JPWO2009145067A1 - Pain therapeutic agent and use thereof - Google Patents

Abstract

Provided are a therapeutic agent and a therapeutic method for pain using a polynucleotide comprising a specific base sequence or a double-stranded polynucleotide comprising the polynucleotide as a functional polynucleotide. Thereby, the technique for suppressing pain is implement | achieved.

Description

  The present invention relates to a technique for treating pain, and more particularly, to a polynucleotide that can be used for treating pain and use thereof.

  Pain is classified into acute pain and chronic pain according to duration. Acute pain is pain associated with tissue damage and its duration is limited. Chronic pain continues after healing of the tissue disorder and often has no obvious organic cause. Acute exacerbation pain in chronic pain is similar to acute pain and is thought to be deeply related to endogenous pain substances. Chronic pain includes unbearable chronic pain such as traffic accidents or aftereffects after surgery, end-stage cancer or diabetes. Pain is classified into nociceptive pain, neuropathic pain, and psychogenic pain depending on the cause. These independent pains gradually merge over time, resulting in chronic intractable pain that is difficult to classify.

  Neuropathic pain is defined in the 1994 IASP (International Association for the Study of Pain) chronic pain classification as intractable pain due to peripheral or central nerve disorders or dysfunction . Neuropathic pain is a complex syndrome that results from compression, degeneration, and damage to the peripheral and central nervous systems, but cannot be explained by a single pathological process. It has not been elucidated yet. Therefore, the effectiveness of the guideline based on the therapeutic effect regarding neuropathic pain is not guaranteed except for a part.

  Neuropathic pain is pain that newly occurs after a wound has healed, unlike initial pain (acute pain) caused by a wound or the like. As neuropathic pain, there is known a symptom (called “allodynia”) that induces severe pain even by a stimulus that does not usually cause pain. Nonsteroidal anti-inflammatory analgesics and narcotic analgesics that are effective against acute pain have no effect on neuropathic pain, and a definitive treatment for neuropathic pain is not yet known. If neuropathic pain can be suppressed, it is possible to reduce the patient's physical pain and improve QOL in clinical terms.

  Pain applied to the periphery is transmitted to the dorsal horn of the spinal cord through sensory nerves (primary neurons) that exist in the dorsal root ganglion (DRG), and to secondary neurons in the dorsal horn I and II layers of the spinal cord. Transmitted through the synapse, secondary neurons transmit their signals to the central (brain). Perceptual signals from the periphery are mostly transmitted to the dorsal horn via the fourth and fifth DRGs, so we have a chronic pain model in which the DRG peripheral side of the fifth lumbar spinal nerve of the rat is ligated, and Then, an acute pain model was prepared by injecting the dead tuberculosis bacterium (Complete Freund's Adjuvant: CFA) causing allergic reaction into the plantar of the rat, and events in DRG were analyzed in detail. As a result, the expression level of brain-derived neurotrophic factor (BDNF) mRNA that plays a role in complementing and enhancing synaptic transmission is increased by CFA stimulation, in particular, exon 1 variant. It was found that the number was remarkably increased (see Non-Patent Document 1).

Kobayashi et al., Brain Res. 1206, 13-19 (2008) Aid et al., J. Neurosci. Res. 85, 525-535 (2007) Yang et al., Cancer Res. 65, 219-225 (2005)

  However, although it has been shown that, as a result of stimulation, the expression level of exon 1 variant of BDNF is increased, the mechanism of pain generation and the technique for suppressing pain are still unclear.

  The present invention has been made in view of the above problems, and an object thereof is to provide a technique for suppressing pain.

  Since BDNF may have a function of enhancing the transmission of pain in the spinal cord, it may be possible to suppress pain by suppressing this expression. That is, if the BDNF synthesized in the primary neurons can be suppressed, the pain applied at the periphery may be reduced. However, if BDNF is suppressed systemically, memory and learning ability in the center are lowered, which impairs all nerve activities and eventually causes neurodegenerative diseases. Therefore, there is a need for a technique that suppresses BDNF synthesis in primary neurons without causing systemic BDNF suppression. However, a technique that suppresses the expression of BDNF in the periphery has not been known so far.

  The present inventors paid attention to the gene structure of BDNF. The detailed gene structure of BDNF was clarified by a group of Timmusk et al. Of Tallinn University in 2007, and consists of 9 exons across 8 introns (see Non-Patent Document 2). The translation region encoding the protein is mainly exon 9, and is thought to express different variants under different transcriptional controls. Since variants are expressed specifically in tissues and cells, if there is a variant specific to nerves that convey pain, we thought that by suppressing only that variant, we could reduce pain while reducing side effects. .

  Further, the present inventors have used a chronic pain model in which the peripheral side of the rat spinal cord 5th lumbar nerve DRG is ligated, and it is exon that expression increases in response to ligation stimulation strongly in the peripheral nerve tissue DRG. It was found to be one variant (see Non-Patent Document 1).

