KR102113090B1 - Composition for preventing or treating neuropathic pain comprising nanoparticle containing an agent for inhibiting expression of p38 or a gene encoding the same - Google Patents
Composition for preventing or treating neuropathic pain comprising nanoparticle containing an agent for inhibiting expression of p38 or a gene encoding the same Download PDFInfo
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- KR102113090B1 KR102113090B1 KR1020180090489A KR20180090489A KR102113090B1 KR 102113090 B1 KR102113090 B1 KR 102113090B1 KR 1020180090489 A KR1020180090489 A KR 1020180090489A KR 20180090489 A KR20180090489 A KR 20180090489A KR 102113090 B1 KR102113090 B1 KR 102113090B1
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- neuropathic pain
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Abstract
본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자에 관한 것으로서, 더욱 상세하게는 상기 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는 신경병증성 통증 예방, 개선 또는 치료용 조성물과, 신경병증성 통증 치료방법에 관한 것이다. 본 발명에 따른 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제를 함유하는 나노입자는 생체적합성 및 생분해성이 우수하며, 세포 및 동물 수준에서 신경병증성 통증을 감소시키는 효과가 우수한바, 신경병증성 통증의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.The present invention relates to nanoparticles containing an agent that inhibits the expression of p38 or a gene encoding the same, and more specifically, neurons comprising a nanoparticle containing an agent that inhibits the expression of the gene encoding the p38 or the same It relates to a composition for preventing, improving or treating pathological pain, and a method for treating neuropathic pain. Nanoparticles containing p38 according to the present invention or an agent that inhibits the expression of a gene encoding the same are excellent in biocompatibility and biodegradability, and have an excellent effect of reducing neuropathic pain at the cellular and animal level. It can be used in various fields in the prevention, improvement and treatment of sexual pain.
Description
본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자에 관한 것으로서, 더욱 상세하게는 상기 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는 신경병증성 통증 예방, 개선 또는 치료용 조성물과, 신경병증성 통증 치료방법에 관한 것이다.The present invention relates to nanoparticles containing an agent that inhibits the expression of p38 or a gene encoding the same, and more specifically, neurons comprising a nanoparticle containing an agent that inhibits the expression of the gene encoding the p38 or the same It relates to a composition for preventing, improving or treating pathological pain, and a method for treating neuropathic pain.
일반적으로 통증은 삶을 위협하거나 강한 외부자극에 대한 방어기작으로 나타나는 생리적 반응 중 하나로서 위험에서 신체를 보호하는 중요한 역할을 하는 것으로 알려져 있다. 통증은 조직손상에 의한 침해성 통증(nociceptive pain)과 조직손상을 수반하지 않지만 신경계 이상반응으로 나타나는 신경병증성 통증(neuropathic pain)으로 구분된다. 침해성 통증은 조직손상에 의한 염증반응이 수반되어 통증과 밀접한 관계가 있는 것으로 알려져 있으며 일반적으로 손상된 부위가 치유되면서 자연히 소멸된다. 신경병증성 통증은 외상(trauma)이나 염증, 허혈성 손상, 또는 대사산물 등 다양한 원인에 의해 신경계가 손상될 때 유발되는 만성적 신경질환이다. Pain is generally known to play an important role in protecting the body from danger as one of the physiological reactions that appear as a threat to life or a defense mechanism against strong external stimuli. Pain is divided into nociceptive pain due to tissue damage and neuropathic pain, which does not involve tissue damage, but is manifested as an adverse reaction of the nervous system. Invasive pain is known to be closely related to pain due to an inflammatory reaction caused by tissue damage, and generally disappears naturally as the damaged area is healed. Neuropathic pain is a chronic neurological disease caused when the nervous system is damaged by various causes such as trauma or inflammation, ischemic damage, or metabolites.
신경병증성 통증은 아무런 자극 없이 생기는 자발통(spontaneous pain)과 외부자극에 의해 유발되는 통각과민(hyperalgesia) 또는 이질통(allodynia)등의 증상을 수반하는 것이 특징이다. 통각과민은 유해자극에 대한 과도한 비정상적인 통증반응으로 정의되며, 이질통은 무해한 자극에 의해 유발되는 통증반응으로 알려져 있다. 신경병증성 통증은 기존의 진통제로는 통증을 완화시키기 어려운 것으로 알려져 있다. 신경병증성 통증 억제제로는 칼슘채널에 작용하는 가바펜틴(Gabapentin), 프리가발린(Pregabalin)과 같은 항경련제, 나트륨 채널에 작용하는 카바마제핀(Carbamazepine)과 같은 항경련제; SNRI(Serotonin Norepinephrine Reuptake Inhibitor) 계열 항우울제; 응급 시에만 사용되는 아편류 등의 4가지 계열이 임상적으로 사용되고 있다.Neuropathic pain is characterized by accompanying symptoms such as spontaneous pain that occurs without any irritation and hyperalgesia or allodynia caused by external stimulation. Hyperalgesia is defined as excessive abnormal pain response to harmful stimuli, and allodynia is known as a pain response caused by harmless stimuli. Neuropathic pain is known to be difficult to relieve pain with conventional painkillers. Neuropathic pain inhibitors include anticonvulsants such as Gabapentin and Pregabalin acting on calcium channels, and anticonvulsants such as Carbamazepine acting on sodium channels; SNRI (Serotonin Norepinephrine Reuptake Inhibitor) series antidepressant; Four types, such as opiates, used only in emergencies, are used clinically.
특히 완전한 혹은 부분적인 척수신경의 손상은 척수손상 환자들 중 약 70% 정도의 대다수에서 손상 후 몇 달 이내에 신경병증성 통증을 유발하며 궁극적으로 오랫동안 지속하는 만성적인 신경병증성 통증으로 발전하게 된다. 척수손상 후 발생하는 이러한 통증은 경미한 저림에서부터 자살을 시도할 정도로 극심한 통증까지 그 양상이 다양하며, 대개 만성 통증으로 이어짐으로써 일상생활이 불가능할 정도로 환자들을 괴롭힌다. 더구나, 신경병증성 통증은 통증 자체뿐만 아니라 수면장애, 불안, 우울증과 같은 정신적 스트레스를 동반하기 때문에 환자의 삶의 질을 위협할 뿐만 아니라, 막대한 치료비용이 지출되어(척수손상 환자의 경우 환자 개인당 연간 2,500만 원으로 평생 약 10억 원) 환자 및 가족을 넘어 심각한 사회, 경제적 문제로 대두되고 있다.Particularly, partial or partial spinal nerve damage causes neuropathic pain within a few months after injury in a majority of about 70% of patients with spinal cord injury and ultimately develops long-lasting chronic neuropathic pain. These pains that occur after spinal cord injury can range from mild tingling to extreme pain to attempt suicide, and usually lead to chronic pain, which annoys patients to the point where daily life is impossible. Moreover, neuropathic pain not only threatens the patient's quality of life because it is accompanied by psychological stress such as sleep disorders, anxiety, and depression as well as the pain itself. Annually 25 million won, about 1 billion won per lifetime) It is becoming a serious social and economic problem beyond patients and families.
이에 본 발명자들은 전술한 바와 같은 종래기술의 문제점을 해결하기 위하여, p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 개발함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention by developing a nanoparticle containing an agent that suppresses the expression of a gene encoding p38 or the same, in order to solve the problems of the prior art as described above.
따라서 본 발명의 목적은, p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는, 신경병증성 통증 예방, 개선 또는 치료용 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating neuropathic pain, comprising nanoparticles containing an agent that suppresses expression of p38 or a gene encoding the same.
본 발명의 다른 목적은, 상기 조성물을 인간을 제외한 개체에 주입하는 단계를 포함하는 신경병증성 통증 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating neuropathic pain comprising injecting the composition into an individual other than a human.
상기 목적을 달성하기 위하여, 본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는, 신경병증성 통증 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating neuropathic pain, comprising nanoparticles containing an agent that suppresses expression of p38 or a gene encoding the same.
또한 본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는, 신경병증성 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving neuropathy, comprising nanoparticles containing an agent that suppresses the expression of p38 or a gene encoding the same.
또한 본 발명은 상기 약학적 조성물을 인간을 제외한 개체에 주입하는 단계를 포함하는 신경병증성 통증 치료방법을 제공한다.In addition, the present invention provides a method of treating neuropathic pain comprising injecting the pharmaceutical composition into an individual other than a human.
본 발명에 따른 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제를 함유하는 나노입자는 생체적합성 및 생분해성이 우수하며, 세포 및 동물 수준에서 신경병증성 통증을 감소시키는 효과가 우수한바, 신경병증성 통증의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.Nanoparticles containing p38 according to the present invention or an agent that inhibits the expression of a gene encoding the same are excellent in biocompatibility and biodegradability, and have an excellent effect of reducing neuropathic pain at the cellular and animal level. It can be used in various fields in the prevention, improvement and treatment of sexual pain.
도 1은 BV2 세포에서 p38 siRNA의 p38 발현 및 염증관련 유전자 억제 효과를 확인한 결과를 나타내는 도이다.
도 2는 본 발명에 따른 p38 siRNA가 포함된 PLGA 나노입자의 특성을 분석한 결과를 나타내는 도이다.
도 3은 본 발명에 따른 p38 siRNA가 포함된 PLGA 나노입자의 세포 독성을 확인한 결과를 나타내는 도이다.
도 4는 본 발명에 따른 p38 siRNA가 포함된 PLGA 나노입자의 실험관 내 및 체내에서 세포 섭취를 분석한 결과를 나타내는 도이다.
도 5는 본 프레이 필라멘트를 이용한 신경병증성 통증 동물 모델의 통증 행동 시험 결과를 나타내는 도이다.
도 6은 신경병증성 통증 동물 모델에서 p38의 신호전달경로 및 신경병증성 통증에 의한 미세아교세포 활성화를 확인한 결과를 나타내는 도이다.1 is a view showing the results of confirming the p38 expression and inflammation-related gene suppression effect of p38 siRNA in BV2 cells.
2 is a view showing the results of analyzing the properties of the PLGA nanoparticles containing p38 siRNA according to the present invention.
3 is a view showing the results of confirming the cytotoxicity of the PLGA nanoparticles containing p38 siRNA according to the present invention.
4 is a view showing the results of analyzing the cell uptake in vitro and in the body of the PLGA nanoparticles containing p38 siRNA according to the present invention.
5 is a diagram showing the results of a pain behavior test of a neuropathic pain animal model using the present Frey filament.
FIG. 6 is a diagram showing the results of confirming microglia activation by p38 signaling pathways and neuropathic pain in a neuropathic pain animal model.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는, 신경병증성 통증 예방, 치료 또는 치료용 약학적 조성물을 제공한다. 상기 조성물은 약학적 조성물 및 식품 조성물을 포함한다.According to an aspect of the present invention, the present invention provides a pharmaceutical composition for preventing, treating or treating neuropathic pain, comprising nanoparticles containing an agent that inhibits expression of p38 or a gene encoding the same. The composition includes a pharmaceutical composition and a food composition.
