KR20200113511A - Composition for prevention, improving or treatment of chronic pain comprising PAT4 - Google Patents
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- KR20200113511A KR20200113511A KR1020190033788A KR20190033788A KR20200113511A KR 20200113511 A KR20200113511 A KR 20200113511A KR 1020190033788 A KR1020190033788 A KR 1020190033788A KR 20190033788 A KR20190033788 A KR 20190033788A KR 20200113511 A KR20200113511 A KR 20200113511A
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Abstract
Description
본 발명은 PAT4를 포함하는 만성통증 질환의 예방, 개선 또는 치료용 조성물에 관한 것으로서, 보다 상세하게는 신경병증성 통증의 예방, 개선 또는 치료를 위한 식품 또는 약학적 조성물에 관한 것이다.The present invention relates to a composition for preventing, ameliorating or treating chronic pain diseases including PAT4, and more particularly, to a food or pharmaceutical composition for preventing, improving or treating neuropathic pain.
통증은 부상이나 수술 후의 수백만의 사람들 및 관절염, 암 및 당뇨병과 같은 질환을 겪는 사람들에게 영향을 미친다. 통각수용(Nociception)(유해 자극 또는 위해 자극의 검출)은 중대한 생물학적 목적을 제공한다. 통증은 발생부위, 원인, 성질 및 발생기전 등을 고려하여 구분할 수 있다. 예를 들면, 통각 수용성 통증은 병태 생리학 기전에 의한 분류로서 조직 손상에 따라 발생하고, 유해한 자극에 비례하여 통증이 증가하는 경향을 보인다.Pain affects millions of people after injury or surgery, and people with diseases such as arthritis, cancer and diabetes. Nociception (detection of harmful or harmful stimuli) serves a significant biological purpose. Pain can be classified in consideration of the location, cause, nature, and mechanism of occurrence. For example, nociceptive pain is classified according to pathophysiological mechanisms and occurs according to tissue damage, and the pain tends to increase in proportion to harmful stimulation.
통증은 그 지속기간에 따라서는 급성과 만성통증으로 나눌 수 있다. 대표적인 만성통증 질환으로는 신경병증성 통증, 복합부위 통증 증후군, 대상포진 후 신경통, 척추 수술 후 통증 증후군 등이 있다. 만성통증은 단순히 급성통증의 연장선이 아니며, 통증의 정도는 원래의 손상 정도와 비례하지 않고 자극이 없이도 자발적으로 일어날 수 있다. 통증의 기간과 강도가 환자의 기능이나 생활에 유해한 효과를 일으키거나 또는 정상적인 조직 치유기간, 보통 3개월을 넘어서서 지속되는 경우는 만성통증으로 정의하며 증상이 아닌 하나의 질병으로 간주한다. 만성통증은 무자비하고 자기-제어적이지 않으며 초기 부상 후 수년 및 수십 년 까지도 지속될 수 있다. 만성통증은 성질에 있어서 주로 신경병성이며 말초 또는 중추신경계 손상을 포함할 수 있다.Pain can be divided into acute and chronic pain depending on its duration. Representative chronic pain diseases include neuropathic pain, complex site pain syndrome, neuralgia after shingles, and pain syndrome after spinal surgery. Chronic pain is not simply an extension of acute pain, and the degree of pain is not proportional to the original degree of damage and can occur spontaneously without stimulation. When the duration and intensity of pain causes a harmful effect on the patient's function or life, or continues beyond the normal tissue healing period, usually 3 months, it is defined as chronic pain and is regarded as a disease, not a symptom. Chronic pain is ruthless, not self-controlling, and can persist for years and even decades after the initial injury. Chronic pain is primarily neuropathic in nature and may include peripheral or central nervous system damage.
