to 25 bp. In the shRNA and microRNA, a partial gene of the LPA1 gene is recombined into an appropriate viral vector and administered as a virus systemically or to the brain or the spinal cord, whereby the LPA1 gene is knocked down. The length of the partial gene is preferably 60 to 70 bp in each case.
The aforementioned antagonist antibody against LPA1 refers to an antibody that binds specifically to LPA1 and inhibits the signaling from LPA1 by binding thereto. This antagonist antibody is preferably an antibody that recognizes an extracellular domain of LPA1 and the like.
As mentioned herein, “antibodies” include natural type antibodies such as polyclonal antibodies and monoclonal antibodies, chimeric antibodies, humanized antibodies and single-stranded antibodies produced using gene recombination technology, human antibodies that can be produced using human antibody-producing transgenic animals and the like, antibodies prepared by phage display and binding fragments thereof.
A binding fragment means a partial region of one of the above-described antibodies; specifically, for example, F(ab′)2, Fab′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), dAb (single domain antibody) and the like can be mentioned (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996).
The class of antibody is not particularly limited; antibodies of any isotypes such as IgG, IgM, IgA, IgD and IgE are encompassed. Preferably, the class is IgG or IgM, and in view of the ease of purification and the like, IgG is more preferable.
A polyclonal antibody or a monoclonal antibody can be produced by a known ordinary method of production. Specifically, for example, an immunogen, along with Freund's Adjuvant as required, is given for immunization to a mammal, for example, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, horse or bovine, preferably a mouse, rat, hamster, guinea pig, goat, horse or rabbit, in the case of a polyclonal antibody, or to a mouse, rat or hamster in the case of a monoclonal antibody.
Specifically, a polyclonal antibody can be produced as described below. An immunogen is injected to a mouse, rat, hamster, guinea pig, goat, horse or rabbit, preferably to a goat, horse or rabbit, more preferably to a rabbit, subcutaneously, intramuscularly, intravenously, into a footpad or intraperitoneally, once to several times, whereby the animal is immunologically sensitized. Normally, 1 to 5 immunizations are performed at intervals of about 1 to 14 days from initial immunization, and about 1 to 5 days after final immunization, a serum is acquired from the immunologically sensitized mammal.
Although the serum can be used as a polyclonal antibody, it is preferably isolated and/or purified by ultrafiltration, ammonium sulfate fractionation, the euglobulin precipitation method, the caproic acid method, the caprylic acid method, ion exchange chromatography (DEAE or DE52 and the like), or affinity column chromatography using an anti-immunoglobulin column, a protein A/G column, an immunogen-crosslinked column or the like.
A monoclonal antibody is produced by preparing a hybridoma from a cell that produces the antibody, obtained from the above-described immunologically sensitized animal, and a myeloma-series cell (myeloma cell) not having the capability of autoantibody production, cloning the hybridoma, and selecting a clone that produces a monoclonal antibody that exhibits specific affinity for the immunogen used to immunize the mammal.
Specifically, a monoclonal antibody can be produced as described below. An immunogen is injected once to several times, or transplanted, to a mouse, rat or hamster (including transgenic animals created to produce an antibody derived from another animal, like human antibody-producing transgenic mice) subcutaneously, intramuscularly, intravenously, into a footpad or intraperitoneally, whereby the animal is immunologically sensitized. Normally, 1 to 4 immunizations are performed at intervals of about 1 to 14 days from initial immunization, and about 1 to 5 days after final immunization, antibody-producing cells are acquired from the immunologically sensitized the mammal.
Preparation of a hybridoma (fusion cell) that secretes a monoclonal antibody can be performed according to the method of Kohler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) or a modified method based thereon. Specifically, the hybridoma is prepared by cell-fusion of an antibody-producing cell contained in a spleen, lymph node, bone marrow, tonsil or the like, preferably in a spleen, acquired from a mammal immunologically sensitized as described above, and a myeloma cell not having the capability of autoantibody production, preferably derived from a mammal such as a mouse, rat, guinea pig, hamster, rabbit or human, more preferably from a mouse, rat or human.
Examples of useful myeloma cells for the cell fusion include the mouse-derived myeloma P3/X63-AG8.653 (653; ATCC No. CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2), PA1, F0 or BW5147, the rat-derived myeloma 210RCY3-Ag.2.3., and the human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15.
Screening for a hybridoma clone that produces a monoclonal antibody can be performed by culturing a hybridoma in, for example, a microtiter plate, and measuring the reactivity of the culture supernatant of proliferative hybridoma in a well to the immunogen used in the immunological sensitization, by, for example, an enzyme immunoassay such as ELISA.
The hybridoma may be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and a centrifugal supernatant of the culture broth may be used as a monoclonal antibody solution. It is also possible to inject this hybridoma into the abdominal cavity of the animal from which it is derived to thereby produce ascites fluid, and to use the thus-obtained ascites fluid as a monoclonal antibody solution. The monoclonal antibody, like the above-described polyclonal antibody, is preferably isolated and/or purified.