본 발명에 있어서, “p38”은 세포내 신호전달을 담당하는 MAP인산화효소슈퍼패밀리에 속하는 세린/트레오닌인산화효소의 일종으로, 암 억제 유전자(tumor suppresor gene)로 알려져 있다.In the present invention, "p38" is a type of serine / threonine phosphatase belonging to the MAP phosphorylase superfamily responsible for intracellular signaling, and is known as a tumor suppresor gene.
본 발명에 있어서, “p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제”는 생물학적 시료에서 p38 또는 이를 코딩하는 mRNA에 특이적으로 결합하여, 이의 발현을 억제함으로써 척수 내 세포의 p38의 인산화 감소 및 염증관련 인자의 발현을 억제하여 신경병증성 통증을 감소시킬 수 있는 물질을 의미한다.In the present invention, "an agent that inhibits the expression of p38 or a gene encoding it" specifically binds p38 or mRNA encoding it in a biological sample, thereby inhibiting its expression, thereby reducing the phosphorylation of p38 in cells in the spinal cord and Refers to a substance capable of reducing neuropathic pain by suppressing the expression of inflammation-related factors.
상기 제제는 siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids), 안티센스 올리고뉴클레오타이드, 항체, 앱타머, 추출물 및 화합물로 이루어진 군으로부터 선택된 1 종 이상이다. 보다 바람직하게는 상기 유전자에 특이적으로 결합하는 siRNA이나, 이에 제한되지 않는다.The agents include siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, antibodies, aptamers, extracts and compounds. It is one or more selected from the group consisting of. More preferably, the siRNA specifically binds to the gene, but is not limited thereto.
본 발명에 있어서, "siRNA"는 이중가닥의 RNA가 다이서에 의해 절단되어 생성되는 21-25 뉴클레오티드 크기의 작은 RNA 조각으로 상보적인 서열을 갖는 mRNA에 특이적으로 결합하여 발현을 억제하는 것을 말한다. 본 발명의 목적상, mRNA에 특이적으로 결합하여 상기 유전자의 발현을 억제하는 것을 의미한다. siRNA는 화학적으로 또는 효소학적으로 합성될 수 있다. siRNA의 제조방법으로는 특별히 한정되지 않으며, 당업계에 공지된 방법을 사용할 수 있다.In the present invention, "siRNA" is a small RNA fragment of 21-25 nucleotides that is produced by cutting double-stranded RNA by a dicer and specifically binding to mRNA having a complementary sequence to suppress expression. . For the purposes of the present invention, it is meant to specifically inhibit mRNA expression by binding specifically to mRNA. siRNA can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and methods known in the art can be used.
본 발명에서 사용되는 siRNA는 그 자체로 폴리뉴클레오타이드 페어링을 갖는 완전한 형태, 즉 시험관에서 siRNA를 직접 합성한 두 형질전환 과정을 거쳐 세포 안으로 도입되는 형태이거나, 생체 내에 투여된 후 이러한 형태를 갖도록 하나의 단일쇄 올리고뉴클레오타이드 단편과 이의 역방향(reverse) 상보물이 스페이서에 의해 분리된 단일쇄 폴리뉴클레오타이드로부터 유도될 수 있는 형태, 예를 들어 siRNA가 세포 안에서 발현되도록 제조된 siRNA 발현 벡터 또는 PCR-유도된 siRNA 발현 카세트를 형질전환 또는 감염(infection) 과정을 거쳐 세포 안으로 도입되는 형태일 수 있다. siRNA를 제조하고 세포 또는 동물로 도입하는 방법의 결정은 목적 및 표적 유전자 산물의 세포 생물학적 기능에 따라 달라질 수 있다.The siRNA used in the present invention is a complete form having a polynucleotide pairing itself, that is, a form introduced into cells through two transformation processes in which siRNA is directly synthesized in vitro, or a single form to have such a form after administration in vivo. Single-stranded oligonucleotide fragments and their reverse complements can be derived from single-chain polynucleotides separated by spacers, e.g. siRNA expression vectors or PCR-derived siRNAs designed to express siRNA in cells. The expression cassette may be in the form of being introduced into cells through a transformation or infection process. The determination of how siRNAs are prepared and introduced into cells or animals can depend on the purpose and cellular biological function of the target gene product.
본 발명의 일 실시예에서, p38 또는 이를 코딩하는 유전자의 발현을 억제하기 위한 제제로서 siRNA인 5'-CCCAGATGCCGAAGATGAA-3'(서열번호 1)을 사용하였으나, 이에 제한되지 않는다.In one embodiment of the present invention, pRNA or a 5'-CCCAGATGCCGAAGATGAA-3 'siRNA (SEQ ID NO: 1) was used as an agent for inhibiting the expression of a gene encoding the same, but is not limited thereto.
본 발명에 있어서, "shRNA"는, 45 내지 70 뉴클레오티드의 길이를 가지는 단일가닥의 RNA로서 타겟유전자 siRNA 염기서열의 sense와 상보적인 nonsense 사이에 3-10 개의 염기 링커를 연결하는 올리고 DNA를 합성한 후 플라스미드 벡터에 클로닝하거나 또RNA (short hairpin RNA)가 만들어지고 세포 내의 Dicer에 의해 siRNA로 전환되어 RNAi 효과를 나타내는 것을 의미한다. shRNA(small hairpin RNA)를 포함한 발현 컨스트럭트/벡터는 당분야에서 공지된 방법으로 제조할 수 있다.In the present invention, "shRNA" is a single-stranded RNA having a length of 45 to 70 nucleotides, and synthesized oligo DNA linking 3-10 base linkers between sense of target gene siRNA sequence and nonsense complementary. It means that it is cloned into a plasmid vector or short RNA RNA (short hairpin RNA) is produced and converted into siRNA by Dicer in the cell to show the RNAi effect. Expression constructs / vectors including small hairpin RNA (shRNA) can be prepared by methods known in the art.
본 발명에 있어서 "miRNA(microRNA)"는, mRNA의 3' 비번역 영역(UTR) 부위와의 염기결합을 통해 전사 후 억제자 역할을 하는 대략 22nt의 비번역 RNA를 의미한다. miRNA의 제조방법으로는 특별히 한정되지 않으며, 당업계에 공지된 방법을 사용할 수 있다. In the present invention, "miRNA (microRNA)" means an untranslated RNA of approximately 22 nt that acts as a suppressor after transcription through base binding with a 3 'untranslated region (UTR) region of mRNA. The method for producing miRNA is not particularly limited, and methods known in the art can be used.
본 발명에 있어서, "안티센스 올리고뉴클레오티드"는 상기 miRNA에 상보적으로 결합하여 발현을 억제하는 염기서열로서, 이에 제한되지는 않으나 안티센스 RNA, 안티센스 DNA 및 안타고니스트(antagonist) mRNA를 포함한다.In the present invention, "antisense oligonucleotide" is a base sequence that inhibits expression by complementarily binding to the miRNA, but is not limited thereto, and includes antisense RNA, antisense DNA, and antagonist mRNA.
본 발명에 있어서, "항체"는 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 p38에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는, 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함된다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다.In the present invention, "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to p38 of the present invention, and such an antibody clones each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene. Obtained and can be prepared by conventional methods from the obtained protein. This includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, and any polyclonal antibody, monoclonal antibody, or antigen binding agent is included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.
본 발명의 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며 Fab, F(ab'), F(ab') 2 및 Fv 등이 있다.Antibodies of the invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F (ab '), F (ab') 2, and Fv.
본 발명에 있어서, “나노입자”는 약물을 중심으로 하여 고분자의 코팅층이 형성되어 이루어진 입자를 의미하는 것으로서, 편의상 "고분자의 종류 나노입자"의 형태로 표기할 수 있다. 예를 들면, 고분자의 종류가 폴리 락티드-코-글리콜라이드(PLGA)인 경우에는 PLGA 나노입자로 표기된다.In the present invention, “nanoparticle” refers to particles formed by forming a coating layer of a polymer with a drug as its center, and for convenience, may be expressed in the form of “polymer type nanoparticle”. For example, when the type of polymer is polylactide-co-glycolide (PLGA), it is indicated as PLGA nanoparticles.
상기 약물을 둘러싸는(즉, 코팅하는) 고분자로는 당업계에 공지된 생분해성 고분자(biodegradable polymer)가 이용될 수 있다. 또한, 오랜 시간 동안 체내에서 머물기 위해 다양한 분자량의 폴리에텔렌글리콜과 결합되어 있는 생분해성 고분자를 사용한다. 그 예로는 폴리스티렌-코-에틸렌글리콜, 폴리에틸렌이민-코-에틸렌글리콜, 폴리포스파겐-코-에틸렌글리콜, 폴리락타이드-코-에틸렌글리콜, 폴리락티드-코-글리콜라이드-코-에틸렌글리콜, 폴리카프로락톤-코-에틸렌글리콜, 폴리안하이드라이드-코-에틸렌글리콜, 폴리말릭산-코-에틸렌글리콜 및 이의 유도체, 폴리알킬시아노아크릴레이트-코-에틸렌글리콜, 폴리하이드로옥시부틸레이트-코-에틸렌글리콜, 폴리카르보네이트-코-에틸렌글리콜 및 폴리오르소에스테르-코-에틸렌글리콜, 폴리에틸렌글리콜, 폴리-L-라이신-코-에틸렌글리콜, 폴리글리콜라이드-코-에틸렌글리콜, 폴리메틸메타아크릴레이트-코-에틸렌글리콜, 폴리비닐피롤리돈-코-에틸렌글리콜, 및 이의 공중합체로 이루어진 군으로부터 선택된 것이 이용될 수 있다. 상기 고분자들은 낮은 독성뿐만 아니라 탁월한 생체 적합성을 보여주는 것으로 알려져 있다[A. J. Domb, et al. Handbook of biodegradable polymers, Harwood academic publishers, USA, 1997).As the polymer surrounding the drug (ie, coating), a biodegradable polymer known in the art may be used. In addition, biodegradable polymers combined with polyethylene glycols of various molecular weights are used to stay in the body for a long time. Examples include polystyrene-co-ethylene glycol, polyethyleneimine-co-ethylene glycol, polyphosphagen-co-ethylene glycol, polylactide-co-ethylene glycol, polylactide-co-glycolide-co-ethylene glycol, Polycaprolactone-co-ethylene glycol, polyanhydride-co-ethylene glycol, polymalic acid-co-ethylene glycol and derivatives thereof, polyalkylcyanoacrylate-co-ethylene glycol, polyhydrooxybutylate-co -Ethylene glycol, polycarbonate-co-ethylene glycol and polyorthoester-co-ethylene glycol, polyethylene glycol, poly-L-lysine-co-ethylene glycol, polyglycolide-co-ethylene glycol, polymethyl meta What is selected from the group consisting of acrylate-co-ethylene glycol, polyvinylpyrrolidone-co-ethylene glycol, and copolymers thereof can be used. These polymers are known to show excellent biocompatibility as well as low toxicity [A. J. Domb, et al. Handbook of biodegradable polymers, Harwood academic publishers, USA, 1997).
본 발명의 바람직한 구체예에서, 상기 고분자는 미국 FDA에 의해 승인된 PLGA를 이용하였으나, 이에 제한되지 않는다.In a preferred embodiment of the present invention, the polymer uses PLGA approved by the US FDA, but is not limited thereto.