다양한 통증의 종류 중, 외견상으로는 상처나 동통의 원인이 없는데도 발생하는 통증이 바로 신경성 동통, 신경병증성 통증(neuropathic pain)이다. 신경병증성 통증은 암, 자가면역 질환 또는 대상포진 감염에 의한 신경 압박(nerve compression), 신경 외상(nerve trauma)과 같은 신경세포의 손상이나 신경계의 이상으로 생기는 통증을 총칭하며, 이는 정상적인 상태에서는 통증을 유발하지 않는 자극에도 통증반응을 보이는 이질통(allodynia)과 통증자극에 대해 더욱 민감한 반응을 보이는 통각과민(hyperalgesia), 또한, 자극이 없는 상태에서도 화끈거리거나 타는듯한 감각(spontaneous burning sensation)을 느끼는 등의 임상적 특성을 보이는 만성통증이다. 이와 같은 신경병증성 통증은 이로움을 주는 급성 통증과는 달리 통증을 전달하는 신경계의 변형으로 인해 질병 이상의 통증을 가져오게 된다.Among the various types of pain, neuropathic pain and neuropathic pain are pain that occurs even though there is no apparent cause of injury or pain. Neuropathic pain refers to pain caused by damage to nerve cells or abnormalities in the nervous system such as nerve compression and nerve trauma caused by cancer, autoimmune disease or shingles infection. Allodynia and hyperalgesia, which exhibits a more sensitive response to pain stimuli, and a spontaneous burning sensation, even in the absence of stimulation. It is a chronic pain with clinical characteristics such as feeling. Unlike acute pain, which is beneficial, neuropathic pain like this causes more than a disease due to deformation of the nervous system that transmits pain.
전통적으로 이러한 신경병증성 통증은 신경세포의 변성에 의해 야기되는 감각뉴런(sensory neuron)의 과흥분성(hyperexcitability)에 기인한다고 생각되어져 왔으며, 이러한 감각뉴런의 과흥분성의 원인으로는, 1) 신경세포 손상에 의한 세포주변 환경의 항상성 (extraneuronal homeostasis)의 변화, 2) 신경세포 손상에 따른 감각뉴런 세포막 이온통로 발현 및 이온통로 조절 단백질 활성의 변화, 3) 중추 시냅스 재조정 (synaptic reorganization)에 의한 새로운 통증전달 회로의 형성, 4) 억제성 인터뉴런의 사멸에 의한 감각신경세포의 과흥분과 같은 몇 가지 원인들이 가설로 제기되었다.Traditionally, this neuropathic pain has been thought to be due to the hyperexcitability of sensory neurons caused by the degeneration of neurons, and the cause of the hyperexcitability of these sensory neurons is 1) neurons. Changes in extraneuronal homeostasis due to damage, 2) changes in sensory neuron cell membrane ion channel expression and ion channel regulation protein activity due to nerve cell damage, 3) new pain due to synaptic reorganization Several causes have been hypothesized, such as the formation of transmission circuits, and 4) hyperexcitement of sensory neurons by the death of inhibitory interneurons.
현재 신경병증성 통증에는 신경흥분성이나 감각축삭전도성을 감소시키는 물질을 투여하는데, 항간질약, 국소리도카인, 진통제, 비스테로이드 소염 진통제, 오피아드 등 다양한 약물을 사용하고 있다. 그러나 여전히 신경병증성 통증을 효과적으로 치료할 수 있는 물질에 대한 연구가 많이 이루어지지 않았으며, 약에 따른 효능과 부작용 차이로 인하여 신경병증성 통증을 치료하기 위한 약물의 선택이 어려워, 새로운 신경병증성 통증 치료제 개발에 대한 필요성이 있다.Currently, substances that reduce nerve excitability or sensory axonal conductivity are administered to neuropathic pain, and various drugs such as antiepileptic drugs, topical lidocaine, analgesics, non-steroidal anti-inflammatory drugs, and opiad are used. However, there are still not many studies on substances that can effectively treat neuropathic pain, and it is difficult to select a drug to treat neuropathic pain due to the difference in efficacy and side effects depending on the drug. There is a need for the development of therapeutic agents.