A chimeric antibody can be produced with reference to, for example, “Jikken Igaku (extra issue), Vol. 6, No. 10, 1988”, JP-B-HEI-3-73280 and the like; a humanized antibody can be prepared with reference to, for example, JP-T-HEI-4-506458, JP-A-SHO-62-296890 and the like; a human antibody can be prepared with reference to, for example, “Nature Genetics, Vol. 15, p. 146-156, 1997”, “Nature Genetics, Vol. 7, p. 13-21, 1994”, JP-T-HEI-4-504365, International Patent Application Publication WO94/25585, “Nikkei Science, June issue, pages 40 to 50, 1995”, “Nature, Vol. 368, p. 856-859, 1994”, JP-T-HEI-6-500233) and the like.
In preparing an antibody by phage display, an antibody such as Fab can easily be obtained from a phage library prepared for antibody screening, by, for example, recovering and concentrating a phage with affinity for the antigen by biopanning. For the preparation of an antibody by phage display, see “Nature, Vol. 348, p. 552-554, 1990”, “Phage display a laboratory manual” In cold spring harbor laboratory press, 2001”, and “Antibody Engineering—a Practical Approach, IRL Press, Oxford, 1996”.
F (ab′)2 and Fab′ can be produced by treating an immunoglobulin with the proteinase pepsin or papain, respectively. Fab can be produced by screening a Fab expression phage library in the same manner as the above-described method of antibody preparation by phage display.
The aforementioned soluble LPA1 is a decoy receptor that binds to LPA but does not allow the signaling into cells to occur. Soluble LPA1 has the function of suppressing the signaling from LPA1 mainly by trapping LPA in the blood to reduce the amount of LPA bound to LPA1. As such, soluble LPA1 can be prepared by a publicly known gene engineering technique on the basis of the amino acid sequence that constitutes an extracellular domain of the receptor.
The aforementioned low-molecular compound is exemplified by the compounds disclosed in WO2002/062389, WO2003/007991, WO2003/024402 (JP-T-2005-508319) or WO2005/032494 (JP-T-2007-508324) and the like. As commercially available antagonists, ammonium (S)-mono-{2-octadeca-9-enoylamino-3-[4-(pyridin-2-ylmethoxy)-phenyl]-propyl}phosphate, ammonium (S)-mono-[3(4-benzyloxy-phenyl)-2-octadeca-9-enoylamino-propyl]phosphate, and the like can be mentioned.
A therapeutic or prophylactic agent of the present invention comprises an autotaxin-inhibiting substance as an active ingredient.
In the present invention, autotaxin (ATX) is also referred to as ENPP (ectonucleotide pyrophosphatase/phosphodiesterase) 2; it is an enzyme that catalyzes the synthesis of LPA from lysophosphatidylcholine (LPC), which is a constituent of the cell membrane, possessing lysophospholipase D (LPLD) activity. The gene that encodes ATX may have a base sequence derived from any animal. For example, for developing a therapeutic drug for humans, the human ATX gene is preferred. When using mice, which are readily utilizable laboratory animals, the analytical results from the mice are well expected to reflect pathologic conditions in other mammals, including humans; therefore, it is also preferable to use the mouse ATX gene. Herein, the human ATX gene is based on the base sequence disclosed in Genbank Accession No. NM—006209 (SEQ ID NO:9). The mouse ATX gene is based on the base sequence disclosed in Genbank Accession No. NM—015744 (SEQ ID NO:11). ATX homologues derived from other animals can be identified using HomoloGene (www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, a particular human base sequence is applied to BLAST (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, www.ncbi.nlm.nih.gov/BLAST/), and the accession number of the identical (exhibiting the highest Score, an E-value of 0 and 100% Identity) sequence is acquired. The accession number is input to UniGene (www.ncbi.nlm.nih.gov/UniGene/), and the thus-obtained UniGene Cluster ID (a number shown by Hs.) is input to HomoloGene. From the resulting list of gene homologue correlations between the genes of other biological species and the human gene, genes of other biological species can be selected as genes corresponding to the human gene shown by the particular base sequence (homologues).
Likewise, in the present invention, the human ATX protein is based on the amino acid sequence disclosed in Genbank Accession No. NM—006209 (SEQ ID NO:10). The mouse ATX protein is based on the amino acid sequence disclosed in Genbank Accession No. NM—015744 (SEQ ID NO:12).
In the present invention, an ATX inhibiting substance generically refers to substances that inhibit the expression of ATX and substances that inhibit the enzyme activity of ATX. Substances that inhibit the enzyme activity of ATX include not only substances that interact with ATX to inhibit the enzyme activity thereof, but also substances that inhibit the binding of ATX and LPA and the like. A substance that inhibits the expression of ATX can be selected by measuring the expression level of the mRNA or protein of ATX in ATX-expressing cells, and confirming a significant reduction in the expression level compared with a control not containing the substance. A substance that inhibits the enzyme activity of ATX can be selected by adding the substance to a system containing ATX and a substrate therefor (a labeled substrate, for example, fluorescently labeled FS-3 (Echelon Biosciences Company)), and confirming a significant reduction in the amount of the marker leaving the substrate compared with a control not containing the substance.
ATX inhibiting substances include inhibitory substances that have been shown to possess inhibitory activity to date, or all substances whose inhibitory activity can be confirmed using the model of stress-induced generalized pain described below. Specifically, ones selected from the group consisting of an antisense nucleic acid, siRNA, viral vector-recombined shRNA, microRNA and inhibitory antibody against ATX and a low-molecular compound that inhibits the enzyme activity of ATX can be mentioned as examples. These may be natural substances or artificially synthesized substances.