본 발명에 따른 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자의 내부에 존재하는 약물은 p38 억제제로서, 약물이 고분자 나노입자의 내부 임의의 위치에 위치하고 있는 것이다. p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 척수관 주사할 경우 고분자가 서서히 분해되면서 담지되어 있는 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 방출되기 때문에 오랫동안 높은 약물 농도를 유지할 수 있다.The drug present inside the nanoparticle containing the p38 or the agent for inhibiting the expression of the gene encoding the same according to the present invention is a p38 inhibitor, and the drug is located anywhere inside the polymer nanoparticle. When a spinal canal injection of nanoparticles containing an agent that inhibits the expression of p38 or a gene encoding it, the polymer slowly decomposes, and the drug that suppresses the expression of the supported p38 or gene encoding the gene is released for a long time. Concentration can be maintained.
본 발명에 있어서, “예방”은 본 발명의 조성물에 의해 병인을 제거하거나 조기 발견하여 해당질환을 막는 모든 행위를 의미한다.In the present invention, "prevention" means all actions to prevent the disease by removing or prematurely detecting the etiology by the composition of the present invention.
본 발명에 있어서, “치료”는 본 발명의 조성물에 의해 질환의 증세가 호전되는 모든 행위를 의미한다.In the present invention, "treatment" refers to all actions in which symptoms of a disease are improved by the composition of the present invention.
본 발명에 있어서, “개선”은 본 발명의 조성물에 의해 생체 대사, 세포 또는 기관의 기능이 이롭게 변경되는 모든 행위를 의미한다.In the present invention, "improvement" means any action in which the function of a biological metabolism, cell or organ is advantageously changed by the composition of the present invention.
본 발명의 조성물은 상기 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자와 함께 신경병증성 통증의 예방, 치료 또는 개선 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다.The composition of the present invention may further contain one or more known active ingredients having the effect of preventing, treating, or improving neuropathic pain together with the nanoparticles containing the p38 or an agent that suppresses the expression of the gene encoding the same. have.
본 발명의 바람직한 구체예에 따르면, p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자는 미세아교세포(microglia cell), 뉴런 세포(neuronal cell) 및 성상세포(astrocytic cell)로 이루어진 군에서 선택되는 1종 이상을 타겟하는 것이 바람직하나, 이에 제한되지 않는다.According to a preferred embodiment of the present invention, nanoparticles containing an agent that inhibits the expression of p38 or a gene encoding the same are composed of microglia cells, neuronal cells, and astrocytic cells. It is preferable to target one or more selected from the group, but is not limited thereto.
본 발명의 바람직한 구체예에 따르면, 본 발명의 조성물은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 0.1 내지 1000mg/ml의 농도로 포함하는 것이 바람직하며, 5 내지 20mg/ml의 농도로 포함하는 것이 더 바람직하다.According to a preferred embodiment of the present invention, the composition of the present invention preferably contains p38 or nanoparticles containing an agent that inhibits the expression of the gene encoding it at a concentration of 0.1 to 1000 mg / ml, and 5 to 20 mg / It is more preferable to include in a concentration of ml.
본 발명의 일 구체예에서, 본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는 신경병증성 통증 예방 또는 치료용 약학적 조성물을 제공한다.In one embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating neuropathic pain comprising nanoparticles containing p38 or an agent that suppresses the expression of a gene encoding the same.
본 발명의 약학적 조성물은 투여를 위하여 약학적으로 허용 가능한 담체를 1종 이상 포함할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 이상의 성분을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화 할 수 있다. 더 나아가 당 분야의 적정한 방법으로 각 질환에 따라 또는 성분에 따라 제제화 할 수 있다.The pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable carriers can be used by mixing one or more of these components: saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and antioxidants, buffers if necessary. , Other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added in addition to formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be formulated according to each disease or component in an appropriate manner in the art.
상기 약학적 조성물은 추가적인 성분을 더 포함할 수 있다. 상기 추가적인 성분의 예로는 유당, 탈크, 전분, 스테아린산 마그네슘, 결정성 셀룰로오스, 락토오스, 젤라틴, 만니톨, 과옥소산, 이성화당, 폴리비닐알코올, 붕산 및 탄산나트륨이 있다.The pharmaceutical composition may further include additional components. Examples of such additional ingredients are lactose, talc, starch, magnesium stearate, crystalline cellulose, lactose, gelatin, mannitol, peroxanoic acid, isomerized sugar, polyvinyl alcohol, boric acid and sodium carbonate.
상기 약학적 조성물은 경구 투여 또는 비경구 투여할 수 있으며, 신속한 효과를 위하여 비경구 투여하는 것이 바람직하며, 척수강에 척수관 주사되는 것이 더 바람직하다. 비경구 투여를 하는 경우, 정맥내 주입, 근육내 주입, 관절내(intra-articular) 주입, 활액내(intra-synovial) 주입, 수망강내 주입, 간내(intrahepatic) 주입, 병변내(intralesional) 주입 또는 두 개강내(intracranial) 주입 등으로 투여할 수 있다. 상기 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다.The pharmaceutical composition may be administered orally or parenterally, it is preferable to administer parenterally for rapid effect, it is more preferable to be injected into the spinal canal. For parenteral administration, intravenous infusion, intramuscular infusion, intra-articular infusion, intra-synovial infusion, intramedullary infusion, intrarahepatic infusion, intralesional infusion, or It can be administered by intracranial injection. Suitable dosages of the pharmaceutical compositions can be variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. have.
본 발명의 일 구체예에서, 본 발명은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자를 포함하는, 신경병증성 예방 또는 개선용 식품 조성물을 제공한다.In one embodiment of the present invention, the present invention provides a food composition for preventing or improving neuropathy, comprising nanoparticles containing an agent that inhibits expression of p38 or a gene encoding the same.
본 발명의 식품 조성물은 건강기능식품, 식품 첨가제 또는 식이보조제일 수 있다. 본 발명의 조성물이 식품 첨가제인 경우, p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제를 그대로 첨가하거나, 다른 식품 또는 식품 성분과 함께 혼합하여 사용되는 등 통상적인 방법에 따라 적절하게 사용될 수 있다.The food composition of the present invention may be a health functional food, food additive or dietary supplement. When the composition of the present invention is a food additive, p38 or an agent that suppresses expression of a gene encoding the same may be added as it is, or may be suitably used according to a conventional method, such as being used in combination with other foods or food ingredients.
구체적인 예로, 식품 또는 음료의 제조 시에는 본 발명의 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제는 원료에 대하여 15중량% 이하, 바람직하게는 10중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하여 장기간 섭취할 경우에는 상기 범위 이하의 양으로 첨가될 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. 상기 식품의 종류에는 특별한 제한은 없으나, 본 발명의 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제를 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료, 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.As a specific example, in the preparation of food or beverage, p38 of the present invention or an agent that suppresses expression of a gene encoding the same is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, for health and hygiene purposes or for long-term intake for health control purposes, it may be added in an amount below the above range, and since there is no problem in terms of safety, the active ingredient may also be used in an amount above the above range. have. There is no particular limitation on the type of the food, but examples of foods to which p38 of the present invention or an agent that suppresses the expression of the gene encoding the same can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, There are ramen, other noodles, gum products, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc. and includes all healthy foods in the ordinary sense.
본 발명의 식품 조성물이 음료로 제조될 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등의 추가 성분을 포함할 수 있다. 상기 천연 탄수화물로는 포도당, 과당 등의 모노사카라이드; 말토오스, 수크로오스 등의 디사카라이드; 덱스트린, 사이클로덱스트린 등의 천연 감미제; 사카린, 아스파르탐 등의 합성 감미제 등이 사용될 수 있다. 상기 천연 탄수화물은 본 발명의 식품 조성물 총 중량에 대하여 0.01 내지 10중량%, 바람직하게는 0.01 내지 0.1중량%로 포함된다.When the food composition of the present invention is prepared as a beverage, it may include various components such as various flavoring agents or natural carbohydrates, such as a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame can be used. The natural carbohydrate is contained in 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight relative to the total weight of the food composition of the present invention.
본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 포함할 수 있으며, 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있으나 이에 제한되지 않는다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 상기의 첨가제 비율은 크게 제한되지는 않으나, 본 발명의 식품 조성물 총 중량에 대하여 0.01 내지 0.1중량% 범위내로 포함되는 것이 바람직하다.The food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid Carbonation agents used in beverages, and the like, and may include, but are not limited to, natural fruit juice, fruit juice beverage, and flesh for preparing vegetable beverages. These ingredients can be used independently or in combination. The additive ratio is not particularly limited, but is preferably included in the range of 0.01 to 0.1% by weight relative to the total weight of the food composition of the present invention.
건강 및 위생을 목적으로 하거나 건강 조절을 목적으로 하는 장기간의 섭취인 경우, 본 발명의 식품 조성물은 안전성 면에서 아무런 문제가 없기 때문에 장기간 복용이 가능하다.In the case of long-term intake for health and hygiene purposes or for health control purposes, the food composition of the present invention can be taken for a long time because there is no problem in terms of safety.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 약학적 조성물을 인간을 제외한 개체에 주입하는 단계를 포함하는 신경병증성 통증 치료방법을 제공한다.According to another aspect of the present invention, the present invention provides a method of treating neuropathic pain comprising injecting the pharmaceutical composition into an individual other than a human.
본 발명의 바람직한 구체예에 따르면, 상기 약학적 조성물은 개체의 척수에 주입되는 것이 바람직하나, 이에 제한되지 않는다.According to a preferred embodiment of the present invention, the pharmaceutical composition is preferably injected into the spinal cord of an individual, but is not limited thereto.
본 발명에 따른 신경병증성 통증 치료방법은 p38 또는 이를 코딩하는 유전자의 발현을 억제하는 제제가 함유된 나노입자가 척수 내 세포의 p38의 인산화 및 염증관련인자의 발현을 감소시킴으로써, 신경병증성 통증을 치료할 수 있다.In the neuropathic pain treatment method according to the present invention, nanoparticles containing an agent that inhibits expression of p38 or a gene encoding the same decreases the expression of p38 phosphorylation and inflammation-related factors in cells in the spinal cord, thereby causing neuropathic pain. Can cure it.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실험예Experimental Example 1. p38 1.p38 siRNA가siRNA 형질도입된 BV2 세포주 준비 Preparation of transduced BV2 cell lines
마우스 미세아교세포인 BV2를 불활화된 10% FBS 및 1% 항생제가 포함된 DMEM 배지를 이용하여 5% CO2 및 37℃ 조건에서 배양하였다. 배양된 BV2 세포에 p38 siRNA를 2일 동안 p38 siRNA를 농도별(10, 20 및 50 nM)로 처리하여 형질도입(transfection)시켰다. 상기 p38 siRNA의 서열은 5'-CCCAGATGCCGAAGATGAA-3'(서열번호 1)이며, 이하 실험예에서는 모두 동일한 서열의 p38 siRNA를 사용하였다. 상기 형질도입은 Lipofectamine™ RNAiMAX Transfection Reagent(ThermoFisher Scientific)을 이용하였으며, 제조사의 매뉴얼에 따라 수행하였다. p38 siRNA가 형질도입된 BV2 세포는 형질도입 48시간 후, 정량적 RT-PCR(quantitative real-time PCR) 및 웨스턴 블롯팅(western blotting)을 수행하였다. 대조군은 스크램블드 siRNA 20μM을 2일 동안 처리하여 형질도입시킨 것을 이용하였다. BV2, a mouse microglia, was cultured at 5% CO 2 and 37 ° C. using DMEM medium containing 10% FBS and 1% antibiotic. The cultured BV2 cells were transfected by treating p38 siRNA with p38 siRNA for 2 days by concentration (10, 20 and 50 nM). The sequence of the p38 siRNA is 5'-CCCAGATGCCGAAGATGAA-3 '(SEQ ID NO: 1), and in the following experimental examples, p38 siRNA of the same sequence was used. The transduction was performed using Lipofectamine ™ RNAiMAX Transfection Reagent (ThermoFisher Scientific), and was performed according to the manufacturer's manual. BV38 cells transduced with p38 siRNA were subjected to quantitative RT-PCR (quantitative real-time PCR) and western blotting 48 hours after transduction. As a control group, 20 μM of scrambled siRNA was treated for 2 days and then transduced.