이에 본 발명자들은 부작용이 적고 지속시간이 긴 만성통증 치료제에 대해 연구하던 중 PAT4가 만성통증 동물 모델에서 기계적 역치를 증가 및 전염증 매개인자의 발현 억제를 통해 만성통증을 감소시킨다는 것을 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors of the present invention confirmed that PAT4 decreases chronic pain by increasing the mechanical threshold and suppressing the expression of pro-inflammatory mediators in an animal model of chronic pain while researching a therapeutic agent for chronic pain with a low side effect and a long duration. Was completed.
따라서 본 발명의 목적은, 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방, 개선 또는 치료용 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating chronic pain diseases comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명의 다른 목적은, 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는 만성통증 질환의 치료방법을 제공하는 것이다.Another object of the present invention is of a chronic pain disease comprising administering a pharmaceutical composition for preventing or treating chronic pain disease comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient It is to provide a treatment method.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating chronic pain diseases comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
또한 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving chronic pain diseases comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
또한 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는 만성통증 질환의 치료방법을 제공한다.In addition, the present invention provides a method for treating chronic pain diseases comprising administering a pharmaceutical composition for preventing or treating chronic pain diseases comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient. to provide.
본 발명에 따른 PAT4는 미세아교세포를 타겟하여 전염증 매개인자의 발현을 억제, 활성산소종 생성 억제, 미세아교세포의 활성을 억제 및 기계적 역치를 증가시키는 효과를 가지고 있고, 그 효과가 종래 만성통증 치료제보다 오랫동안 지속되는바, PAT4는 만성통증의 치료제로서 만성통증의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.PAT4 according to the present invention has the effect of inhibiting the expression of pro-inflammatory mediators by targeting microglia, inhibiting the production of reactive oxygen species, inhibiting the activity of microglia, and increasing the mechanical threshold. Since it lasts longer than a pain treatment, PAT4 is a treatment for chronic pain and can be used in various ways in the field of prevention, improvement, and treatment of chronic pain.
도 1은 마우스 미세아교세포주에서 본 발명에 따른 PAT4가 전염증 매개인자의 발현을 억제하는 효과를 확인한 결과를 나타내는 도이다.
도 2는 신경병증성 통증 동물 모델에서 본 발명에 따른 PAT4가 타겟하는 세포를 확인한 결과를 나타내는 도이다.
도 3은 신경병증성 통증 동물 모델에서 본 발명에 따른 PAT4의 진통 감소 및 미세아교세포 활성 억제 효과를 확인한 결과를 나타내는 도이다.
도 4 및 5는 신경병증성 동물 모델에서 본 발명에 따른 PAT4의 신경병증성 통증 감소 효과를 확인한 결과를 나타내는 도이다.
도 6은 무릎 관절염 통증 모델에서 PAT4의 통증 감소 효과를 확인한 결과를 나타낸 도이다.1 is a diagram showing the results of confirming the effect of inhibiting the expression of pro-inflammatory mediators of PAT4 according to the present invention in a mouse microglia cell line.
2 is a diagram showing the results of confirming the cells targeted by PAT4 according to the present invention in an animal model of neuropathic pain.
3 is a diagram showing the results of confirming the analgesic reduction and microglia activity inhibition effect of PAT4 according to the present invention in an animal model of neuropathic pain.
4 and 5 are diagrams showing the results of confirming the neuropathic pain reduction effect of PAT4 according to the present invention in a neuropathic animal model.