The aforementioned ATX antisense nucleic acid refers to, for example, a single-stranded nucleic acid complementary to at least a part of base sequence selected from among the base sequence of SEQ ID NO:9 or 11 or the base sequence that encodes the amino acid sequence of SEQ ID NO:10 or 12. The nucleic acid may be a naturally derived or artificial nucleic acid, or may be one based on DNA or RNA. The length of the antisense nucleic acid is normally about 15 bases to the same as the full length of the mRNA, preferably about 15 to about 30 bases long. The complementarity of the antisense nucleic acid does not always need to be 100%, and may be of a degree that allows complementary binding to the DNA or RNA of ATX in a living organism.
The aforementioned siRNA refers to a double-stranded RNA synthesized artificially to cause the degradation of the mRNA transcribed from the ATX gene (RNA interference) on the basis of the base sequence thereof, or a vector capable of supplying the double-stranded RNA in a living organism, in order to suppress the expression of the ATX gene. Using the siRNA or siRNA expression vector of the present invention, it is possible to have an action to suppress generalized pains by reducing the expression of ATX to inhibit the production of LPA by ATX. To construct an siRNA or siRNA expression vector, a publicly known method can be used (Ui-Tei K, et al., Nucleic Acids Res. 2004; 32: 936-948; Miyagishi M, and Taira K, Nature biotechnology 2002; 20: 497-500). The length of the siRNA is preferably 19 to 27 bp, more preferably 21 to 25 bp. In the shRNA and microRNA, a partial gene of the ATX gene is recombined into an appropriate viral vector and administered as a virus systemically or to the brain or the spinal cord, whereby the ATX gene is knocked down. The length of the partial gene is preferably 60 to 70 bp in each case.
The aforementioned inhibitory antibody against ATX refers to an antibody that binds specifically to ATX and inhibits the enzyme activity of ATX by binding thereto. Inhibitory antibodies against ATX include the antibodies described in the literature (Nakamura K, et al., Clin Chim Acta. 2008 February; 388 (1-2):51-8. Epub 2007 Oct. 11.) and the like. “Antibody” in an inhibitory antibody against ATX is as explained for the “antibody” in the aforementioned LPA1 antagonist antibody.
As the aforementioned low-molecular compound that inhibits the enzyme activity of ATX, sphingosine 1-phosphatase (S1P), lysophosphatidic acid (LPA), FTY720 (S1P analogue), cyclic PA (natural type analogue of LPA), BrP-LPA (bromomethylenephosphonate LPA), [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826) and the like can be mentioned.
The disease targeted by a therapeutic or prophylactic agent of the present invention is generalized pain syndrome. Generalized pain syndrome generically refers to diseases accompanied by systemic chronic pain for which the cause has not been clarified and no therapies have been established.
Encompassed by the aforementioned generalized pain syndrome are fibromyalgia, chronic fatigue syndrome, hypersensitivity colitis (or temporomandibular disorders) and the like. The onset of these diseases is reportedly profoundly related to a stress. In particular, fibromyalgia, chronic fatigue syndrome, hypersensitivity colitis and the like, which are presumably involved by the action of LPA in the central nervous system, are preferred as targets of treatment by the present invention. In particular, the majority of patients with fibromyalgia have a history of experiencing an injury (e.g., whiplash injury, surgery) in the past, and the involvement of the action of LPA in the hypothalamus is likely. LPA seems to be contributory to the formation and maintenance of pains in the aforementioned diseases.
Therefore, it is preferable that a therapeutic or prophylactic agent of the present invention be administered to the central nervous system for ensuring the effect thereof to be exhibited in full. A method is also available wherein cerebral transfer is promoted by systemic administration in a collagen formulation and the like.
Although any route of administration is acceptable to deliver a therapeutic or prophylactic agent of the present invention to the central nervous system, parenteral administrations are preferred. Parenteral administrations include intravenous injection (drip infusion), subcutaneous administration, intradural administration, intrameningeal administration, spinal epidural administration, spinal subarachnoidal administration, intra-lateral-cerebroventricular administration, intracisternal administration and the like; because of the possible involvement of the upper parts of the brain, intra-lateral-cerebroventricular administration and intracisternal administration are more preferred.
A therapeutic or prophylactic agent of the present invention may contain a pharmaceutically acceptable carrier, depending on the choice and route of administration of the active ingredient LPA1 antagonist or autotaxin-inhibiting substance. Those skilled in the art are able to choose as appropriate a carrier suitable for the situation. Examples of carriers that can be chosen include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, Aerosil, talc, and sodium lauryl sulfate; preservatives such as sodium benzoate, sodium hydrogen sulfite, methyl paraben, and propyl paraben; pH regulators such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; solvents such as water, physiological saline, ethanol, and propylene glycol; isotonizing agents such as glucose, sodium chloride, and potassium chloride; base waxes such as cacao butter, polyethylene glycol, and refined kerosene; and the like. These carriers are not limited to a single action, and can be used for the purpose of having a plurality of actions.
The ratio of the aforementioned active ingredient contained in a therapeutic or prophylactic agent of the present invention can be set as appropriate, as far as the desired effect is obtained; the ratio is normally 0.01 to 100% by weight, preferably 0.1 to 99.9% by weight, more preferably 0.5 to 99.5% by weight.