  Based on their own achievements obtained so far, the present inventors have made intensive efforts and as a result, completed the present invention. That is, the polynucleotide according to the present invention is a polynucleotide comprising any one of the following base sequences (a) to (g): (a) the base sequence represented by SEQ ID NO: 18 (B) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 18 and includes at least one of the base sequences shown in SEQ ID NOs: 13, 15, and 16; (c) shown in SEQ ID NO: 19; (D) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 19 and includes at least one of the base sequences shown in SEQ ID NOs: 21 to 23; (e) SEQ ID NO: 20 (F) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 20 and contains at least one of the base sequences shown in SEQ ID NOs: 24-26; Nucleotide sequence shown in (g) any one of SEQ ID NO 13,15,16,21～26 each time.

  The polynucleotide according to the present invention is also characterized in that it is one of the following (I) to (VI): (I) 1 or several bases are present in the base sequence represented by SEQ ID NO: 18 A polynucleotide comprising a nucleotide sequence deleted or substituted or added, or a partial sequence thereof, and comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 13, 15, and 16; (II) shown in SEQ ID NO: 18 A polynucleotide capable of hybridizing under stringent conditions to a polynucleotide comprising a complementary sequence of the nucleotide sequence comprising at least one of the nucleotide sequences shown in SEQ ID NOs: 13, 15 and 16 ; (III) 1 or several bases are deleted, substituted or added to the base sequence shown in SEQ ID NO: 19 A polynucleotide comprising a base sequence or a partial sequence thereof and comprising at least one of the base sequences represented by SEQ ID NOs: 21 to 23; (IV) a polynucleotide comprising a complementary sequence of the base sequence represented by SEQ ID NO: 19 A polynucleotide that can hybridize under stringent conditions, comprising at least one of the nucleotide sequences represented by SEQ ID NOs: 21 to 23; (V) a nucleotide sequence represented by SEQ ID NO: 20 A polynucleotide comprising a base sequence in which one or several bases are deleted, substituted or added, or a partial sequence thereof, and comprising at least one of the base sequences shown in SEQ ID NOs: 24-26; and (VI) Stringent to a polynucleotide comprising a complementary sequence of the base sequence shown in SEQ ID NO: 20 A polynucleotide capable of hybridizing under conditions include at least one of the nucleotide sequences shown in SEQ ID NO: 24-26, the polynucleotide.

  The double-stranded polynucleotide according to the present invention is characterized by comprising the above-mentioned polynucleotide and a polynucleotide paired with the polynucleotide.

  The therapeutic agent according to the present invention is characterized by containing the above-mentioned polynucleotide or double-stranded polynucleotide in order to treat pain. In the present invention, pain treatment may be performed prophylactically before onset or therapeutically after onset.

  The therapeutic kit according to the present invention is characterized by comprising the above-mentioned polynucleotide or double-stranded polynucleotide in order to treat pain. In the present invention, pain treatment may be performed prophylactically before onset or therapeutically after onset.

  The treatment method according to the present invention is characterized by including the step of administering the polynucleotide or the double-stranded polynucleotide to a subject in order to treat pain. In the present invention, pain treatment may be performed prophylactically before onset or therapeutically after onset.

  If this invention is used, the expression of the exon 1 variant of BDNF can be suppressed. Furthermore, if this invention is used, pain can be suppressed.

  Other objects, features, and advantages of the present invention will be fully understood from the following description. The advantages of the present invention will become apparent from the following description with reference to the accompanying drawings.

It is a figure which shows the outline of the structure of the BDNF gene, and the constructed reporter plasmid. It is a figure which shows the promoter activity investigated using the constructed reporter plasmid. It is a figure which shows the arrangement | sequence of the synthesized double stranded polynucleotide. It is a figure which shows the promoter activity investigated using the synthetic | combination double stranded polynucleotide. It is a figure which shows the outline of the treatment regimen using the polynucleotide based on this invention. It is a figure which shows the expression inhibitory effect of the exon 1 variant of BDNF by the polynucleotide based on this invention. It is a figure which shows the outline of the treatment regimen using the polynucleotide based on this invention. It is a figure which shows the pain suppression effect by the polynucleotide based on this invention. It is a figure which shows the chronic pain inhibitory effect by the polynucleotide based on this invention. It is a figure which shows the chronic pain inhibitory effect by the polynucleotide based on this invention. It is a figure which shows the chronic pain inhibitory effect by the polynucleotide based on this invention. It is a figure which shows the arrangement | sequence of the double stranded polynucleotide of the BDNF promoter area | region which concerns on this invention. It is a figure which shows the promoter activity investigated using the double stranded polynucleotide which concerns on this invention.

[1] Functional polynucleotide As described later in Examples, the present inventors obtained three types of promoter regions of exon 1 of BDNF, and then called S3-AS2 (SEQ ID NOs: 1 and 4). Was found to have promoter activity throughout, particularly that the S3-S4 region (SEQ ID NOs: 18, 20) had strong promoter activity. Furthermore, among the S3-S4 regions, the C region, the E region, and the F region (SEQ ID NOs: 13, 15, 16, 24 to 26) can suppress the expression of exon 1 variant of BDNF and suppress pain. DNA could be provided.