6 is a diagram showing the results of confirming the pain reduction effect of PAT4 in the knee arthritis pain model.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방, 개선 또는 치료용 조성물을 제공한다.According to an aspect of the present invention, the present invention provides a composition for preventing, improving or treating chronic pain diseases comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명에 따른 PAT4는 전염증 매개인자의 발현을 억제, 활성산소종 생성 억제 및 기계적 역치를 증가시킴으로써 만성통증을 감소시키는 효과가 있다. 또한 종래 만성통증의 치료제로 알려져 있는 가바펜틴(gabapentin)의 약효 지속시간이 약 1일인 반면, 본 발명에 따른 PAT4는 진통 감소 효과의 지속시간이 21일 이상이다.PAT4 according to the present invention has the effect of reducing chronic pain by inhibiting the expression of pro-inflammatory mediators, inhibiting the production of reactive oxygen species, and increasing a mechanical threshold. In addition, the duration of the drug gabapentin (gabapentin), which is known as a treatment for chronic pain, has a duration of about 1 day, whereas the PAT4 according to the present invention has a duration of the analgesic reduction effect of 21 days or more.
본 발명의 구체예에서, 상기 PAT4는 이의 기능적 동등물을 포함한다. In an embodiment of the present invention, the PAT4 includes a functional equivalent thereof.
상기 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 적어도 80% 이상의, 바람직하게는 90%, 더욱 바람직하게는 95%이상의 서열 상동성(즉, 동일성)을 갖는 것으로 예를 들면, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%의 서열 상동성을 갖는 것을 포함하며, 서열번호 1의 PAT4와 실질적으로 동질의 생리활성을 나타내는 펩타이드를 말한다. 또한, 본 발명의 PAT4에는 이의 천연형 아미노산 서열을 갖는 단백질뿐만 아니라 이의 아미노산 서열 변이체가 또한 본 발명의 범위에 포함된다. PAT4의 변이체란 PAT4의 천연 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 단백질을 의미한다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 상기 PAT4 또는 이의 변이체는 천연에서 추출하거나 합성(Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963) 또는 DNA 서열을 기본으로 하는 유전자 재조합 방법에 의해 제조될 수 있다(Sambrook et al, Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, 2판, 1989).The "functional equivalent" refers to at least 80% or more, preferably 90%, more preferably 95% or more of sequence homology with the amino acid sequence represented by SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids (ie , Identity), for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 It includes those having sequence homology of %, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, and refers to a peptide that exhibits substantially the same physiological activity as PAT4 of SEQ ID NO: 1. In addition, in PAT4 of the present invention, not only proteins having its native amino acid sequence but also amino acid sequence variants thereof are also included in the scope of the present invention. A variant of PAT4 refers to a protein having a sequence different from the natural amino acid sequence of PAT4 and one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not totally alter the activity of the molecule are known in the art. The PAT4 or a variant thereof may be extracted from nature or synthesized (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963) or prepared by a method of genetic recombination based on a DNA sequence (Sambrook et al. , Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).
또한 상기 서열 상동성은 단백질을 구성하는 아미노산 서열의 유사한 부분을 비교하기 위해 사용되는 일반적인 표준 방법에 의해 결정할 수 있다. BLAST 또는 FASTA와 같은 컴퓨터 프로그램은 두 개 이상의 단백질을 각각 구성하는 아미노산이 최적으로 매칭(matching) 되도록 정렬한다(하나 또는 두 서열의 전장서열을 따라 또는 하나 또는 두 서열의 예측된 부분을 따라). 상기 프로그램은 디펄트 오프닝 패널티(default opening penalty) 및 디펄트 갭 페널티(default gap penalty)를 제공하며 컴퓨터 프로그램과 함께 연계되어 사용될 수 있는 PAM250(표준 스코링 매트릭스; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp. 3, 1978)와 같은 스코링 매트릭스를 제공한다. 예를 들어, 백분율로 표시되는 서열 상동성은 다음과 같이 계산할 수 있다: 일치하는 서열(indentical matches)의 총 수에 100을 곱한 다음 대응되는 스팬(machted span) 내의 보다 긴 서열의 길이와두 서열을 정렬하기 위해 보다 긴 서열내로 도입된 갭(gaps)의 수의 합으로 나눈다.In addition, the sequence homology can be determined by a general standard method used to compare similar portions of amino acid sequences constituting proteins. Computer programs such as BLAST or FASTA align the amino acids that make up two or more proteins, respectively, so that they are optimally matched (along the full length of one or two sequences, or along the predicted portions of one or two sequences). The program provides a default opening penalty and a default gap penalty, PAM250 (standard scoring matrix; Dayhoff et al., in Atlas of Protein, which can be used in conjunction with a computer program). Sequence and Structure,
상기에서 "실질적으로 동질의 생리활성"이란 항염증 활성과, 만성통증의 개선, 예방 또는 치료 활성을 의미한다. In the above, "substantially homogeneous physiological activity" means anti-inflammatory activity and an improvement, prevention or treatment activity of chronic pain.