Although the dosage of a therapeutic or prophylactic agent of the present invention varies depending on the choice of active ingredient, the recipient's body weight and age, symptoms, and the like, and cannot be generalized, it can be chosen over the range from 0.0001 mg to 1000 mg per kg body weight in a single dose.
Recipients of a promotive or prophylactic drug of the present invention include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, bovines, horses, sheep, monkeys, and humans.
Although the dosing frequency of a therapeutic or prophylactic agent of the present invention is not particularly limited, it is normally about 1 to 5 times a day. Since it is highly likely that LPA is contributory to the formation and maintenance of pains in the aforementioned diseases to be treated, preference is given to long-term use for about several days to 1 month. This dosing frequency allows the achievement of preventing the formation and maintenance of pains. Note that in the event of a recurrence of the aforementioned disease after a considerable time interval, the therapeutic or prophylactic agent of the present invention can be administered again.
For the LPA1 antagonist or autotaxin-inhibiting substance that is an active ingredient of a therapeutic or prophylactic agent of the present invention, the effect thereof can be confirmed using an animal model of generalized pain disease created by the method described below. In the animal model described herein, pain threshold value reductions are observed for at least 2 to 3 weeks after completion of repeated stress loading. Pain threshold values are expressed by PWL values (paw withdrawal latency (seconds)) as determined by a mechanical stimulation induction test method (paw pressure test). If the PWL value decreases significantly compared with non-stress-loaded animals as controls, it is judged that the chronic pain persists; if the PWL level rises to the same level as the controls, it is judged that the chronic pain has healed.
Any non-human animal can be used. Suitable animals include laboratory animals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, and monkeys; and farm animals such as bovines, sheep, horses, and pigs; preference given to mice because a wide variety of disease models have been established to date, and also because they permit easy utilization for gene engineering.
Model of Generalized Pain Disease with SART (ICS) Stress
The SART stress model, in which normal temperature and a cold environment are repeatedly loaded, is commonly known as a model of autonomic imbalances of the parasympathetic nerve dominant type; a wide variety of abnormalities, such as persistent hypotension, immune balance abnormalities, and hyperalgesia, have been reported. Meanwhile, the SART stress model is said to be also useful as an animal model of generalized pain syndrome, including fibromyalgia. In the method of creating a model by SART stress, the rearing environment for animals such as mice is repeatedly altered between room temperature (around 24° C.) and cold temperature (around 4° C.); this is desirably referred to as intermittent cold stress (ICS), so as to distinguish it from constant cold stress (CCS stress), which represents conditions only involving cold temperature stress without returning to room temperature as control stress that does not induce chronic pains. As described in Examples, ICS stress is more strictly defined than traditionally reported SART stress. Likewise, how to create a model based on CCS stress is also described in Examples.
EXAMPLES
The present invention is hereinafter described in further detail by means of the following examples, by which, however, the present invention is not limited anyway.
Production Example 1
SART (ICS) Stress Loading Group
Exposure to SART (ICS) stress was achieved by repeatedly altering extremely different rearing environmental temperatures. Temperature settings were established at room temperature (24° C.) and cold temperature (4° C.). Rearing cages were placed in environments set at the respective temperatures; the animals were reared, with transfer of the mice only every 30 minutes in the daytime (from 10:00 to 16:30), and at the cold temperature in the nighttime (from 16:30 to 10:00 on the following day). This repeated stress was given for 3 days from 16:30 on the day before stress loading to 10:00 on day 3 of stress loading. A solid food and agar for water supply were always kept in the cage during the above-described stress loading period, to maintain an environment allowing the animals to have free access thereto.
The mice used for a normal control group were mice reared in the same way as the stress loading group, except that the animals were reared constantly at 24° C. during the above-described stress loading period.
Example 1
A hypersensitive response induction experiment with ICS stress was applied to mice previously dosed with an antisense oligodeoxynucleotide that complementarily binds to the lysophosphatidic acid (hereinafter, denoted as LPA) receptor gene to inhibit the expression thereof (hereinafter, denoted as AS-ODN) as an animal model of generalized pain disease, and the stress exposure of Production Example 1 was performed.
LPA1 AS-ODN-treated Mice
Ten to twelve C57BL/6J mice at 6 weeks of age (20 to 22 g) were used in a normal control group and an ICS stress loading group.
Using “5′-AGCTGCCATGACAGTGCTGT-3′ (SEQ ID NO:5)” as AS-ODN, BLAST search was performed; it was confirmed that the sequence thereof was specific for the LPA1 receptor. For comparative control, mice dosed with a missense oligodeoxynucleotide having a noncomplementary sequence (hereinafter, denoted as MS-ODN) and mice dosed with artificial cerebro-spinal fluid (ACSF) as Vehicle were used. Using “5′-AGCAGCGTTGTCACTGCAGT-3′ (SEQ ID NO:6)” as MS-ODN, BLAST search was performed; it was confirmed that the sequence thereof was not similar to other receptors.
SART (ICS) stress based on Production Example 1 above was loaded on the mice after being given ODN and ACSF, after which pain threshold values were measured, and changes over days were examined.