  The present invention provides functional polynucleotides for treating pain. The functional polynucleotide according to the present invention can suppress the expression of exon 1 variant of BDNF. Moreover, the pain suppression effect was demonstrated by the DNA produced using the functional polynucleotide according to the present invention.

  As used herein, “functional polynucleotide” is intended to be “polynucleotide specific for exon 1 variant of BDNF”. The functional polynucleotide according to the present invention is preferably a double-stranded polynucleotide comprising a polynucleotide (first polynucleotide) described later and a second polynucleotide paired with the polynucleotide, There may be only the first polynucleotide or the second polynucleotide. Moreover, the 1st polynucleotide and the 2nd polynucleotide may be included in the aspect which is hard to be decomposed | disassembled in the living body. Such an embodiment is known in the art. Furthermore, the first polynucleotide and / or the second polynucleotide may be provided as a vector inserted therein.

  As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and is deoxyribonucleotide (abbreviated as A, G, C and T) or. Shown as the sequence of ribonucleotides (abbreviated as A, G, C and U).

  The short polynucleotides are called dinucleotides (dimers) and trinucleotides (trimers), and the long ones are represented by the number of nucleotides polymerized such as 30-mers or 100-mers. The polynucleotide according to the present invention may be produced as a fragment of a longer polynucleotide or may be chemically synthesized.

  The functional polynucleotide according to the present invention may exist in the form of RNA (eg, mRNA, siRNA, RNAi, microRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA or RNA can be single-stranded or double-stranded. The DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

  The functional polynucleotide according to the present invention can be easily prepared according to any technique known in the art, and may be chemically synthesized or recombinantly expressed, for example. Moreover, such functional polynucleotide may be chemically modified to suppress degradation in vivo or to promote stability in vivo.

  The functional polynucleotide according to the present invention is preferably 20 to 300 bases long, more preferably 25 to 200 bases long, and further preferably 30 to 100 bases long. When the functional polynucleotide according to the present invention is double-stranded, the pair of polynucleotides forming the double-strand does not have to be perfectly matched, and an unpaired portion such as a mismatch may exist. . In addition, the double-stranded polynucleotide may or may not protrude at the end portion, and when protruding, the number of bases is not particularly limited as long as it does not inhibit the function as a functional polynucleotide. In addition, the functional polynucleotide according to the present invention can be single-stranded or double-stranded DNA or RNA, and if it is a polynucleotide that can specifically inhibit the expression of exon 1 variant of BDNF, It may be a double-stranded RNA strand or a mixed polynucleotide of DNA and RNA.

  In one aspect, the first polynucleotide is (i) a polynucleotide comprising the base sequence represented by SEQ ID NO: 18. In another aspect, the first polynucleotide is (ii) a polynucleotide consisting of a base sequence represented by at least one of SEQ ID NOs: 13, 15, and 16.

  In addition, since the polynucleotide comprising the C region, the E region and the F region maintains the effect of the polynucleotide comprising the S3-S4 region, it is a fragment (partial fragment) of the polynucleotide comprising the S3-S4 region. However, those skilled in the art who have read this specification will easily understand that those including the base sequence of at least one of the C region, E region and F region can also be included within the scope of the present invention. That is, the first polynucleotide consists of (iii) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 18 and includes at least one of the base sequences shown in SEQ ID NOs: 13, 15, and 16 It can also be a polynucleotide.

  Furthermore, in the polynucleotide comprising the S3-S4 region, the base sequence of other regions may be slightly different as long as at least one base sequence of the C region, E region and F region is retained. Are easily understood by those skilled in the art who have read this specification. That is, the first polynucleotide comprises (iv) a base sequence in which one or several bases have been deleted, substituted or added to the base sequence shown in SEQ ID NO: 18, or a partial sequence thereof. Hybridizes under stringent conditions to a polynucleotide comprising at least one of the base sequences shown in 13, 15 and 16, or (v) a polynucleotide comprising a complementary sequence of the base sequence shown in SEQ ID NO: 18 And a polynucleotide comprising at least one of the base sequences shown in SEQ ID NOs: 13, 15 and 16.

  The promoter region of exon 1 of BDNF is highly conserved among rats, humans and mice. The effects similar to those of rats are shown in the above-described region called S3-AS2, S3-S4 region, C region, E region, and F region in humans. That is, in one aspect, the first polynucleotide is (i) ′ a polynucleotide having the base sequence represented by SEQ ID NO: 20. In another aspect, the first polynucleotide is (ii) ′ a polynucleotide comprising the base sequence represented by at least one of SEQ ID NOs: 24-26. Still further, the first polynucleotide comprises (iii) 'a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 20 and includes at least one of the base sequences shown in SEQ ID NOs: 24-26. (Iv) 'consists of a base sequence in which one or several bases are deleted, substituted or added to the base sequence shown in SEQ ID NO: 20, or a partial sequence thereof, and SEQ ID NO: 24 It can hybridize under stringent conditions to a polynucleotide comprising at least one of the nucleotide sequences represented by -26, or (v) 'a polynucleotide comprising the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 20 The polynucleotide may be a polynucleotide containing at least one of the base sequences shown in SEQ ID NOs: 24-26.