또한 상기 기능적 동등물의 범위에는 서열번호 1의 아미노산 서열로 표시되는 PAT4의 기본골격; 및 항염증, 만성통증의 개선, 예방 또는 치료 활성을 유지하면서 구성하는 아미노산의 일부 화학 구조가 변형된 유도체;가 포함된다. 예를 들어 단백질의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경이 이에 포함된다.In addition, the functional equivalent range includes the basic skeleton of PAT4 represented by the amino acid sequence of SEQ ID NO: 1; And derivatives in which some chemical structures of amino acids constituting while maintaining anti-inflammatory, chronic pain improvement, prevention or treatment activity are modified. This includes structural changes to alter the stability, storage, volatility or solubility of proteins, for example.
본 발명에 있어서, “만성통증 질환”은 몸의 한 곳 또는 여러 곳에 영향을 주고 3개월 이상 지속되는 다소 심한 고통을 의미하며, 시간이 지남에 따라 그 강도가 달라질 수 있는 통증 증후군을 포함한다. In the present invention, "chronic pain disease" refers to a rather severe pain that affects one or several parts of the body and lasts for 3 months or more, and includes a pain syndrome whose intensity may vary over time.
본 발명의 구체예에서, 상기 만성통증 질환은 신경병증성 통증, 관절염에 의한 만성통증, 복합부위 통증 증후군, 대상포진 후 신경통 및 척추 수술 후 통증 증후군으로 이루어진 군에서 선택되는 1종 이상의 질환인 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the chronic pain disease is one or more diseases selected from the group consisting of neuropathic pain, chronic pain due to arthritis, complex site pain syndrome, neuralgia after shingles, and pain syndrome after spinal surgery. Although preferred, it is not limited thereto.
본 발명에 있어서, “신경병증성 통증(neuropathic pain)”은 외상(trauma)이나 염증, 허혈성 손상, 또는 대사산물 등 다양한 원인에 의해 신경계가 손상될 때 유발되는 만성적 신경질환을 의미한다. In the present invention, "neuropathic pain" refers to a chronic neurological disease caused when the nervous system is damaged by various causes, such as trauma, inflammation, ischemic damage, or metabolites.
본 발명의 구체예에서, 상기 신경병증성 통증은 말초 신경 손상 또는 중추 신경 손상에 의해 유도된 것이 바람직하다. 또한 상기 신경병증성 통증은 말초신경계 이상 또는 손상, 척수신경 손상을 포함한 다발성경화증, 척수부상, 통각과민, 감각과민, 신경병증, 당뇨성 신경장애, 신경염, 신경통, 작열통, 이질통, 포진후신경통, 요추 신경근압박, 암과 관련된 통증, 알코올 중독, 비정형 안면통증, 포진성 신경통, 중풍후통증, HIV 연관 신경병증, 골관절염 및 환상사지통증으로 이루어진 군에서 선택되는 1종 이상의 질환을 포함하는 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the neuropathic pain is preferably induced by peripheral nerve injury or central nerve injury. In addition, the neuropathic pain may include abnormalities or damage to the peripheral nervous system, multiple sclerosis including spinal nerve damage, spinal cord injury, hyperalgesia, hypersensitivity, neuropathy, diabetic neuropathy, neuritis, neuralgia, burning pain, allodynia, postherpetic neuralgia, It is preferable to include at least one disease selected from the group consisting of lumbar nerve muscle compression, cancer-related pain, alcoholism, atypical facial pain, herpes neuralgia, post-stroke pain, HIV-related neuropathy, osteoarthritis, and phantom limb pain. , Is not limited thereto.