실험예Experimental Example 2. 정량적 RT- 2. Quantitative RT- PCRPCR (quantitative real time (quantitative real time PCRPCR ))
Hybrid-R 키트(GeneAll, Seoul, Korea)를 이용하여 동물모델의 척수의 동측성 배면각(ipsilateral dorsal horn)(L4-L5 segment, 0.7 cm) 또는 BV2 세포로부터 전체RNA를 분리하였다. 분리된 전체RNA에 TOPscript RT DryMix(Enzynomics, 대전, 한국) 20μl를 반응시켜 cDNA를 합성하였다. 합성된 cDNA를 이용하여 정량적 RT-PCR을 수행하였다. 구체적으로, 상기 정량적 RT-PCR은 95℃에서 10분, 그 후 95℃에서 15초, 60℃에서 1분을 40회 반복하였다. 또한 상기 정량적 RT-PCR에 사용된 프라이머는 다음과 같다;GAPDH, forward: 5′- CTC ATG ACC ACA GTC CAT GC -3′, reverse: 5′- TTC AGC TCT GGG ATG ACC TT -3′; IL-1β, forward: 5′- CAG CAG CAT CTC GAC AAG AG -3′, reverse: 5′- CAT CAT CCC ACG AGT CAC AG -3′; TNF-α, forward: 5′- AGA TGT GGA ACT GGC AGA GG -3′, reverse: 5′- CCC ATT TGG GAA CTT CTC CT -3′; IL-6, forward: 5′- CCG GAG AGG AGA CTT CAC AG -3′, reverse: 5′- ACA GTG CAT CAT CGC TGT TC -3′; iNOS forward: 5′- TCT GTG CCT TTG CTC ATG ACA -3′, reverse: 5′- TGC TTC GAA CAT CGA ACG TC -3′; COX-2 forward: 5′- CAG TAT CAG AAC CGC ATT GCC -3′, reverse: 5′- GAG CAA GTC CGT GTT CAA GGA-3′. 각 표적 유전자의 mRNA 발현 수준은 GAPDH mRNA의 발현 수준으로 정규화 하였으며, 그 값은 2- ΔΔCt 방법으로 계산하였다(K. J. Livak and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Delta Delta C(T)) Method. Methods. 2001;25:402-8)Total RNA was isolated from the ipsilateral dorsal horn (L4-L5 segment, 0.7 cm) or BV2 cells of the spinal cord of an animal model using a Hybrid-R kit (GeneAll, Seoul, Korea). CDNA was synthesized by reacting 20 μl of TOPscript RT DryMix (Enzynomics, Daejeon, Korea) with the isolated total RNA. Quantitative RT-PCR was performed using the synthesized cDNA. Specifically, the quantitative RT-PCR was repeated 40 times for 10 minutes at 95 ° C, then 15 seconds at 95 ° C, and 1 minute at 60 ° C. In addition, the primers used for the quantitative RT-PCR are as follows: GAPDH, forward: 5'- CTC ATG ACC ACA GTC CAT GC -3 ', reverse: 5'- TTC AGC TCT GGG ATG ACC TT -3'; IL-1β, forward: 5′- CAG CAG CAT CTC GAC AAG AG-3 ′, reverse: 5′- CAT CAT CCC ACG AGT CAC AG-3 ′; TNF-α, forward: 5'- AGA TGT GGA ACT GGC AGA GG -3 ', reverse: 5'- CCC ATT TGG GAA CTT CTC CT -3'; IL-6, forward: 5'- CCG GAG AGG AGA CTT CAC AG -3 ', reverse: 5'- ACA GTG CAT CAT CGC TGT TC -3'; iNOS forward: 5'- TCT GTG CCT TTG CTC ATG ACA -3 ', reverse: 5'- TGC TTC GAA CAT CGA ACG TC -3'; COX-2 forward: 5'- CAG TAT CAG AAC CGC ATT GCC -3 ', reverse: 5'- GAG CAA GTC CGT GTT CAA GGA-3'. The mRNA expression level of each target gene was normalized to the expression level of GAPDH mRNA, and the value was calculated by 2 - ΔΔCt method (KJ Livak and TD Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2 ( .. - Delta Delta C (T )) Method Methods 2001; 25: 402-8)
실험예Experimental Example 3. 3. 웨스턴Western 블롯팅Blotting (western blotting)(western blotting)
실험예 1에서 준비된 p38 siRNA가 형질도입된 BV2 세포를 단백질 추출 버퍼(PRO-PREP, Intron, Daejeon, Republic of Korea)와 함께 배양하여 세포 용해물을 준비하였다. 브래드포드 분석(Bradfordassay, BIO-RAD, Hercules, CA)을 통해 세포 용해물의 단백질량을 정량하였다. 세포 용해물 20μg을 12% SDS-폴리아크릴아마이드 겔로 분리하였으며, 이를 니트로셀룰로오스 멤브레인에 트랜스퍼하였다. 상기 멤브레인을 1차 항체(anti-p38, 1:1000, Cell Signaling Technology, Danvers, MA; anti-β actin, Sigma-Aldrich)와 함께 배양한 후 세척하였다. 세척된 멤브레인에 HRP가 결합되어 있는 2차 항체(anti-rabbit-HRP or anti-mouse-HRP, KOMA, Seoul, Republic of Korea)를 첨가하여 배양한 후 세척하였다. 세척된 멤브레인을 ECL 용액(enhanced chemiluminescence solution, GE Healthcare, Little Chalfont, UK)과 반응시켰으며, 이를 ChemiDoc Touch imaging system(BIO-RAD)을 통해 신호를 검출하였다. Cell lysates were prepared by culturing the BV2 cells transduced with p38 siRNA prepared in Experimental Example 1 together with a protein extraction buffer (PRO-PREP, Intron, Daejeon, Republic of Korea). The protein amount of cell lysates was quantified through Bradford assay (BIO-RAD, Hercules, CA). 20 μg of cell lysate was separated with a 12% SDS-polyacrylamide gel, which was transferred onto a nitrocellulose membrane. The membrane was incubated with primary antibodies (anti-p38, 1: 1000, Cell Signaling Technology, Danvers, MA; anti-β actin, Sigma-Aldrich) and washed. To the washed membrane, HRP-coupled secondary antibody (anti-rabbit-HRP or anti-mouse-HRP, KOMA, Seoul, Republic of Korea) was added and cultured, followed by washing. The washed membrane was reacted with an ECL solution (enhanced chemiluminescence solution, GE Healthcare, Little Chalfont, UK), and the signal was detected through a ChemiDoc Touch imaging system (BIO-RAD).
실험예Experimental Example 4. p38 4. p38 siRNA를siRNA 포함하는 Containing PLGAPLGA (( polypoly (lactic-co-(lactic-co- glycolicglycolic acid) 나노입자 제조 acid) nanoparticle preparation
PLGA 나노입자를 제조하기 위하여, p38 siRNA가 20μM의 농도로 포함된TE7.5(Sigma-Aldrich) 버퍼 및 PLGA(Corbion, Amsterdam, the Netherlands) 25mg가 포함된 디클로로메탄(dichloromethane, DCM) 800μl를 준비하였다. 상기 p38 siRNA의 서열은 5'-CCCAGATGCCGAAGATGAA-3'(서열번호 1)이다. 상기 TE7.5 버퍼 200μl를 디클로로메탄에 한 방울씩 첨가하여 혼합물을 제조하였다. 상기 혼합물을 초음파 처리하여(UP100H ultrasonic processor, Hielscher Ultrasonics GmbH, Teltow, Germany) 1차 W1/O 에멀젼으로 유화시켰다. 2% PVA1500(w/v) 2ml을 1차 W1/O 에멀젼에 첨가한 후 초음파 처리하여 W1/O/W2 이중 에멀젼을 제조하였다. 제조된 W1/O/W2 이중 에멀젼을 2% PVA1500(w/v) 6ml에 희석하였으며, DCM을 증발시키기 위하여 실온에서 3시간 동안 교반시켰다. 또한 4℃에서 10분 동안 38,000g에서 초원심분리(OptimaTM Max Ultracentrifuge, Beckman COULTER, Brea, CA)하여 PLGA 나노입자를 수집하였다. 수집된 PLGA 나노입자를 탈이온화된 RNase 프리-워터로 2회 세척하였다. 세척된 PLGA 나노입자를 물에 재현탁시킨 후 동결건조 시켰다. 세포 및 척수에서 PLGA 나노입자를 추적하기 위하여, PLGA 나노입자에 쿠마린 6을 1mg/ml의 농도로 포함시켰다. 대조군은 스크렘블드 siRNA가 포함된 PLGA 나노입자를 이용하였다.To prepare PLGA nanoparticles, 800 μl of dichloromethane (DCM) containing 25 mg of TE7.5 (Sigma-Aldrich) buffer and 25 mg of PLGA (Corbion, Amsterdam, the Netherlands) containing p38 siRNA at a concentration of 20 μM was prepared. Did. The sequence of the p38 siRNA is 5'-CCCAGATGCCGAAGATGAA-3 '(SEQ ID NO: 1). The mixture was prepared by adding 200 μl of the TE7.5 buffer dropwise to dichloromethane. The mixture was sonicated (UP100H ultrasonic processor, Hielscher Ultrasonics GmbH, Teltow, Germany) to emulsify into a primary W1 / O emulsion. 2 ml of 2% PVA1500 (w / v) was added to the primary W1 / O emulsion, followed by sonication to prepare a W1 / O / W2 double emulsion. The prepared W1 / O / W2 double emulsion was diluted in 6 ml of 2% PVA1500 (w / v) and stirred for 3 hours at room temperature to evaporate DCM. In addition, PLGA nanoparticles were collected by ultracentrifugation (OptimaTM Max Ultracentrifuge, Beckman COULTER, Brea, CA) at 38,000 g for 10 minutes at 4 ° C. The collected PLGA nanoparticles were washed twice with deionized RNase pre-water. The washed PLGA nanoparticles were resuspended in water and then lyophilized. To trace PLGA nanoparticles in cells and spinal cord,
Zetasizer Nano ZS(Malvern instruments, Malvern, UK)를 이용하여, 완성된 PLGA 나노입자의 크기 및 제타 전위를 측정하였다. 또한 PLGA 나노입자의 무결성(integrity) 및 크기를 투과 전자 현미경(TEM) 및 주사 전자 현미경(SEM)을 통해 측정하였다.The size and zeta potential of the finished PLGA nanoparticles were measured using Zetasizer Nano ZS (Malvern instruments, Malvern, UK). In addition, the integrity and size of the PLGA nanoparticles were measured by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
실험예Experimental Example 5. 5. MTTMTT 분석을 통한 세포 독성 확인 Confirmation of cytotoxicity through analysis
MTT 분석을 통해 상기 실험예 4의 p38 siRNA가 포함된 PLGA 나노입자의 세포 독성을 확인하였다. 구체적으로, BV2 세포를 96웰 플레이트에 5x103cells/well로 접종하여 배양하였다. 배양 1일 경과 후, BV2 세포에 p38 siRNA가 포함된 PLGA 나노입자를 각각 10, 20, 50, 100, 200μg/ml의 농도로 처리하여 24시간동안 배양하였다. PLGA 나노입자가 처리된 BV2 세포에 MTT 용액(100 μl of 5 mg/ml solution)을 첨가하고, 37℃에서 4시간 동안 배양하였다. 배양 후 각 웰의 배지를 제거한 후 DMSO 100ml을 첨가하여 15분 동안 반응시켰다. 세포생존률은 마이크로플레이트microplate reader (Sunrise, Tecan Austria GmbH, Grodig, Austria)를 이용하였으며, 540nm에서의 흡광도를 측정하였다.Through MTT analysis, cytotoxicity of PLGA nanoparticles containing p38 siRNA of Experimental Example 4 was confirmed. Specifically, BV2 cells were cultured by inoculating 96-well plates at 5x10 3 cells / well. After 1 day of culture, PL2 nanoparticles containing p38 siRNA were treated with BV2 cells at concentrations of 10, 20, 50, 100, and 200 μg / ml, respectively, and cultured for 24 hours. MTT solution (100 μl of 5 mg / ml solution) was added to BV2 cells treated with PLGA nanoparticles, and cultured at 37 ° C. for 4 hours. After incubation, the medium of each well was removed, and 100 ml of DMSO was added to react for 15 minutes. Cell viability was measured using a microplate microplate reader (Sunrise, Tecan Austria GmbH, Grodig, Austria), and absorbance at 540 nm was measured.