  Similar effects can be expected in mice. In one aspect, the first polynucleotide is (i) ″ a polynucleotide consisting of the base sequence represented by SEQ ID NO: 19. In another aspect, the first polynucleotide is (ii) ″ a polynucleotide comprising a base sequence represented by at least one of SEQ ID NOs: 21 to 23. Still further, the first polynucleotide comprises (iii) '' a partial sequence of the base sequence shown in SEQ ID NO: 19 and a base sequence containing at least one of the base sequences shown in SEQ ID NOs: 21 to 23 (Iv) '' consisting of a base sequence in which one or several bases are deleted, substituted or added to the base sequence shown in SEQ ID NO: 19, or a partial sequence thereof, Hybridization under stringent conditions to a polynucleotide containing at least one of the nucleotide sequences shown in Nos. 21 to 23, or (v) '' a polynucleotide consisting of a complementary sequence of the nucleotide sequence shown in SEQ ID No. 19 A polynucleotide that can be soybeans, and can include a polynucleotide containing at least one of the base sequences shown in SEQ ID NOs: 21 to 23.

  As described later in Examples, the C region, the E region, and the F region (SEQ ID NOs: 13, 15, and 16) provide DNA that can suppress the expression of exon 1 variant of BDNF and suppress pain. Obtained. The S3-AS4, S4-AS5, S5-AS2 regions are similar to the fact that the polynucleotide comprising the C region, E region, and F region capable of suppressing pain maintains the effect of suppressing the expression of exon 1 variant. S3-AS4, S4-AS5, S5-AS2 because the effect of suppressing the expression of exon 1 variant is maintained even in the case of a polynucleotide fragment (partial fragment) comprising Those skilled in the art who have read this specification will readily appreciate that polynucleotide fragments comprising regions may also be included within the scope of the present invention.

  The functional polynucleotide according to the present invention has the above-described configuration, and thus DNA or RNA that can specifically inhibit the expression of exon 1 variant of BDNF without inhibiting the expression of variants other than BDNF exon 1 variant. Can be provided.

  Hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

  As used herein, the term “stringent hybridization conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate. (Containing pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) overnight at 42 ° C., then 0.1 × at about 65 ° C. It is intended to clean the filter in the SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated. A polynucleotide that hybridizes to such “part” of a polynucleotide is also useful as a probe for detection.

[II] Treatment of pain The present invention provides a technique for treating pain. More specifically, the present invention provides a therapeutic agent for pain, containing the functional polynucleotide described above. The present invention also provides a pain treatment kit comprising the functional polynucleotide described above. Furthermore, the present invention provides a method for treating pain, comprising the step of administering the functional polynucleotide described above to a subject. As used herein, the term “treatment” is intended to alleviate or eliminate symptoms, and includes anything that can be done prophylactically (before onset) or therapeutically (after onset).

  The subject to be treated using the present invention is not particularly limited as long as it is a disease that develops pain, but is preferably classified as neuropathic pain or CRPS (complex regional pain syndrome). PHN (Post-Herpetic Neuralgia), trigeminal neuralgia, causalgia thalamic pain, diabetic neuropathy, cancer pain with aspects of chronic pain, orthopedic chronic pain (vertebral canal stenosis, deformed spine) Disease, cervical shoulder pain, etc.) and post-operative pain. The site of administration is also not particularly limited, but may preferably be spinal subarachnoid administration or dorsal root ganglion direct injection.

  The therapeutic agent according to the present invention is a composition containing the functional polynucleotide described above as an active ingredient, and is known to those skilled in the art such as a buffer solution and / or an adjuvant depending on the purpose, ingredient, etc. Contains optional ingredients. The therapeutic kit according to the present invention is a kit comprising the functional polynucleotide described above as a main component, and can be used by those skilled in the art such as buffers and / or adjuvants depending on the purpose, components, and the like. Any known component is provided as a further component. As used herein, a “composition” is intended to include an embodiment in which a plurality of components are contained in one substance, and such a plurality of components are provided separately as individual substances. May be certified as a “kit”. The term “kit” is intended for packaging with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material. Preferably, it includes instructions for using the material. As used herein in the context of a kit, “comprising” is intended to mean being contained within any of the individual containers that make up the kit. Further, the kit according to the present invention may be a kit provided with the components in one composition separately or a kit provided with a plurality of compositions.