본 발명의 구체예에서, PAT4는 미세아교세포(microglia cell), 뉴런 세포(neuronal cell) 및 성상세포(astrocytic cell)로 이루어진 군에서 선택되는 1종 이상을 타겟하는 것이 바람직하며, 보다 바람직하게는 미세아교세포를 타겟하나, 이에 제한되지 않는다.In an embodiment of the present invention, PAT4 is preferably targeting at least one selected from the group consisting of microglia cells, neuronal cells, and astrocytic cells, and more preferably It targets microglia, but is not limited thereto.
본 발명에 있어서, “예방”은 본 발명의 조성물에 의해 병인을 제거하거나 조기 발견하여 해당질환을 막는 모든 행위를 의미한다.In the present invention, "prevention" refers to any action that prevents the disease by removing the etiology or early detection 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 any action 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 metabolism of a living body, a function of a cell or an organ is advantageously changed by the composition of the present invention.
본 발명의 조성물은 상기 PAT4와 함께 만성통증의 예방, 개선 도는 치료 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 an effect of preventing, improving or treating chronic pain together with the PAT4.
본 발명의 구체예에서, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 예방 또는 치료용 약학적 조성물을 제공한다.In an embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating chronic pain, comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
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 contain one or more pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and at least one of these ingredients, and if necessary, antioxidants and buffers. , Other conventional additives such as bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, and pills, capsules, granules, or tablets. Furthermore, it can be formulated according to each disease or component by an appropriate method 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 ingredients. Examples of such additional ingredients are lactose, talc, starch, magnesium stearate, crystalline cellulose, lactose, gelatin, mannitol, peroxic 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, preferably parenterally for rapid effect, and more preferably injected into the spinal canal. For parenteral administration, intravenous injection, intramuscular injection, intra-articular injection, intra-synovial injection, intrathecal injection, intrahepatic injection, intralesional injection, or It can be administered by intracranial injection. The appropriate dosage of the pharmaceutical composition may be prescribed in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity of the patient. have.
본 발명의 구체예에서, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 예방 또는 개선용 식품 조성물을 제공한다.In an embodiment of the present invention, the present invention provides a food composition for preventing or improving chronic pain, including PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명의 식품 조성물은 건강기능식품, 식품 첨가제 또는 식이보조제일 수 있다. 본 발명의 조성물이 식품 첨가제인 경우, PAT4를 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, a food additive or a dietary supplement. When the composition of the present invention is a food additive, PAT4 may be added as it is, or may be appropriately used according to a conventional method, such as being used by mixing with other foods or food ingredients.
구체적인 예로, 식품 또는 음료의 제조 시에는 본 발명의 PAT4는 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 PAT4를 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 manufacture of food or beverage, PAT4 of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term consumption for health and hygiene purposes or for health control purposes, it may be added in an amount below the above range, and there is no problem in terms of safety, so the active ingredient can be used in an amount above the above range. have. There is no particular limitation on the type of food, but examples of foods to which PAT4 of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream. There are dairy products, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc., and all health foods in the usual sense are included.
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 additional ingredients such as various flavoring agents or natural carbohydrates, like 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 may be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on 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 adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid. It may include a carbonation agent used in beverages, and may include flesh for the production of natural fruit juice, fruit juice beverage, and vegetable beverage, but is not limited thereto. These components may be used independently or in combination. The ratio of the additives is not largely limited, but is preferably included in the range of 0.01 to 0.1% by weight based on 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 the purpose of health and hygiene or for the purpose of health control, 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 다른 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 PAT4를 유효성분으로 포함하는 만성통증 질환의 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는 만성통증 질환의 치료방법을 제공한다.According to another aspect of the present invention, the present invention comprises the step of administering a pharmaceutical composition for preventing or treating chronic pain diseases comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient to an individual other than humans. Provides a method of treating chronic pain diseases.