실험예Experimental Example 6. 6. PLGAPLGA 나노입자의 Nanoparticle 실험관Test tube 내(in vitro) 및 체내(in In vitro and in vivovivo ) 섭취 분석) Intake analysis
24웰 플레이트에 BV2(murine microglial cell line), HT22(murine neuronal cell line) 및 U87MG(human astrocytic cell line) 세포를 1X104cells/well로 접종하였다. 쿠마린 6가 포함된 PLGA 나노입자는 PBS에 5mg/ml의 농도로 재부유시켰으며, 재부유된 용액 10μl을 각 웰에 첨가하여 3시간동안 배양하였다. 24-well plates in the BV2 (murine microglial cell line), (murine neuronal cell line) and the (human astrocytic cell line) U87MG HT22 cells were inoculated with 1X10 4 cells / well. PLGA
배양 후 형광 현미경을 이용하여, 각 세포에서 PLGA 나노입자의 세포질 섭취를 조사하였다. After cultivation, the cytoplasmic uptake of PLGA nanoparticles in each cell was examined using a fluorescence microscope.
또한 척수에서 PLGA 나노입자의 표적을 확인하기 위하여, 해밀턴 주사기로 쿠마린 6이 포함된 PLGA 나노입자 10μl를 마우스의 척수강 내에 주사하였다. 주사한 후 2일째에 척수를 분리하였으며, 두께 30μm인 4-6번 요추(L4-6) 동결절편을 제조하였다. 상기 동결절편을 anti-NeuN(neuronal marker; 1: 500, Millipore, Burlington, MO), anti-GFAP(astrocytic marker; 1: 5,000, DAKO, Troy, MI) 또는 anti-OX42 (microglial marker; 1: 500, BIO-RAD) 항체와 면역염색하여 척수 내 쿠마린 6이 포함된 PLGA 나노입자의 위치를 시각화하였다.Further, in order to confirm the target of PLGA nanoparticles in the spinal cord, 10 μl of PLGA
실험예Experimental Example 7. 신경병증성 통증 동물모델 제조 7. Neuropathic pain animal model manufacturing
7-1. 동물 실험7-1. Animal experiment
신경병증성 통증 동물 모델을 제조하기 위하여, 대한 바이오링크(DBL, 충북)에서 Sprague-Dawley 래트(6 wks males, 150-200 g)fmf 구입하여 1주일동안 새로운 환경에 적응시켰다. 모든 동물은 12시간의 밝음/어두움 주기에서 1 케이지 당 3마리씩 사육하였으며, 음식 및 물에 대한 자유로운 접근이 가능한 환경에서 사육하였다. 본 실시예의 동물실험은 충남대학교의 동물 관리 및 이용위원회(CNUH-A017-0017)의 승인을 받았으며, 식약처의 통증 연구의 윤리 가이드라인에 따라 수행되었다.To prepare a neuropathic pain animal model, Sprague-Dawley rats (6 wks males, 150-200 g) fmf were purchased from Daehan Biolink (DBL, Chungbuk) and adapted to a new environment for one week. All animals were bred 3 animals per cage in a 12 hour light / dark cycle, and kept in an environment with free access to food and water. The animal experiment of this example was approved by the Animal Care and Use Committee of Chungnam National University (CNUH-A017-0017), and was performed according to the Ethics Guidelines for Pain Research at the Food and Drug Administration.
7-2. L5 7-2. L5 SNLSNL (L5 spinal nerve ligation) 유도된 신경병증성 통증 동물 모델 제조(L5 spinal nerve ligation) induced neuropathic pain animal model preparation
실험예 7-1의 Sprague-Dawley 래트를 아이소플루레인(isoflurane, 2% in oxygen mixture; Hana pharm, Kyung-gi, Republic of Korea)으로 마취하고, 시각적으로 L4 및 L5 척추 신경을 확인할 수 있도록 왼쪽 6번 횡돌기(left lumbar (L) 6 transverse process)를 제거하였다. 이어서 왼쪽 L5 척추 신경을 분리하고 3-0 실크실로 타이트하게 묶었다. 완전히 지혈한 후에 상처를 봉합하였다. 대조군은 실험군과 동일한 방법으로 수술하되, SNL을 유도하지 않은 것이다.The Sprague-Dawley rat of Experimental Example 7-1 was anesthetized with isoflurane (2% in oxygen mixture; Hana pharm, Kyung-gi, Republic of Korea) and left to visually identify L4 and L5 spinal nerves. The left lumbar (L) 6 transverse process was removed. The left L5 vertebral nerve was then isolated and tightly tied with 3-0 silk thread. The wound was closed after complete hemostasis. The control group was operated in the same manner as the experimental group, but did not induce SNL.
실험예Experimental Example 8. 본 8. Pattern 프레이Frey 필라멘트( filament( vonvon freyfrey filament)를 이용한 통증 행동 시험 Filament)
통증 행동 시험은 J. M. Chung, H. K. Kim and K. Chung, Segmental spinal nerve ligation model of neuropathic pain. Methods Mol Med. 2004;99:35-45에 기재된 방법으로 수행하였다. 기계적 이질통(Mechanical allodynia)은 수술 후 3, 7, 10, 12 및 14일 째에 본 프레이 필라멘트(NC12775-99, Touch Test® Sensory Evaluators, 20-Piece Hand Kit, North Coast Medical & Rehabilitation Products, Camino Arroyo Gilroy, CA)를 이용하여 측정하였다. 구체적으로, 동물모델의 뒷발의 발바닥 표면에 본 프레이 필라멘트를 1 내지 2초 동안 3회 적용하였으며, 업-다운 테스트 패러다임을 통해 각 발의 역치를 측정하였다. 역치 측정 시 기준선을 설정하기 위하여 10-g 자극을 사용하였으며, 신경병증성 통증의 발생을 판단하기 위하여 4-g의 자극을 먼저 적용하였다. 자극을 적용하였을 시 긍정적 응답이 발생하는 경우 더 작은 본 프레이 헤어를 사용하였다. 이와 반대로, 부정적인 응답이 발생하는 경우 더 높은 힘을 가하였다.The pain behavior test was conducted by JM Chung, HK Kim and K. Chung, Segmental spinal nerve ligation model of neuropathic pain . Methods Mol Med . 2004; 99: 35-45. Mechanical allodynia was seen on 3, 7, 10, 12 and 14 days after surgery (NC12775-99, Touch Test® Sensory Evaluators, 20-Piece Hand Kit, North Coast Medical & Rehabilitation Products, Camino Arroyo Gilroy, CA). Specifically, this Frey filament was applied to the plantar surface of the hind paw of the animal model three times for 1-2 seconds, and the threshold of each paw was measured through an up-down test paradigm. A 10-g stimulus was used to establish a baseline when measuring the threshold, and a 4-g stimulus was first applied to determine the occurrence of neuropathic pain. If a positive response occurs when stimulation is applied, a smaller bone fry hair is used. Conversely, higher forces were applied in the event of a negative response.
실험예Experimental Example 9. 9. 척수관Spinal cord 주사( injection( IntrathecalIntrathecal injection) injection)
척수관 주사는 J. L. Hylden and G. L. Wilcox, Intrathecal morphine in mice: a new technique. Eur J Pharmacol. 1980;67:313-6에 기재된 방법으로 수행하였다. Spinal canal injections include JL Hylden and GL Wilcox, Intrathecal morphine in mice: a new technique . Eur J Pharmacol . 1980; 67: 313-6.
먼저, 래트를 수술대에 눕힌 후 아이소플루레인을 이용하여 마취시켰다. 해밀톤 주사기(50 μl; Reno, NV)를 이용하여, 마취된 래트의 5번 요추(L5) 및 6번 요추(L6)의 사이의 요추강에 PLGA 나노입자 용액(p38 억제제 또는 p38 siRNA가 포함된 PLGA 나노입자가 PBS에 10mg/ml의 농도로 재부유시킨 것) 20μl를 주사하였다. 대조군은 스크렘블드 siRNA 및 p38 억제제인 SB20303580을 이용하였다. 척수관 주사가 완료되었을 시 꼬리-플릭 반응(tail-flick response, TFR)이 관찰되었다.First, rats were placed on the operating table and anesthetized using isoflurane. PLGA nanoparticle solution (p38 inhibitor or p38 siRNA) in the lumbar cavity between the 5th and 5th lumbar (L5) and 6th lumbar (L6) of anesthetized rats using a Hamilton syringe (50 μl; Reno, NV) 20 μl of PLGA nanoparticles were resuspended in PBS at a concentration of 10 mg / ml). As a control, scrambled siRNA and p38 inhibitor SB20303580 were used. A tail-flick response (TFR) was observed when spinal cord injection was complete.