As a general method for administering a therapeutic agent, there are two methods of local administration and systemic administration. However, even if an attempt is made to suppress BDNF for the treatment of pain, it is highly likely that side effects are caused by suppressing all of BDNF throughout the body. Therefore, when applying the BDNF suppression technique which can usually be assumed easily to pain treatment, it is necessary to limit the range of BDNF to be suppressed. When the therapeutic agent according to the present invention is used, any administration method is possible as shown in the following (1) to (2):
(1) Since human DRG has a diameter of about 1 cm, the functional polynucleotide can be easily introduced directly by local administration to DRG. It is also possible to insert a catheter in the vicinity of a nerve that transmits pathological pain and continuously administer the therapeutic agent according to the present invention over a long period of time.
(2) The expression of a BDNF variant is controlled by a tissue-specific or cell-specific expression mechanism. Further, since BDNF is specific to nerve cells, the therapeutic agent according to the present invention is considered to be harmless to cells other than nerve cells, and the expression of BDNF is very high in nerve cells in the absence of pain stimulation. Therefore, it is considered that the therapeutic agent according to the present invention is unlikely to affect the expression of other variants. Therefore, even if the functional polynucleotide according to the present invention is systemically administered, it is considered that knockdown can be performed only with a target tissue that develops pain.

  Examples of the method for introducing the therapeutic agent according to the present invention include a method for introducing a polynucleotide solution, a method for introducing using a transfection reagent, and a method for introducing using a virus (eg, herpes virus, adenovirus, etc.). It is not limited to these.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Example 1: Identification of transcriptional regulatory region]
The base sequences upstream of BDNF exon 1 of human, rat and mouse were aligned to find a place where homology was conserved. Primers S3, S4, S5, AS2 (SEQ ID NOs: 7 to 10) are set in a region common to human, rat, and mouse, and the promoter region of human and rat BDNF exon 1 is amplified by PCR, then length (SEQ ID NO: 1; human S3-AS2, SEQ ID NO: 2; human S4-AS2, SEQ ID NO: 3; human S5-AS2, SEQ ID NO: 4; rat S3-AS2, SEQ ID NO: 5) Rat S4-AS2, SEQ ID NO: 6; rat S5-AS2). It was confirmed that there was no mutation in the base sequence of the promoter region obtained by sequence analysis. Each promoter was inserted into a firefly luciferase reporter plasmid pGL4.14 (Promega) (FIG. 1). The firefly luciferase reporter plasmid (0.18 μg) having each promoter and the Renilla luciferase reporter plasmid pTK-hRG (0.02 μg) for correcting the introduction efficiency were ratified using the transfection reagent Effectene (QIAGEN). It was introduced into the glioma cell line C6 cells. As a control, a pGL4.14 vector having no promoter was used. Dual-Luciferase assay (Promega) was performed 72 hours after introduction. The cell transfer efficiency at the time of transfection was corrected by dividing the firefly luciferase activity value of each promoter by the Renilla luciferase activity value which is an internal control. It was shown to have strong promoter activity at least between S3 and S4 (FIG. 2).

[Example 2: Inhibitory effect of transcription ability of transcription regulatory region by double-stranded polynucleotide]
Dividing between the promoter regions S3 and S4 of rat BDNF exon 1 (rat S3-S4 region: SEQ ID NO: 18) into seven overlapping fragments (fragments A to G: SEQ ID NOs: 11 to 17, respectively) Sense strand DNA and antisense strand DNA were synthesized (FIG. 3). Double-stranded DNA was prepared by mixing the sense strand and the antisense strand of each fragment and naturally cooling in 70 mM NaCl from 70 ° C. Firefly luciferase reporter plasmid pGL4.14 (0.18 μg) having human S3-AS2 and rat S3-AS2 in the presence of 50 nM of each double-stranded DNA fragment (AG), and Renilla luciferase reporter plasmid pTK-hRG (0.02 μg) was introduced into rat glioma cell line C6 cells under the same conditions, and a Dual-Luciferase assay was performed 24 hours after the introduction (FIG. 4). As a result, it was found that all DNA fragments A to G have transcription repressive ability. In particular, it was confirmed that DNA fragments C, E, and F have the ability to strongly suppress the transcriptional activity of the promoter S3-AS2 region. The nucleotide sequences between the promoter regions S3 and S4 of mouse and human BDNF exon 1 are shown in SEQ ID NOs: 19 and 20, respectively. Moreover, the mouse | mouth base sequence corresponding to said C, E, and F is shown to sequence number 21-23, and a human base sequence is shown to sequence number 24-26.

[Example 3: Large-scale preparation of functional polynucleotide]
The above three types of DNA fragments C, E, and F were used. Both are double-stranded DNAs of 35 base pairs.

  First, the pCRII-TOPO plasmid was cut into blunt ends at the EcoRV restriction enzyme site, and double-stranded DNA of each 5 'phosphorylated DNA fragment was inserted using DNA ligase. Since this DNA fragment exists between the binding sites of the M13 primer (FW and RV) in the pCRII-TOPO plasmid, using this plasmid as a template, the M13 primer (FW and RV) is used and the sequence of the DNA fragment is included. A total of 261 base pairs was amplified by PCR.