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 만성통증 치료방법은 PAT4가 미세아교세포를 타겟하여 전염증 매개인자의 발현을 억제, 활성산소종 생성 억제, 미세아교세포의 활성을 억제 및 기계적 역치를 증가시킴으로써, 만성통증을 치료할 수 있다.In the method for treating chronic pain according to the present invention, PAT4 targets microglia to inhibit the expression of pro-inflammatory mediators, inhibit the production of reactive oxygen species, inhibit the activity of microglia, and increase the mechanical threshold, thereby treating chronic pain. I can.
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 for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.
실험예 1. 신경병증성 통증 동물 모델 제조Experimental Example 1. Neuropathic Pain Animal Model Preparation
만성통증 질환으로는 신경병증성 통증, 복합부위 통증 증후군, 대상포진 후 신경통, 척추 수술 후 통증 증후군 등이 있다. 만성통증의 일종인 신경병증성 통증에 대한 동물 모델을 제조하였으며, 이를 이용하여 만성통증 치료제의 활성을 검증하였다.Chronic pain diseases include neuropathic pain, complex site pain syndrome, neuralgia after shingles, and pain syndrome after spinal surgery. An animal model for neuropathic pain, which is a type of chronic pain, was prepared, and the activity of a chronic pain treatment was verified using this.
1-1. 동물 실험1-1. Animal testing
신경병증성 통증 동물 모델을 제조하기 위하여, 대한 바이오링크(DBL, 충북)에서 Sprague-Dawley 래트(6 wks males, 150-200 g)를 구입하여 1주일 동안 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-A019-P0001)의 승인을 받았으며, 식약처의 통증 연구의 윤리 가이드라인에 따라 수행되었다.In order to prepare an animal model of neuropathic pain, Sprague-Dawley rats (6 wks males, 150-200 g) were purchased from Daehan Biolink (DBL, Chungbuk) and adapted to a new environment for one week. All animals were reared at a rate of 3 per cage in a 12-hour light/dark cycle, and were reared 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-A019-P0001), and was performed according to the ethical guidelines for pain research of the Ministry of Food and Drug Safety.
1-2. L5 SNL(L5 spinal nerve ligation) 유도된 신경병증성 통증 동물 모델 제조1-2. L5 SNL (L5 spinal nerve ligation)-induced neuropathic pain animal model manufacturing
실험예 1-1의 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. 제조된 신경병증성 통증 동물 모델은 기계적 역치가 낮아 외부 자극이 없이도 기계적 이질통(Mechanical allodynia)이 지속적으로 발생한다.Sprague-Dawley rats of Experimental Example 1-1 were anesthetized with isoflurane (2% in oxygen mixture; Hana pharm, Kyung-gi, Republic of Korea), and left to visually check the L4 and L5 spinal nerves. The 6th transverse process (left lumbar(L) 6 transverse process) was removed. Then, the left L5 spinal nerve was separated and tied tightly with 3-0 silk thread. After complete hemostasis, the wound was closed. The manufactured neuropathic pain animal model has a low mechanical threshold, resulting in continuous mechanical allodynia without external stimulation.
대조군은 실험군과 동일한 방법으로 수술하되, SNL을 유도하지 않은 것이다.The control group operated in the same manner as the experimental group, but did not induce SNL.
실험예 2. 본 프레이 필라멘트(von frey filament)를 이용한 통증 행동 시험Experimental Example 2. Pain behavior test using von frey 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 behavioral 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 observed on
실험예 3. 척수관 주사(Intrathecal injection)Experimental Example 3. Intrathecal 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 injection was performed by JL Hylden and GL Wilcox, Intrathecal morphine in mice: a new technique . Eur J Pharmacol . 1980;67:313-6.