실험예Experimental Example 10. 면역조직화학 10. Immunohistochemistry
래트를 소디움펜토바비탈(100 mg/kg, i.p.)로 마취시킨 후 헤파린화된 PBS(pH 7.4)를 관류(perfuse)시켰다. 그 후, 연동 펌프를 사용하여, 래트에 4% 파라포름알데하이드(Merck, Darmstadt, Wetzlar, Germany)(in PBS)를 20ml/min의 속도로 10분 동안 관류하였다. 관류를 마친 후 래트의 척수(L4-L6) 절편을 즉시 채취하여 고정액에 밤새도록 담근 후, 10 내지 30% 수크로오스 용액에 담갔다. 2일 후 척수 절편을 크라이오스탯(CM1950, Leica Biosystems, Germany)을 이용하여 30μm의 두께로 절단하였으며, 절단된 절편을 저장 버퍼 (30% 글리세롤, 30% 에틸렌글리콜 in PBS)에 담가 4℃로 유지하였다. 이중 면역염색을 위하여, 유지된 절편을 블로킹 버퍼(5% 닭 혈청, 0.3% Triton X-100in PBS)와 실온에서 1시간동안 반응시켰다. 그 후 절편과 1차 항체 (OX42, 1 : 500, BIORAD, # MCA275G, Phospho-p38 MAPK, 1 : 1,000, Cell signaling, # 9211S;NeuN, 1 : 500, Millipore; (1 : 400, Jackson ImmunoResearch, West Grove, PA)를 함께 배양한 후 세척하였다. 절편을 1차 항체와 동일한 버퍼에 희석된 Cy3 또는 FITC (1:400, Jackson ImmunoResearch, West Grove, PA)와 접합된 이차 항체와 함께 배양한 후 세척하여 준비하였다. 준비된 절편을 DAPI(5μg/ml)로 대조 염색하였으며, 마운팅 배지(Biomeda, Foster city, CA)를 포함하는 슬라이드에 장착하였다. 면역 염색된 절편의 이미징은 공초점 현미경(TCS SP8, Leica)을 이용하였다.Rats were anesthetized with sodium pentobarbital (100 mg / kg, i.p.) and then perfused with heparinized PBS (pH 7.4). Then, using a peristaltic pump, rats were perfused with 4% paraformaldehyde (Merck, Darmstadt, Wetzlar, Germany) (in PBS) at a rate of 20 ml / min for 10 minutes. After perfusion, the spinal cord (L4-L6) sections of the rats were immediately collected and soaked overnight in the fixative solution, and then immersed in a 10-30% sucrose solution. After 2 days, spinal cord sections were cut to a thickness of 30 μm using a cryostat (CM1950, Leica Biosystems, Germany), and the cut sections were immersed in a storage buffer (30% glycerol, 30% ethylene glycol in PBS) at 4 ° C. Maintained. For double immunostaining, the retained sections were reacted with blocking buffer (5% chicken serum, 0.3% Triton X-100in PBS) for 1 hour at room temperature. Sections and primary antibodies (OX42, 1: 500, BIORAD, # MCA275G, Phospho-p38 MAPK, 1: 1,000, Cell signaling, # 9211S; NeuN, 1: 500, Millipore; (1: 400, Jackson ImmunoResearch, West Grove, PA) were incubated and washed, and the sections were incubated with a secondary antibody conjugated with Cy3 or FITC (1: 400, Jackson ImmunoResearch, West Grove, PA) diluted in the same buffer as the primary antibody. The prepared sections were counter stained with DAPI (5 μg / ml) and mounted on slides containing mounting media (Biomeda, Foster city, CA.) Imaging stained sections were imaged using a confocal microscope (TCS SP8). , Leica).
실시예Example 1. BV2 세포에서 p38 1. p38 in BV2 cells siRNA의siRNA p38 발현 및 염증관련 유전자 억제 효과 p38 expression and inflammation-related gene suppression effect
p38 siRNA가 포함된 PLGA 나노 입자의 제조 및 신경병증성 동물 모델에의 상기 나노 입자의 적용에 앞서, p38 siRNA가 미세아교세포에서 p38 유전자를 특이적으로 표적할 수 있는지 여부를 검증하였다. Prior to the preparation of PLGA nanoparticles containing p38 siRNA and application of the nanoparticles to neuropathic animal models, it was verified whether p38 siRNA can specifically target the p38 gene in microglia.
1-1. p38 siRNA가 미세아교세포에서 p38 MAPK의 mRNA를 실제로 녹다운(knockdown)시킬 수 있는지 확인하였다. 구체적으로, 상기 실험예 1에서 개시한 바와 같이, 대조군인 스크렘블드 siRNA(20nM) 또는 p38 siRNA(10, 20 및 50nM)를 BV2 세포에 2일 동안 처리한 후 정량적 RT-PCR을 수행하였다. 정량적 RT-PCR 수행 결과는 도 1A에 나타내었다. 1-1. It was confirmed that p38 siRNA can actually knock down p38 MAPK mRNA in microglia. Specifically, as described in Experimental Example 1, the control group was treated with scrambled siRNA (20nM) or p38 siRNA (10, 20 and 50nM) for 2 days in BV2 cells, and then quantitative RT-PCR was performed. The results of quantitative RT-PCR are shown in Figure 1A.
도 1A에 나타낸 바와 같이, p38 siRNA가 형질도입된 실험군의 p38 mRNA 수준은 스크렘블드 siRNA가 형질도입된 대조군보다 약 50% 감소되는 것을 확인하였다.As shown in FIG. 1A, it was confirmed that the p38 mRNA level of the experimental group in which the p38 siRNA was transduced was reduced by about 50% compared to the control in which the scrambled siRNA was transduced.
1-2. 1-1과 동일한 p38 siRNA 또는 스크렘블드 siRNA가 형질도입된 BV2 세포의 p38 단백질의 발현을 웨스턴 블롯팅을 통해 확인하였으며, 그 결과를 도 1B에 나타내었다. 1-2. Expression of p38 protein of BV2 cells transduced with p38 siRNA or scrambled siRNA identical to 1-1 was confirmed by Western blotting, and the results are shown in FIG. 1B.
도 1B에 나타낸 바와 같이, p38 siRNA가 형질도입된 BV2 세포는 스크렘블드 siRNA가 형질도입된 BV2 세포보다 p38 단백질의 발현이 50% 감소되는 것을 확인하였다.As shown in FIG. 1B, it was confirmed that the expression of p38 protein was reduced by 50% in BV2 cells transduced with p38 siRNA than BV2 cells transduced with scrambled siRNA.
1-3. 지질다당류(lipopolysaccharide,LPS) 또는 ATP와 같은 자극에 의해 활성화된 미세아교세포에서 p38 MAPK 신호 전달 계통은 염증 관련 매개체의 전사 개시와 관련이 있다고 알려져 있다. 따라서 p38 siRNA 형질도입에 의한 p38 발현 감소가 활성화된 미세아교세포에서 염증관련인자인 TNF-α, iNOS, Nrf2 및 COX-2를 포함한 이들 유전자의 mRNA 발현을 하향 조절할 수 있는지 확인하였으며, 그 결과를 도 1C에 나타내었다. 1-3. In microglia activated by stimulation such as lipopolysaccharide (LPS) or ATP, the p38 MAPK signaling pathway is known to be involved in the initiation of transcription of inflammatory mediators. Therefore, it was confirmed that the expression of mRNA of these genes including TNF-α, iNOS, Nrf2 and COX-2, which are inflammation-related factors, can be down-regulated in microglia in which p38 expression reduction by p38 siRNA transduction is activated. It is shown in Figure 1C.
도 1C에 나타낸 바와 같이, 스크렘블드 siRNA 및 LPS 처리군에서 염증관련인자의 mRNA 발현은 LPS를 처리하지 않은 스크렘블드 siRNA 처리군보다 3 내지 30배 증가되었다. 그러나 p38 siRNA 및 LPS 처리군은 염증관련인자의 mRNA 발현이 통계 유의적으로 감소하였다.As shown in Fig. 1C, mRNA expression of inflammatory factors in the scrambled siRNA and LPS treated groups was increased by 3 to 30 fold compared to the scrambled siRNA treated group without LPS treatment. However, in the p38 siRNA and LPS treatment groups, the mRNA expression of inflammation-related factors was significantly decreased.
본 실시예에서, p38 siRNA는 미세아교세포에서 p38 MAPK의 mRNA 및 단백질 발현을 억제하며, LPS에 의해 활성화된 미세아교세포에서 염증관련인자의 발현을 억제한다는 것을 확인하였다.In this example, it was confirmed that p38 siRNA suppresses mRNA and protein expression of p38 MAPK in microglia, and suppresses expression of inflammation-related factors in microglia activated by LPS.
실시예Example 2. p38 2. p38 siRNA가siRNA 포함된 included PLGAPLGA 나노입자의 특성 Characteristics of nanoparticles
p38 siRNA를 척수의 미세아교세포에 효율적으로 전달하기 위하여, 효능, 비용, 안전성 및 유전자 전달 시스템 선택의 편의성 등을 고려하였다. 그 결과, PLGA 공중합체를 p38 siRNA 전달 물질로 선정하였다. 상기 PLGA 공중합체는 미국 FDA(Food and Drug Administration)에서 우수한 생분해성 및 생체적합성을 인정받은 물질이다. 구체적으로, 상기 실험예 4에서 개시한 바와 같이, p38 siRNA가 포함된 PLGA 나노 입자는 이중 PLGA 나노입자에 친수성인 p38 siRNA를 캡슐화하기 위해 초음파 처리(sonication)에 의한 에멀젼(W/O/W) 방법을 사용하였다. 제조된 p38 siRNA가 포함된 PLGA 나노입자의 크기 및 제타 전위를 측정하였다. 또한 상기 p38 siRNA가 포함된 PLGA 나노입자의 무결성(integrity) 및 크기를 투과 전자 현미경(TEM) 및 주사 전자 현미경(SEM)을 통해 측정하였다. p38 siRNA가 포함된 PLGA 나노입자의 크기, 제타전위 및 무결성을 측정한 결과는 도 2에 나타내었다. In order to efficiently deliver p38 siRNA to the microglial cells of the spinal cord, efficacy, cost, safety, and convenience of selecting a gene delivery system were considered. As a result, the PLGA copolymer was selected as the p38 siRNA delivery material. The PLGA copolymer is a material recognized for excellent biodegradability and biocompatibility by the US Food and Drug Administration (FDA). Specifically, as disclosed in Experimental Example 4, p38 siRNA-containing PLGA nanoparticles are emulsions (W / O / W) by sonication to encapsulate p38 siRNAs that are hydrophilic to double PLGA nanoparticles. Method was used. The size and zeta potential of the PLGA nanoparticles containing the prepared p38 siRNA were measured. In addition, the integrity and size of the PLGA nanoparticles containing the p38 siRNA were measured by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The results of measuring the size, zeta potential and integrity of the PLGA nanoparticles containing p38 siRNA are shown in FIG. 2.
도 2A 및 B에 나타낸 바와 같이, siRNA가 포함된 PLGA 나노입자의 평균 크기는 153.1±68.08nm이며, 제타전위는 25.0±5.08mV임을 확인하였다. 또한 투과전자현미경(TEM) 및 주사전자현미경(SEM)을 이용하여 siRNA가 포함된 PLGA 나노입자의 무결성 및 캡슐 형태인 것을 확인하였다(도 2C). 2A and B, it was confirmed that the average size of the PLGA nanoparticles containing siRNA was 153.1 ± 68.08nm, and the zeta potential was 25.0 ± 5.08mV. In addition, it was confirmed that the integrity and capsule form of PLGA nanoparticles containing siRNA using a transmission electron microscope (TEM) and a scanning electron microscope (SEM) (FIG. 2C).