  The amplified PCR product was purified and concentrated, and the DNA concentration was adjusted to 17.4 pM (3 μg / μL). Further, equal amounts of the prepared DNA fragments C, E, and F were mixed to prepare a solution containing each at a concentration of 5.8 pM (1 μg / μL). This mixed solution has a total DNA concentration of 17.4 pM (3 μg / μL). As a control DNA fragment, a pCRII-TOPO plasmid not containing the sequences of DNA fragments C, E, and F was used as a template, M13 primer was used, and a total of 225 base pairs between the primers was amplified by PCR. The obtained PCR product was purified and concentrated to adjust the DNA concentration to 17.4 pM (2.6 μg / μL) and used as control DNA.

[Example 4: Functional polynucleotide administration experiment 1]
In order to confirm whether or not the DNA fragment administered subarachnoidally suppresses the expression of exon 1 variant of BDNF, the tip of the catheter was compared to an untreated rat (6 week old male SD rat). It inserted so that it might reach the 4th lumbar nerve DRG vicinity. The other end of the catheter was removed from the back of the head through the skin. In order to avoid pain and inflammation associated with the insertion operation, an interval of about 1 week was left before the next treatment (7 days after catheter insertion was regarded as day 0 (FIG. 5)).

  A solution of DNA fragments C, E, F or a control DNA solution (17.4 pM each) not containing the sequences of DNA fragments C, E, F was placed under the arachnoid of 3 rats each 12 hours before and 0 hours. At that time, 20 μL each was injected.

  The rat fourth lumbar DRG was excised 36 hours after the second administration (ie, 0 hour), and the expression level of exon 1 variant of the BDNF gene was analyzed by RT-PCR. The amount of the BDNF gene transcript was corrected using the expression of the GAPDH gene as an internal standard. Specifically, RNA extraction from tissues was performed using RNeasy Lipid Tissue Mini Kit (QIAGEN). Total RNA (3-5 μg) obtained from one DRG was reverse-transcribed using a Ready-To-Go T-Primed First-Strand Kit (Amersham). The obtained cDNA was diluted to 1/10 and used for quantification of each exon of BDNF. Further, the GAPDH mRNA or actin mRNA for internal standard was used after further dilution with 1/10. Using a PCR fragment incorporated into the pCRII-TOPO vector, the base sequence was confirmed in advance and a calibration curve for quantification was prepared. LightCycler (Roche) was used for the quantitative PCR apparatus, and primers, reaction solutions and PCR programs were set as shown in Tables 1-3. Takara product was used for the SYBR Green I solution for quantitative PCR. A reaction solution containing no template was used as a negative control.

  As shown in FIG. 6, the amount of exon 1 variant mRNA was significantly decreased in the group administered with DNA fragments C, E, and F compared to the control DNA administered group. This indicates that introduction of DNA fragments C, E, and F into DRG effectively suppresses the expression of exon 1 variant of BDNF.

[Example 5: Functional polynucleotide administration experiment 2]
In Example 4, since it was found that the DNA fragment suppresses the expression of BDNF exon 1 variant of DRG, whether or not the DNA fragment can suppress pain due to inflammation was confirmed. Nine days after insertion of the catheter, 100 μL of complete Freund's adjuvant (CFA), which is an inflammatory pain inducer, was subcutaneously injected into the left hind limb sole, and the same procedure as in Example 4 was performed (FIG. 7).

  A solution in which DNA fragments C, E and F were mixed (17.4 pM; each containing 5.8 pM of DNA fragments C, E and F) or a control DNA solution (17.4 pM) was used. 348 pmol (20 μL) of solution was injected subarachnoidally 2 days before, 1 day before and immediately before CFA injection. Five rats each were used for the mixed solution introduction group and the control DNA solution introduction group.

  Rats were evaluated daily for pain behavior from immediately before administration of DNA fragments to 5 days after CFA injection. Pain behavior was evaluated by the pain threshold (50% response value) for mechanical stimulation. Specifically, the rat was placed on a wire net surrounded by a 10 × 20 cm square wall and left for 30 minutes or more until it was rested. Under the wire mesh, five types of nylon filaments (von Frey filaments) with different thicknesses were gently pressed against the center of the sole of the hind limbs in order from the thin one, and the presence or absence of escape behavior due to stimulation was observed. The threshold indicating escape behavior was determined by the method of Chaplan et al. The threshold indicates the stiffness of the filament in grams, and the lower the value, the more sensitive it is to pain.

  As shown in FIG. 8, the DNA fragment administration group significantly suppressed pain compared to the control DNA administration group. In particular, after 3 days and 4 days after stimulation, the effect of the DNA fragment was significantly observed. From this experiment, it was revealed that DNA fragments C, E, and F have a pain suppressing effect and that the pain suppressing effect can be exhibited even if administered in advance.

[Example 6: Functional polynucleotide administration experiment 3]
A chronic pain model was created by ligating the distal side of the fifth lumbar nerve DRG of the rat spinal cord, and at the same time, the tip of the catheter was placed in the vicinity of the fourth lumbar nerve DRG. According to Example 4, the other end of the catheter was removed from the back of the head through the skin.