Example 2
LPA2 AS-ODN-treated Mice
Ten to twelve C57BL/6J mice at 6 weeks of age (20 to 22 g) were used in a normal control group and an ICS stress loading group.
Using “5′-TACTACAACGAGACCATCGG-3′ (SEQ ID NO:7)” as AS-ODN, BLAST search was performed; it was confirmed that the sequence thereof was specific for the LPA1 receptor. For comparative controls, mice dosed with MS-ODN having a noncomplementary sequence and mice dosed with artificial cerebro-spinal fluid (ACSF) as Vehicle were used. Using “5′-CGATACACTAGAACGCTCGA-3′ (SEQ ID NO:8)” as MS-ODN, BLAST search was performed; it was confirmed that the sequence thereof was not similar to other receptors.
SART (ICS) stress based on Production Example 1 above was loaded on the mice after being dosed with ODN and ACSF, after which pain threshold values were measured, and changes over days were examined.
Example 3
In Example 3, chronic pains were evaluated on mice loaded with stress based on Production Example and Examples. Regarding how to evaluate chronic pains, pain threshold values were evaluated over days by a mechanical stimulus-induced pain test. The mechanical stimulus-induced pain test was performed according to the digitized von Frey method.
<Method 1>
Method of Treatment with AS-ODN
AS-ODN was given by intracerebroventricular administration at 10 μg once daily, starting five days before SART (ICS) stress loading. After pretreatment for 5 consecutive days, SART (ICS) stress was started, and intracerebroventricular administration was performed once a day even during the stress loading period. However, there was no administration on day 3 of stress loading, after which administration was discontinued; a total of 7 administrations were performed. The single-dose volume was 5 μl.
In the same way as the above-described method of treatment, MS-ODN and Vehicle were administered 7 times in total before stress loading and during the stress loading period.
<Method 2>
Mechanical Stimulus-Induced Pain Test
On mice acclimated to the environment on a 5 mm×5 mm-mesh net (adaptation-applied), a custom-made plastic chip was applied to a planta of each mouse under a constant pressure from below. At that time, responding weight threshold values were measured digitally.
<Results 1>
FIGS. 1 to 3 show results of measurements of pain threshold values over days in the mice given the LPA1 receptor AS-ODN in Example 1; FIG. 1 shows the observed results for the Vehicle treatment group, FIG. 2 for the AS-ODN treatment group, and FIG. 3 for the MS-ODN treatment group.
In FIGS. 1 to 3, the vertical axis (PWT) indicates measured weight threshold value (g). The transverse axis indicates the number of days that elapsed before the start of ICS stress loading (Pre), during the stress loading period (Stress), and after stress loading (Post stress). In FIGS. 1 to 3, “stress (●)” indicates the stress loading group and “control (◯)” indicates the normal control group.
As shown in FIG. 1, when SART (ICS) stress was given to mice previously treated by Vehicle administration, the mice that exhibited a value of 9.44±0.2 g on the day before stress loading responded at 6.14±0.2 g on day 1 of stress loading. A pain threshold value reduction due to the stress was confirmed, at which time a significant difference was noted compared with the pain threshold value of 10.06±0.3 g for the normal control group. This hypersensitive response persisted for at least 19 days or more, and was chronic.
As shown in FIG. 2, the SART (ICS) stress loading group treated with LPA1 AS-ODN in Example 1 exhibited a value of 9.77±0.4 g on the day before stress loading, with no difference observed from the value for the Vehicle group obtained on the day before stress loading. On day 1 of stress loading, the threshold value for the AS-ODN treatment group was 8.76±0.5 g; the hypersensitive response observed with Vehicle was not observed. Statistical processing revealed no significant difference from the AS-ODN-treated normal control group; rather, the results showed suppression of the threshold value reduction in the Vehicle-treated stress group. Even measurements were made over days, no hypersensitive responses were noted.
As shown in FIG. 3, when SART (ICS) stress was given to mice previously treated by MS-ODN administration, the mice that exhibited a value of 9.59±0.3 g on the day before stress loading responded at 5.68±0.7 g on day 1 of stress loading. A reduction in the pain threshold by the stress was confirmed, at which time a significant difference was noted compared with the pain threshold value of 9.49±0.2 g for the normal control group. This hypersensitive response persisted for at least 19 days or more, and was chronic.
According to the results of Example 1 and Example 3, it was found that when SART (ICS) stress was given to mice previously dosed with LPA1 AS-ODN to suppress the expression, chronic pains, which were observed in both the Vehicle treatment group and the LPA1 MS-ODN treatment group, were suppressed. However, the threshold values for the normal control group dosed with AS-ODN were comparable to those for the Vehicle treatment group and the LPA1 MS-ODN treatment group.
<Results 2>
FIGS. 4 and 5 show results of measurements of pain threshold values over days in the mice dosed with the LPA2 receptor AS-ODN in Example 2; FIG. 4 shows the observed results for the AS-ODN treatment group, and FIG. 5 for the MS-ODN treatment group.
In FIGS. 4 and 5, the vertical axis (PWT) indicates measured weight threshold value (g). The transverse axis indicates the number of days that elapsed before the start of ICS stress loading (Pre), during the stress loading period (Stress), and after stress loading (Post stress). In FIGS. 4 and 5, “stress (●)” indicates the stress loading group, and “control (◯)” indicates the normal control group.