실시예Example 3. p38 3. p38 siRNA가siRNA 포함된 included PLGAPLGA 나노입자의 세포 독성 확인 Confirmation of cytotoxicity of nanoparticles
상기 실험예 5에 개시한 바와 같이, p38 siRNA가 포함된 PLGA 나노입자에 대한 세포 독성을 확인하였다. MTT 분석을 통하여, 미세아교세포 BV2 세포에서 p38 siRNA가 포함된 PLGA 나노입자의 세포 독성을 확인하였다. MTT 분석 결과는 도 3A에 나타내었다.As described in Experimental Example 5, cytotoxicity was confirmed for PLGA nanoparticles containing p38 siRNA. Through MTT analysis, cytotoxicity of PLGA nanoparticles containing p38 siRNA in microglia BV2 cells was confirmed. The results of the MTT analysis are shown in Figure 3A.
도 3A에 나타낸 바와 같이, p38 siRNA가 포함된 PLGA 나노입자는 세포 독성이 거의 없는 것을 확인하였다.3A, it was confirmed that the PLGA nanoparticles containing p38 siRNA had little cytotoxicity.
실시예Example 4. p38 4. p38 siRNA가siRNA 포함된 included PLGAPLGA 나노입자의 Nanoparticle 실험관Test tube 내(in vitro) 및 체내(in In vitro and in vivovivo ) 세포 섭취 분석) Cell uptake analysis
4-1. 4-1. 실험관Test tube 내(in vitro) 세포 섭취 분석 In vitro cell uptake assay
척수에 존재하는 BV2(murine microglial cell line), HT22(murine neuronal cell line) 및 U87MG(human astrocytic cell line)의 p38 siRNA가 포함된 PLGA 나노입자의 세포 섭취를 분석하였다. 세포 섭취 분석에는 녹색 형광을 나타내는 쿠마린 6을 함유하는 p38 siRNA가 포함된 PLGA 나노입자를 이용하였으며, 상기 PLGA 나노입자는 실시간으로 세포 섭취를 추적하였다. 구체적인 실험방법은 실험예 6의 개시와 동일하게 수행하였으며, 세포 섭취 분석 결과는 도 3B에 나타내었다.Cell uptake of PLGA nanoparticles containing p38 siRNA from murine microglial cell line (BV2), murine neuronal cell line (HT22) and human astrocytic cell line (U87MG) present in the spinal cord was analyzed. For cell uptake analysis, PLGA nanoparticles containing p38
도 3B에 나타낸 바와 같이, BV2, HT22 및 U87MG 세포 모두 p38 siRNA가 포함된 PLGA 나노입자를 섭취하는 것을 확인하였다. 특히, 미세아교세포인 BV2 세포가 세포 섭취가 활발한 것을 확인하였다.3B, it was confirmed that all BV2, HT22 and U87MG cells ingest PLGA nanoparticles containing p38 siRNA. In particular, it was confirmed that BV2 cells, which are microglial cells, had active cell uptake.
4-2. 체내(in 4-2. In the body (in vivovivo ) 세포 섭취 분석) Cell uptake analysis
래트에서 쿠마린 6을 함유하는 p38 siRNA가 포함된 PLGA 나노입자의 세포 섭취를 분석하였다. 먼저, 래트에 쿠마린 6을 함유하는 p38 siRNA가 포함된 PLGA 나노입자를 척수관 주사하였다. 척수관 주사 2일 후 척수에에 포함된 세포의 타입을 결정하기 위하여, anti-NeuN(neuron marker), anti-GFAP(astrocyte marker) 및 anti-OX42(microglia marker) 항체를 이용하여 면역염색하였으며, 이를 형광 현미경으로 확인하였다. 구체적인 실험방법은 실험예 6의 개시와 동일하게 수행하였으며, 체내 세포 섭취 분석 결과는 도 3C에 나타내었다.Cell uptake of PLGA nanoparticles containing p38
도 3C에 나타낸 바와 같이, 쿠마린 6을 함유하는 p38 siRNA가 포함된 PLGA 나노입자는 래트 척수에서 모든 타입의 세포를 표적하는 것을 확인하였다. 특히, 미세아교세포의 세포 섭취가 2배 이상 활발한 것을 확인하였다.3C, it was confirmed that PLGA nanoparticles containing p38
본 실시예에서, p38 siRNA가 포함된 PLGA 나노입자는 척수의 다양한 타입의 세포에 섭취되며, 특히 미세아교세포에 다량 섭취되는 것을 확인하였다.In this example, it was confirmed that the PLGA nanoparticles containing p38 siRNA are ingested into various types of cells of the spinal cord, and in particular, are ingested in large amounts in microglia.
실시예Example 5. 본 5. Pattern 프레이Frey 필라멘트( filament( vonvon freyfrey filament)를 이용한 통증 행동 시험 Filament)
5-1. p38 siRNA가 포함된 PLGA 나노입자가 신경병증성 통증을 감소시킬 수 있는지 확인하기 위하여, 상기 실험예 7에 개시된 것과 동일한 SNL(L5 spinal nerve ligation) 유도된 신경병증성 통증 동물 모델을 이용하였다. 래트의 SNL 수술은 본 프레이 필라멘트 시험에서 기준선(> 10g)을 통과 한 래트만을 사용하였다. SNL 수술 후 3, 5, 7, 10, 14 일에 기계적 역치를 측정하여 통증 행동을 분석하였으며, 그 결과는 도 4A에 나타내었다. 5-1. In order to confirm that the PLGA nanoparticles containing p38 siRNA can reduce neuropathic pain, the same LNL spinal nerve ligation (SNL) induced neuropathic pain animal model as described in Experimental Example 7 was used. For rat SNL surgery, only rats that passed the baseline (> 10 g) were used in this Frey filament test. Pain behaviors were analyzed by measuring mechanical thresholds at 3, 5, 7, 10, and 14 days after SNL surgery, and the results are shown in FIG. 4A.
도 4A에 나타낸 바와 같이, SNL 수술 시행 3일 후부터 발생된 통증 행동은 3일차에 크게 감소하였으며, 7일차에 가장 낮았다,As shown in Fig. 4A, the pain behavior generated 3 days after the SNL surgery was significantly reduced on the 3rd day and the lowest on the 7th day.
5-2. SNL 수술에 의해 척수의 미세아교세포가 활성화된다는 것이 잘 알려저 있다. 실제로 미세아교세포가 활성화되는지 여부를 확인하기 위하여, 절편 anti-Ibal 항체를 이용하여 미세아교세포를 면역염색하여 활성도를 분석하였으며, 그 결과를 도 4B에 나타내었다. 5-2. It is well known that microglial cells of the spinal cord are activated by SNL surgery. In order to confirm whether the microglial cells are actually activated, the activity was analyzed by immunostaining the microglial cells using a fragment anti-Ibal antibody, and the results are shown in FIG. 4B.
도 4B에 나타낸 바와 같이, 대조군은 척수 등쪽뿔(dorsal horn)의 부근에서 미세 신경총이 활성화되지 않았으나, 실험군은 척수 등쪽뿔의 동측에서 급격히 활성화되는 것을 확인하였다.As shown in FIG. 4B, the control group did not activate the micro plexus in the vicinity of the spinal dorsal horn, but the experimental group confirmed that it was rapidly activated in the ipsilateral side of the spinal dorsal horn.
5-3. p38 siRNA가 포함된 PLGA 나노입자가 통증 행동을 향상시킬 수 있는지 여부를 확인하기 위하여, SNL 수술 7일째인 신경병증성 동물 모델에 해밀턴 주사기를 이용하여 p38 siRNA가 포함된 PLGA 나노입자를 주입한 후, 기계적 역치를 측정하였다. 음성 대조군은 신경병증성 통증 동물 모델에 스크렘블드 siRNA가 포함된 PLGA 나노입자를 주입하였으며, 양성 대조군은 p38 억제제인 SB20303580을 주입하였다. 기계적 역치를 측정한 결과는 도 4C에 나타내었다. 5-3. To confirm whether the PLGA nanoparticles containing p38 siRNA can improve pain behavior, after injecting PLGA nanoparticles containing p38 siRNA into the neuropathic animal model on the 7th day of SNL surgery using a Hamilton syringe , The mechanical threshold was measured. The negative control group was injected with PLGA nanoparticles containing scrambled siRNA into the neuropathic pain animal model, and the positive control group was injected with the p38 inhibitor SB20303580. The result of measuring the mechanical threshold is shown in Figure 4C.
도 4C에 나타낸 바와 같이, 스크렘블드 siRNA가 포함된 PLGA 나노입자를 주입한 대조군은 통증이 14일 째까지 지속되는 것을 확인하였다. 반면에, p38 siRNA가 포함된 PLGA 나노입자를 주입한 실험군은 통증이 감소된 것을 알 수 있으며, 특히 주입 후 5일 째에 통증이 크게 개선되는 것을 확인하였다.As shown in Figure 4C, the control group injected with PLGA nanoparticles containing scrambled siRNA confirmed that the pain persisted until
본 실시예에서, 신경병증성 통증 동물 모델에 p38 siRNA가 포함된 PLGA 나노입자를 척수관 주사함으로써, 상기 동물 모델에서 SNL에 의해 유발된 통증이 개선된다는 것을 확인하였다.In this example, it was confirmed that the pain induced by SNL in the animal model is improved by injecting PLGA nanoparticles containing p38 siRNA into the neuropathic pain animal model by spinal cord injection.
실시예Example 6. p38의 신호전달경로 및 신경병증성 통증에 의한 미세아교세포 활성화 확인 6. Confirmation of microglia activation by p38 signaling pathway and neuropathic pain
6-1. 상기 실험예 10에 나타낸 바와 같이, p38 siRNA가 포함된 PLGA 나노입자가 신경병증성 동물 모델에서 척수의 미세아교세포의 활성화에 관여 여부를 면역조직화학 염색을 통해 확인하였다. 구체적으로, 신경병증성 통증 동물 모델에 p38 siRNA가 포함된 PLGA 나노입자를 척수관 주사하였다. 척수관 주사 3일 후 면역조직화학 분석을 수행하여, 미세아교세포의 활성화 정도를 확인하였다. 대조군은 신경병증성 통증 동물모델에 스크렘블드 siRNA가 포함된 PLGA 나노입자를 척수관 주사한 것을 이용하였다. 미세아교세포 활성화를 확인하기 위한 면역조직화학 분석 결과는 도 5A에 나타내었다. 6-1. As shown in Experimental Example 10, it was confirmed by immunohistochemical staining whether PLGA nanoparticles containing p38 siRNA are involved in the activation of microglial cells of the spinal cord in a neuropathic animal model. Specifically, PLGA nanoparticles containing p38 siRNA were injected into the spinal canal in an animal model of neuropathic pain. Immunohistochemical analysis was performed 3 days after the injection of the spinal canal to confirm the degree of microglia activation. As a control group, a spinal canal injection of PLGA nanoparticles containing scrambled siRNA in an animal model of neuropathic pain was used. The results of immunohistochemistry analysis to confirm microglia activation are shown in Figure 5A.