  A solution (17.4 pM) in which DNA fragments C, E, and F were mixed (containing 5.8 pM each of DNA fragments C, E, and F) or a control DNA solution (17.4 pM) was used. After confirming the establishment of hyperalgesia due to nerve ligation, the mixed solution was injected subarachnoidally on the 5th, 6th, and 7th days.

  Rats were observed for pain behavior for another week after the third (final) administration on the 7th day immediately before the creation of the chronic pain model. The method is the same as in Example 5.

  FIG. 9 shows a typical embodiment. The data of Day 0 indicates behavioral evaluation immediately before the operation. As shown in FIG. 9 (a), in this rat, the reaction on the affected side recovered to about 80% of the response on the healthy side (non-invasive side) by administration of the DNA fragment (from the 7th day to the 10th day). It was revealed that the effect of the DNA fragment was remarkable. FIG. 9B shows the result of comparing the affected side of the DNA fragment-administered rat shown in FIG. 9A with another control DNA-administered rat, but no pain suppression was observed in the control DNA-administered rat. The results obtained by increasing the number of examples are shown in FIG. There are 4 DNA fragment-administered rats and 3 control-administered rats including the above examples. As shown in FIG. 10, it was revealed that pain was significantly suppressed in the DNA fragment-administered rats compared to the control DNA-administered rats. Only the affected foot feeling pain in order to make the graph easy to see is shown in FIG. According to this experiment, DNA fragments C, E, and F have an inhibitory effect on chronic pain, and that DNA fragment administration has no effect on behavioral evaluation on the healthy side, and is administered after the onset of pain. However, it has become clear that it can exert a pain-suppressing effect, and there are great expectations for clinical application in humans.

[Example 7: Effect of suppressing transcription ability by functional polynucleotide over a wide range of BDNF exon 1 promoter region]
The above experiments revealed that DNA fragments C, E, and F have the effect of suppressing chronic pain. Next, transcription ability suppression by a wide promoter region double-stranded DNA containing DNA fragments C, E, and F The effect was analyzed. The promoter region S3-AS2 of BDNF exon 1 was divided into three to produce three overlapping fragments. Specifically, antisense primers AS4 (SEQ ID NO: 27) and AS5 (SEQ ID NO: 28) were prepared, and rat S3-AS4 (240 bp; SEQ ID NO: 29), S4-AS5 (308 bp; SEQ ID NO: 30), S5- AS2 (432 bp; SEQ ID NO: 6) was amplified by PCR, purified and concentrated (FIG. 12). As a control, between the M13 primers of plasmid pBluescriptKS (246 bp between M4 and RV primers) was amplified by PCR, purified and concentrated. The nucleotide sequences of human promoter regions S3-AS4, S4-AS5, and S5-AS2 corresponding to rat promoter regions S3-AS4, S4-AS5, and S5-AS2 are shown in SEQ ID NOs: 31, 32, and 3, respectively.

  Firefly luciferase reporter plasmid pGL4. Which has human S3-AS2 and rat S3-AS2 in the presence of 0.6 μg of each double-stranded DNA fragment (rat S3-AS4, S4-AS5, S5-AS2, control). 14 (0.18 μg) and Renilla luciferase reporter plasmid pTK-hRG (0.02 μg) were introduced into rat glioma cell line C6 cells, and a Dual-Luciferase assay was performed 24 hours after the introduction. As a positive control, DNA fragment C (0.6 μg) was also used. As a result, it was confirmed that the promoter regions S3-AS4, S4-AS5, and S5-AS2 suppress the transcriptional activity of the promoter S3-AS2 region (FIG. 13). S4-AS5 and S5-AS2 are somewhat less effective when they are used alone, but are considered more effective when administered in combination with S3-AS4.

  As described above, the double-stranded polynucleotide for the expression regulatory region important for gene expression of the BDNF gene significantly suppresses the expression of exon 1 variant of BDNF both in vitro and in vivo, and is used as a functional polynucleotide to suppress pain. It turns out that it is possible. In particular, as shown in Example 1 (FIG. 2), since the DNA sequence between S3-AS2 in the regulatory region upstream of exon 1 has activity throughout, S3-AS2 that actually confirmed the inhibitory effect. It is considered that the suppressive ability can be more effectively exhibited by using not only the double-stranded DNA consisting of a part of the DNA but also the full-length double-stranded DNA of S3-AS2.

  Various preparations relating to acute pain are known, but few preparations effective for chronic pain are known, and none of them targets BDNF. Pain suppression using the present invention is considered to be effective for traffic accidents, aftereffects after surgery, cancer pain, diabetes, and the like. It has also been reported that cancer cells synthesize BDNF after becoming cancerous (see Non-Patent Document 3). Since BDNF has an action of attracting nerves, one of the causes of unbearable pain of cancer may be due to BDNF. Suppression of BDNF expression using the present invention is considered to be effective in the treatment of cancer pain such as terminal cancer and bone metastasis of cancer.