As shown in FIG. 4, the SART (ICS) stress loading group treated with LPA2 AS-ODN in Example 2 exhibited a value of 9.77±0.5 g on the day before stress loading; no difference was observed from the value for the Vehicle group on the day before stress loading, shown in FIG. 1. On day 1 of stress loading, the threshold value for the AS-ODN treatment group was 6.43±0.1 g, showing a reduction in threshold value, i.e., a hypersensitive response. Statistical processing revealed a significant difference compared with the AS-ODN-treated normal control group. This hypersensitive response persisted for at least 19 days.
As shown in FIG. 5, when SART (ICS) stress was given to mice previously treated by MS-ODN administration, the mice that exhibited a value of 9.58±0.3 g on the day before stress loading responded at 5.79±0.1 g on day 1 of stress loading. A reduction in the pain threshold value by the stress was confirmed, at which time a significant difference was noted compared with the pain threshold value of 9.65±0.3 g for the normal control group. This hypersensitive response persisted for at least 19 days, and was chronic.
According to the results of Example 2 and Example 3, it was found that when SART (ICS) stress was given to mice previously dosed with LPA2 AS-ODN to suppress the expression of LPA2, no suppression of SART (ICS) stress-induced pains was noted as in the LPA1 AS-ODN treatment group, which exhibited a hypersensitive response comparable to that for the Vehicle group.
Judging from the results of Example 1 and Example 2, it can be thought that the pains induced by repeated cold temperature stress are more largely involved by the LPA1 receptor than by the LPA2 receptor. For this reason, suppression of stress-induced pains with LPA1 receptor-specific antibodies, antisense nucleic acids, inhibitors and the like is likely. The stress model is believed to be a possible animal experimental model of fibromyalgia, and considering the results of the present invention, it is expected to find an application for the treatment of fibromyalgia.
(Experimental Animals, Experimental Environment and Rearing Environment)
In the Examples below, male C57BL/6J mice at 6 weeks of age (20 to 25 g) were used. These mice were reared in a room set at a room temperature of 22±2° C. and a humidity of 55±5% under natural conditions in both the daytime and nighttime. The mice were allowed to take water (tap water) and a solid food (MF, Oriental Yeast, Tokyo) ad libitum.
Regarding the experimental environment, the experiments were performed using a room in a laboratory under constant temperature (22±2° C.) and constant humidity (55±5%) conditions. The mice to be used in the experiments were transferred to the room by 24 hours before the start of the experiments, reared under natural management in both the daytime and nighttime, and allowed to take a food and tap water ad libitum as described above. The experiments were performed between 10 a.m. and 17 p.m.
All the experiments were performed in compliance with the Guidelines for Animal Experimentation, Nagasaki University, and the method specified by the International Committee on Pain Experiments (Animal Experiment Permission Number: 0706130596).
Example 4
Drug Administration
1) ATX-antisense
ATX-antisense is a drug that suppresses the synthesis of the mRNA of ATX, an LPA synthetase. The ATX-antisense (ATX-AS) used was designed to have the base sequence: “5′-GTC TTG CCA TGC CGA GGG AT-3′ (SEQ ID NO:13)”, and the ATX-missense (ATX-MS) used was designed to have the base sequence: “5′-GTT CTC GCA GTC GCA GGA GT-3′ (SEQ ID NO:14)”. The dosage was 10 μg/5 μl both for the antisense and for the missense.
ATX-antisense (operon, 3170003) was given by intracerebroventricular administration on consecutive days starting five days previously, including days of stress loading (FIG. 8A). Doses were given in solution in artificial cerebro-spinal fluid. For control groups, the same amounts of ATX-missense (operon, 3170004) and the solvent were administered, respectively. Using a 50 μl Hamilton microsyringe and a needle-equipped cannula, a total volume of 5 μl was given by intracerebroventricular administration. A 26-gauge needle for intracerebroventricular administration was used.
2) PLA2 Inhibitor
As a PLA2 inhibitor, Bromoenol lactone (Cayman chemical) was used. Drug administration was performed twice in total at 10 a.m. before performing repeated cold stimulation in stress loading (FIG. 9A).
The whole amount of 5 mg of the drug was dissolved in 200 μl of DMSO and dispensed at 20 nmol/10 μl. 384 μl of artificial cerebro-spinal fluid was added thereto. For a control group, the same amounts of DMSO and artificial cerebro-spinal fluid were mixed and administered. Using a 50 μl Hamilton microsyringe and a needle-equipped cannula, a total volume of 5 μl was given by intracerebroventricular administration. A 26-gauge needle for intracerebroventricular administration was used.
Example 5
Creation of SART (ICS) Model and CCS Model
To create a mouse model of fibromyalgia, ICS (Intermittent Cold Stress) was loaded. Regarding mouse rearing environmental temperatures, the animals were reared with repeated alterations of room temperature (24±2° C.) and low temperature (4±2° C.) every 30 minutes in the daytime, and at low temperature in the nighttime. To avoid excess moisture in the rearing environment, a cage was placed upsidedown on a cage mesh, and a gap was made between the cage and the mesh using fractions of a solid food for ordinary experimentation (MF, Oriental Yeast, Tokyo). The animals were allowed to take a solid food and water in the form of tap water solidified with agar and cut into about 1 cm cubes ad libitum, and reared at constant humidity (55±5%) under natural conditions in both the daytime and nighttime. Two or one mouse was reared in each cage.