도 5A에 나타낸 바와 같이, p38 siRNA가 포함된 PLGA 나노입자를 척수관 주사한 실험군은 척수 등쪽뿔의 동측 측면에서 미세아교세포의 수 및 밀도가 현저히 증가되는 것을 확인하였다.As shown in FIG. 5A, the experimental group in which the PLGA nanoparticle containing p38 siRNA was injected into the spinal canal was confirmed to significantly increase the number and density of microglia on the ipsilateral side of the spinal dorsal horn.
6-2. 신경병증성 통증 동물 모델의 척수 미세아교세포에서 p38의 발현 감소로 인한 p38 하류(downstream) 유전자의 발현을 조사하였다. 구체적으로, 신경병증성 통증 동물 모델에 p38 siRNA가 포함된 PLGA 나노입자를 척수관 주사하였다. 척수관 주사 3일째인 신경병증성 통증 동물 모델의 척수 등쪽뿔의 동측면에서 TNF-α, IL-1β, COX-2 및 iNOS 유전자의 mRNA 발현을 정량적 RT-PCR을 통해 분석하였다. 대조군은 신경병증성 동물 모델에 스크렘블드 siRNA가 포함된 PLGA 나노입자를 척수관 주사한 것이다. 정량적 RT-PCR 분석 결과는 도 5B에 나타내었다. 6-2. Expression of the p38 downstream gene due to decreased expression of p38 in spinal microglial cells of the neuropathic pain animal model was investigated. Specifically, PLGA nanoparticles containing p38 siRNA were injected into the spinal canal in an animal model of neuropathic pain. MRNA expression of TNF-α, IL-1β, COX-2 and iNOS genes on the ipsilateral side of the spinal dorsal horn of the neuropathic
도 5B에 나타낸 바와 같이, 실험군은 척수 등쪽뿔의 동측면에서 TNF-α, IL-1β, COX-2 및 iNOS 유전자의 mRNA 발현이 모두 감소한 반면, 대조군은 상기 유전자들의 발현이 크게 증가한 것을 알 수 있다. As shown in Fig. 5B, the experimental group showed that the mRNA expression of the TNF-α, IL-1β, COX-2 and iNOS genes were all decreased on the ipsilateral side of the spinal dorsal horn, whereas the control group showed that the expression of the genes increased significantly. have.
본 실시예에서 p38 siRNA가 포함된 PLGA 나노입자는 신경병증성 통증 동물 모델의 척수 등쪽뿔 동측면에서 p38의 발현과, 상기 p38의 하류 유전자인 TNF-α, IL-1β, COX-2 및 iNOS 유전자의 발현을 감소시킴으로써 신경병증성 통증을 감소시킨다는 것을 확인하였다.In this embodiment, the PLGA nanoparticles containing p38 siRNA are expressed in p38 on the ipsilateral side of the spinal dorsal cone of the neuropathic pain animal model, and the downstream genes of the p38, TNF-α, IL-1β, COX-2 and iNOS. It was confirmed that the neuropathic pain was reduced by reducing the expression of the gene.
실시예Example 7. 신경병증성 통증 동물 모델에서 p38 7. p38 in a neuropathic pain animal model siRNA가siRNA 포함된 included PLGAPLGA 나노입자의 p38 인산화 확인 Confirmation of p38 phosphorylation of nanoparticles
7-1. 신경병증성 통증 동물 모델의 척수에서 p38의 인산화를 확인하였다. 구체먹으로 SNL 수술 7일째인 신경병증성 통증 동물 모델로부터 척수 조직을 수득하였다. 수득한 척수 조직을 anti-p-p38- 및 anti-OX42-(microglia marker), GFAP(astrocytic marker), NeuN(neuronal marker) 항체를 이용하여 면역 염색하여 척수 조직에서 p38의 인산화를 분석하였다. 척수 조직에서 p38의 인산화를 분석한 결과는 도 6A에 나타내었다. 7-1. Phosphorylation of p38 was confirmed in the spinal cord of the neuropathic pain animal model. Spinal cord tissue was obtained from a neuropathic pain animal model on the 7th day of SNL surgery with a spherical feed. The obtained spinal cord tissue was immunostained using anti-p-p38- and anti-OX42- (microglia marker), GFAP (astrocytic marker), and NeuNal (neuronal marker) antibodies to analyze p38 phosphorylation in spinal cord tissue. The results of analyzing phosphorylation of p38 in spinal cord tissue are shown in FIG. 6A.
도 6A에 나타낸 바와 같이, p-p38 및 OX-42 항체의 이중양성 신호는 척수 등쪽뿔의 미세아교세포에서 다량 검출되었다.As shown in Fig. 6A, the bi-positive signal of the p-p38 and OX-42 antibodies was detected in large amounts in the microglial cells of the spinal dorsal horn.
7-2. 통증 행동의 감소가 신경병증성 통증 동물 모델의 미세아교세포에서 p38의 인산화 감소에 미치는 영향을 조사하였다. 구체적으로, 신경병증성 통증 동물 모델에 p38 siRNA가 포함된 PLGA 나노입자를 척수관 주사하였으며, 척수조직을 채취하였다. 상기 p38 siRNA가 포함하는 척수 조직을 p-p38 및 OX-42 항체로 동시에 면역 염색하였다. 대조군은 신경병증성 통증 동물 모델에 스크렘블드 siRNA를 포함하는 PLGA 나노입자를 척수관 주사한 것을 이용하였다. p38의 인산화 감소에 미치는 영향을 조사 결과는 도 6B에 나타내었다. 7-2. The effect of decreased pain behavior on the reduction of phosphorylation of p38 in microglia in a neuropathic pain animal model was investigated. Specifically, PLGA nanoparticles containing p38 siRNA were injected into the spinal canal and the spinal cord tissue was collected in a neuropathic pain animal model. Spinal cord tissue contained in the p38 siRNA was immunostained simultaneously with p-p38 and OX-42 antibodies. As a control group, a spinal canal injection of PLGA nanoparticles containing scrambled siRNA was used in an animal model of neuropathic pain. The effect of p38 on phosphorylation reduction is shown in Figure 6B.
도 6B에 나타낸 바와 같이, p38 siRNA가 포함된 PLGA 나노입자를 주사한 실험군이 대조군보다 p-p38 및 OX-42 항체의 이중양성 신호가 적게 검출된 것을 알 수 있다.As shown in Figure 6B, it can be seen that the experimental group injected with PLGA nanoparticles containing p38 siRNA detected less bi-positive signals of p-p38 and OX-42 antibodies than the control group.
본 실시예에서, p38 siRNA 나노 입자가 투여된 신경병증성 통증 동물에서 통증 행동의 완화는 미세아교세포에서 p38의 인산화 감소와, p38 관련 신호 전달 경로 및 유전자 발현의 하향 조절(또는 차단)에 의한 것임을 의미한다.In this embodiment, the relief of pain behavior in neuropathic pain animals administered with p38 siRNA nanoparticles is due to reduced phosphorylation of p38 in microglia and down regulation (or blocking) of p38-related signaling pathways and gene expression. It means
종합적으로 본 발명자들은 p38 siRNA를 포함하는 PLGA 나노입자를 개발하고, 상기 p38 siRNA를 포함하는 PLGA 나노입자가 생체적합성 및 생분해성이 우수하며, 세포 및 동물 수준에서 신경병증성 통증을 감소시킨다는 것을 확인하였다. 이는 본 발명의 p38 siRNA를 포함하는 PLGA 나노입자가 신경병증성 통증의 치료제로서 이용될 수 있음을 의미하는 바, 신경병증성 통증의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.Overall, the present inventors have developed PLGA nanoparticles containing p38 siRNA, confirming that the PLGA nanoparticles containing p38 siRNA have excellent biocompatibility and biodegradability, and reduce neuropathic pain at the cellular and animal level. Did. This means that the PLGA nanoparticles comprising the p38 siRNA of the present invention can be used as a therapeutic agent for neuropathic pain, and can be used in various fields in the prevention, improvement and treatment of neuropathic pain.
<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Composition for preventing or treating neuropathic pain comprising nanoparticle containing an agent for inhibiting expression of p38 or a gene encoding the same <130> 1-61P <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> p38 siRNA <400> 1 cccagatgcc gaagatgaa 19 <110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Composition for preventing or treating neuropathic pain comprising nanoparticle containing an agent for inhibiting expression of p38 or a gene encoding the same <130> 1-61P <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> p38 siRNA <400> 1 cccagatgcc gaagatgaa 19
Claims (9)
A pharmaceutical composition for preventing or treating neuropathic pain, comprising poly (lactic-co-glycolic acid) nanoparticles containing a small interference RNA (siRNA) that suppresses the expression of a gene encoding p38.
상기 siRNA는 서열번호 1의 염기서열로 표시되는, 신경병증성 통증 예방 또는 치료용 약학적 조성물.
According to claim 1,
The siRNA is represented by the nucleotide sequence of SEQ ID NO: 1, pharmaceutical composition for preventing or treating neuropathic pain.
상기 조성물은 나노입자를 0.1 내지 1000mg/ml의 농도로 포함하는, 신경병증성 통증 예방 또는 치료용 약학적 조성물.
According to claim 1,
The composition comprises a nanoparticle in a concentration of 0.1 to 1000mg / ml, a pharmaceutical composition for preventing or treating neuropathic pain.
상기 나노입자는 미세아교세포(microglia cell), 뉴런 세포(neuronal cell) 및 성상세포(astrocytic cell)로 이루어진 군에서 선택되는 1종 이상을 타겟하는 것인, 신경병증성 통증 예방 또는 치료용 약학적 조성물.
According to claim 1,
The nanoparticles target one or more selected from the group consisting of microglia cells, neuronal cells and astrocytic cells, pharmaceutical for preventing or treating neuropathic pain Composition.
A food composition for preventing or improving neuropathic pain, comprising poly (lactic-co-glycolic acid) nanoparticles containing small interference RNA (siRNA) that inhibits the expression of the gene encoding p38.
A method of treating neuropathic pain comprising; injecting a composition according to any one of claims 1, 3, 5 and 6 into an individual other than a human.
상기 주입은 척수에 주입되는 것을 특징으로 하는, 신경병증성 통증 치료방법.The method of claim 8,
The injection is characterized in that the injection into the spinal cord, neuropathic pain treatment method.
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| US20120015037A1 (en) * | 2010-07-19 | 2012-01-19 | National Cheng Kuang University | Functionalized nanoparticle, method for preparing the same and application thereof |
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| KR101576606B1 (en) | 2012-12-21 | 2015-12-10 | 경북대학교 산학협력단 | composition comprising lipocalin 2 inhibitor for alleviating neuropathic pain |
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| US20120015037A1 (en) * | 2010-07-19 | 2012-01-19 | National Cheng Kuang University | Functionalized nanoparticle, method for preparing the same and application thereof |
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| Title |
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| Jin, S. 등, The Journal of Neuroscience, 2003년, 23권, 10호, 페이지 4017-4022 (2003.05.15. 공개)* |
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