  Thus, the present inventors have shown that pain can be suppressed by inhibiting the expression of BDNF. Therefore, a substance that inhibits the expression of BDNF can be a pain therapeutic agent. The system created by the present inventors for measuring the increase or decrease in the expression level of BDNF in C6 rat glioma cultured cells is a new technique for screening a novel therapeutic agent for pain. More specifically, this system is the “rat glioma C6 cultured cell into which the luciferase plasmid having the exon 1 upstream region of BDNF has been introduced” used in Examples 1 and 2. In this system, preferably, the luciferase plasmid is transfected into rat glioma C6 cultured cells (gene transfer) and then cultured in the presence of hygromycin, and only the rat glioma C6 cultured cells into which the luciferase plasmid has been introduced are established. The luciferase plasmid-introduced rat glioma C6 cultured cell line may be used. Since the luciferase plasmid has a hygromycin resistance gene, only the introduced cells can be purified by culturing in the presence of hygromycin. The cells to be used are not limited to C6 cells, and may be DRG primary cultured cells or other BDNF producing cells. The method for detecting the expression level of BDNF is not limited to the luciferase assay, but may be RT-PCR, a method using a microarray, a Western blot using an antibody, an ELISA, or the like. Amplification of cDNA may be carried out as necessary. Preferred methods include PCR, LAMP (Loop-mediated Isothermal Amplification) (Eiken Chemical Co., Ltd.), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids). ) Law (Takara Shuzo Co., Ltd.).

  The specific embodiments or examples made in the detailed description section of the invention are merely to clarify the technical contents of the present invention, and are limited to such specific examples and are interpreted in a narrow sense. It should be understood that the invention can be practiced with various modifications within the spirit of the invention and within the scope of the following claims.

  By using the present invention, it becomes possible to treat pain typified by intractable chronic pain, which can greatly contribute to the medical field and the pharmaceutical field.

Claims (8)

A polynucleotide comprising any one of the following base sequences (a) to (g):
(A) the nucleotide sequence shown in SEQ ID NO: 18;
(B) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 18 and includes at least one of the base sequences shown in SEQ ID NOs: 13, 15, and 16;
(C) the nucleotide sequence shown in SEQ ID NO: 19;
(D) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 19 and includes at least one of the base sequences shown in SEQ ID NOs: 21 to 23;
(E) the nucleotide sequence shown in SEQ ID NO: 20;
(F) a base sequence that is a partial sequence of the base sequence shown in SEQ ID NO: 20 and includes at least one of the base sequences shown in SEQ ID NOs: 24-26; and (g) SEQ ID NOs: 13, 15, 16 The base sequence shown in any one of 21-26. A polynucleotide that is any of the following (I) to (VI):
(I) A nucleotide sequence in which one or several bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 18, or a partial sequence thereof, and the nucleotides shown in SEQ ID NOs: 13, 15 and 16 A polynucleotide comprising at least one of the sequences;
(II) a polynucleotide capable of hybridizing under stringent conditions to a polynucleotide comprising a complementary sequence of the base sequence represented by SEQ ID NO: 18, and comprising at least one of the base sequences represented by SEQ ID NOs: 13, 15 and 16 A polynucleotide comprising:
(III) consisting of a base sequence in which one or several bases have been deleted, substituted or added to the base sequence shown in SEQ ID NO: 19 or a partial sequence thereof, of the base sequence shown in SEQ ID NOs: 21 to 23 A polynucleotide comprising at least one;
(IV) a polynucleotide capable of hybridizing under stringent conditions to a polynucleotide comprising a complementary sequence of the base sequence represented by SEQ ID NO: 19, comprising at least one of the base sequences represented by SEQ ID NOs: 21 to 23 Containing a polynucleotide;
(V) consisting of a base sequence in which one or several bases have been deleted, substituted or added to the base sequence shown in SEQ ID NO: 20, or a partial sequence thereof, of the base sequence shown in SEQ ID NOs: 24-26 A polynucleotide comprising at least one; and (VI) a polynucleotide capable of hybridizing under stringent conditions to a polynucleotide comprising a complementary sequence of the base sequence represented by SEQ ID NO: 20, comprising SEQ ID NO: 24 A polynucleotide comprising at least one of the base sequences represented by -26.   A double-stranded polynucleotide comprising the polynucleotide according to claim 1 or 2 and a polynucleotide paired with the polynucleotide.   A therapeutic agent for pain, comprising the polynucleotide according to claim 1 or 2 or the double-stranded polynucleotide according to claim 3.   The therapeutic agent of Claim 4 for preventing the pain before onset.   The therapeutic agent of Claim 4 for preventing the pain after onset.   A kit for treating pain, comprising the polynucleotide according to claim 1 or 2 or the double-stranded polynucleotide according to claim 3.   A method for treating pain, comprising a step of administering the polynucleotide of claim 1 or 2 or the double-stranded polynucleotide of claim 3 to a subject.

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