On the starting day, at 16:30, the mice were transferred to a refrigerator under low temperature conditions (4° C.); they were reared until 10:00 on the following day. At 10:00, the animals were returned to room temperature, after which they were reared until 16:30 with repeated alterations of low temperature conditions and room temperature conditions every 30 minutes. This day was established as the starting day of repeated stress, taken as day 1 of stress loading. From 16:30 to 10:00 on day 2 of stress loading, the animals were reared at 4° C. as on the day before stress loading. On day 2, repeated stress was given in the same way as on day 1. At 10:00 on day 3, the animals were returned to room temperature to finish. The day of completion was taken as P1 (Post Stress day 1).
A control group (Control) was reared constantly at room temperature during the same period (from 16:30 on the day before stress loading to 10:00 on day 3) for 3 days.
CCS model mice were reared constantly at low temperature during the same period (from 16:30 on the day before stress loading to 10:00 on day 3) for 3 days (Molecular Pain 6, 4:52 November 2008).
Example 6
Blood Collection in Measuring Corticosterone Concentrations
Serum corticosterone was collected at P1, P5, P12 and P19. Each mouse was decapitated, and all the blood was gathered. After the collection, the blood was applied to a centrifuge at 3000 rpm for 30 minutes to separate the plasma. The supernatant was collected, frozen in dry ice, and stored at −80° C. until analysis. Blood sampling was performed between 21:00 and 22:00, when the concentration was highest, in synchronization with the rhythm of diurnal change in corticosterone in the blood.
Example 7
How to Evaluate Pain-Related Behavior
The test methods 1) to 2) below were performed to obtain measurements at an interval of about 10 minutes. This was to prevent tissue damage due to the continuous measurement.
1) Automated Digitized Von Frey Test: Mechanical Nociception Test Method
Prior to the experiment, each mouse was placed on a rack on a mesh and covered with a cage from above, and the animal was acclimated to the same environment as the experiment for not less than 1 hour. The tip of a plastic chip was pressed vertically against the center of a hind paw of the mouse from below the rack; when the mouse behaved to withdraw the hind leg, the automatically measured value was read. Intensity of stimulation was set at a level such that the weight would be around 10 g for a normal mouse. Three measurements or more were made, and the mean value was adopted.
2) Hargreaves Test (Thermal Paw-Withdrawal Test): Thermal Nociception Test Method
Prior to the experiment, each mouse was placed on a glass plate and covered with a cage from above, and the animal was acclimated to the same environment as the experiment for not less than 1 hour. A thermal stimulus was given to a hind paw of the mouse; when the mouse behaved to escape the stimulus, the automatically measured value was read. The stimulating beam was set at a level such that the measurement would be around 10 seconds for a normal mouse. To avoid tissue damage, a cutoff time was set at 20 seconds. Three measurements or more were made, and the mean value was adopted.
3) Evaluation of Hyperalgesia Over Days
With the day of completion of ICS loading as P1, pain threshold values were measured on P3, P5, P12 and P19.
Example 8
Statistical Processing
All data were statistically analyzed using Sheffe's F test. If a test by one-way layout analysis of variance revealed a difference between levels, a multiple comparison test was performed to determine the groups between which a difference was present. If one * or # mark is obtained, the significance level was determined to be 5% or less; if two marks are obtained, the significance level was determined to be less 1% or less. All results are expressed as (mean)±S.E.M.
<Results 3>
1) Hypersensitive Responses Over Days to Mechanical Stimulation in ICS
After completion of stress loading, for both the ICS group and the control group, observations were continued over days on the schedule of P1, P3, P5, P12, and P19. Pains were evaluated on a right leg of each mouse using the mechanical nociception test method (FIG. 6). Before performing stress loading, no difference was seen between the threshold values for the control group and the ICS group. After completion of stress loading, there was a significant difference between the ICS group and the control group (P<0.01). At P1, the threshold value was 9.5±0.2 g for the control group and 4.8±0.5 g for the ICS group; until P19, persistent reductions in threshold value were confirmed (control group: 9.1±0.3 g, ICS group: 5.9±0.4 g). This hypersensitive response was of the same degree as the threshold values exhibited by a neuropathic pain model undergoing partial ligation of the sciatic nerve at our laboratory.
The following two observations were obtained during the creation of the models. First, the ICS group seemed to have greater food consumption than the control group, and this was particularly evident just after a change in the temperature environment. This may represent an adaptive phenomenon to prevent body temperature falls by increasing food consumption to have a pyrogenic effect. Second, body weight tended to decrease in the ICS group during the stress loading period. However, the body weight exhibited a recovering tendency after completion of stress loading, the recovery being to the same extent as the control group. Regarding the behavior of other animals, there was no difference from the control group.
2) Corticosterone Concentrations in ICS/CCS
As an index of stress loading, blood corticosterone concentrations were measured. Concentration measuring samples were collected on days 1, 5, 12, and 19 after completion of stress loading. On day 1 after completion of repeated stress loading, the corticosterone concentration for the ICS group was 32.7±4.1 μg/dl, showing a significant elevation compared with the concentration for the control (CCS) group (10.0±2.4 μg/dl) (FIG. 7). However, regarding the changes over days, the concentration elevation for the ICS group was no longer observed on day 5 after completion of stress loading. Thereafter, both the ICS group and the control group exhibited a value of about 10 μg/dl. Judging from this result, it can be thought that the animals were stressed by the repeated cold stress, whereas the animals were not chronically stressed by the continuous cold stress to the extent that caused an elevation of corticosterone concentration.
A difference was found when creating the mouse models of ICS and CCS. Both food consumption and the amount of behavior were larger in ICS. In particular, regarding food consumption, ICS often exhibited the behavior of eating food just after the temperature change was given every 30 minutes. Regarding the amount of behavior, the CCS animals tended to stay at one place, but no abnormal behavior was seen.
3) Responses to Nociceptive Stimulus by Intracerebroventricular Administration of ATX-AS in Male C57BL/6J Mice
Recently, Nishiyori and Ueda reported that hyperalgesia and hyperpathia such as allodynia in an ICS stress model were suppressed over a long period of 4 days by intracerebroventricular administration of a small amount of gabapentin (Molecular Pain 6, 4:52 November 2008). Gabapentin is a drug with Caα2δ-1 as a molecular target. Caα2δ-1 was shown to be expressed in myelinated fiber A in nerve injuries that induce neuropathic pains, and in applying the causal molecule LPA to the spinal subarachnoidal cavity, and to be one of responsible molecules for neuropathic pains (Yasashii Itami Gaku, Hiroshi Ueda, Kazuo Toda, Brain Shuppan, pp. 89-90; Nature Medicine Volume. 10, Number. 7, July 2004, pp. 712-718; Mol Pain. 2008 Apr. 1; 4:11. Review). Hence, the possible production of LPA in the brain was assumed in the ICS stress model, like in a nociceptive neuropathic pain model. In this experiment, the LPA synthetase inhibitor ATX-AS was given by intracerebroventricular administration for consecutive days starting 5 days previously, including the starting day of stress loading, and the effect on the development of hyperpathia was evaluated (FIG. 8A). A mechanical nociception test method was performed on days 1, 3, 5, and 12 after completion of stress loading, and an electrical nociception test method on days 5 and 6. In the two tests, a significant difference (P<0.05) was noted between the artificial cerebro-spinal fluid (ACSF)+stress group and the ACSF group.
3-1) Changes Over Days in Mechanical Noceceptive Responses
In the ACSF control group and the ATX-MS dosing group, significant reductions of mechanical nociceptive response threshold values were observed from P1 to P12 after ICS stress loading. By contrast, in the ATX-AS dosing group, almost no threshold value changes were noted with ICS stress. Comparing the ATX-MS dosing group and the ATX-AS dosing group, a significant difference was observed from P1 to P12.
4) Responses to Nociceptive Stimulation by Intracerebroventricular Administration of Bromoenol Lactone (BEL) in Male C57BL/6J Mice
Bromoenol lactone (BEL) is a PLA2 inhibitor. The pathway of lysophosphatidylcholine (LPC) production from phosphatidylcholine by phospholipase A2 (PLA2) and subsequent production of LPA by ATX is reportedly the most important to the process of LPA production. While hypersensitivity persists in ICS model mice, transient hypersensitivity is seen in the CCS model mice. This leads to the notion that repeated normal-and-low-temperature stimulation is associated with the development of hypersensitivity in the ICS model mice. For this reason, dosing took place at 10 am, just before giving repeated cold stimulation (FIG. 9A). On days 1, 3, and 5 after completion of stress loading, pain threshold values were examined by the mechanical nociception test method (FIG. 9B) and the thermal nociception test method (FIG. 10).
4-1) Changes Over Days in Mechanical Nociceptive Responses
Referring to FIG. 9B, reductions of mechanical nociceptive response threshold values due to ICS stress tended to be suppressed by BEL administration between P1 and P5, with a significant change at P3.
4-2) Changes Over Days in Thermal Nociceptive Responses
Referring to FIG. 10, thermal hyperalgesia due to ICS stress tended to be suppressed by BEL administration between P1 and P5, with a significant change at P3.
Industrial Applicability
According to a therapeutic or prophylactic agent for generalized pain disease of the present invention, it is possible to make a major contribution to the establishment of an effective and reliable therapeutic policy and prophylactic method for generalized pain disease, for which no therapies have been established to date because the cause remains unclear.
This application is based on patent application Nos. 2008-204762 (filing date: Aug. 7, 2008) and 2009-112990 (filing date: May 7, 2009) filed in Japan, the contents of which are incorporated in full herein.

## Claims

1. A method of treating chronic pain comprising administering an effective amount of a low-molecular weight compound that inhibits the enzyme activity of autotaxin by interacting with autotaxin to a subject in need thereof, thereby treating chronic pain in the subject.

2. The method of claim 1, wherein the chronic pain is fibromyalgia, chronic fatigue syndrome, or hypersensitivity colitis.

3. The method of claim 2, wherein the chronic pain is fibromyalgia.

4. The method of claim 2, wherein the chronic pain is chronic fatigue syndrome.

5. The method of claim 2, wherein the chronic pain is hypersensitivity colitis.