구체적으로, 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 농도가 20nmol인 PAT4를 20μl만큼 주사하였으며, 대조군은 PBS를 동량 주입하였다. 척수관 주사가 완료되었을 시 꼬리-플릭 반응(tail-flick response, TFR)이 관찰되었다.Specifically, the rat was laid on an operating table and anesthetized using isoflurane. Using a Hamilton syringe (50 μl; Reno, NV), 20 μl of PAT4 with a concentration of 20 nm was injected into the lumbar cavity between the 5th lumbar spine (L5) and the 6th lumbar spine (L6) of anesthetized rats, and the control group was The same amount of PBS was injected. When the spinal canal injection was completed, a tail-flick response (TFR) was observed.
또한, PAT4가 표적한 세포의 유형을 확인하기 위해 FITC(fluorescein isothiocyanate)가 접합된 TAP2(Peptron, Daejeon, Korea)를 사용하였다.In addition, FITC (fluorescein isothiocyanate) conjugated TAP2 (Peptron, Daejeon, Korea) was used to confirm the type of cells targeted by PAT4.
실험예 4. 정량적 RT-PCR(quantitative real time PCR)Experimental Example 4. Quantitative RT-PCR (quantitative real time PCR)
Hybrid-R 키트(GeneAll, Seoul, Korea)를 이용하여 동물모델의 척수 등쪽뿔의 동측(ipsilateral side of the spinal dorsal horn)(L4-L5 segment, 0.7 cm) 또는 BV2 세포로부터 전체 RNA를 분리하였다. 분리된 전체 RNA에 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;mouse GAPDH, forward: 5’- ACC CAG AAG ACT GTG GAT GG -3’, reverse: 5’- CAC ATT GGG GGT AGG AAC AC -3’; mouse TNF- α, forward: 5’- AGC AAA CCA CCA AGT GGA GGA-3’,reverse: 5’- GCT GGC ACC ACT AGT TGG TTG T -3’;mouse IL-1β, forward: 5’- TTG TGG CTG TGG AGA AGC TGT -3’, reverse: 5’- AAC GTC ACA CAC CAG CAG GTT -3’; mouse IL-6, forward: 5’- TCC ATC CAG TTG CCT TCT TGG -3’, reverse: 5’- CCA CGA TTT CCC AGA GAA CAT G -3’; mouse COX-2, forward: 5’- TGA GTA CCG CAA ACG CTT CT - 3’, reverse: 5’- CTC CCC AAA GAT AGC ATC TGG - 3’ ; mouse iNOS, forward: 5’- GGC AAA CCC AAG GTC TAC GTT - 3’, reverse: 5’- TCG CTC AAG TTC AGC TTG GT - 3’; rat GAPDH, forward: 5′- CTC ATG ACC ACA GTC CAT GC -3′, reverse: 5′- TTC AGC TCT GGG ATG ACC TT -3′; rat TNF-α, forward: 5′- AGA TGT GGA ACT GGC AGA GG -3′, reverse: 5′- CCC ATT TGG GAA CTT CTC CT -3′; rat IL-1β, forward: 5′- CAG CAG CAT CTC GAC AAG AG -3′, reverse: 5′- CAT CAT CCC ACG AGT CAC AG -3′; rat IL-6, forward: 5′- CCG GAG AGG AGA CTT CAC AG -3′, reverse: 5′- ACA GTG CAT CAT CGC TGT TC -3′; rat COX-2 forward: 5′- CAG TAT CAG AAC CGC ATT GCC -3′, reverse: 5′- GAG CAA GTC CGT GTT CAA GGA-3′; rat TLR4 forward: 5’- TGA GCA GTC GTG CTG GTA TC -3’, reverse: 5’- CAG GGC TTT TCT GAG TCG TC -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 side of the spinal dorsal horn (L4-L5 segment, 0.7 cm) or BV2 cells 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) to the isolated total RNA. Quantitative RT-PCR was performed using the synthesized cDNA. Specifically, the quantitative RT-PCR was repeated 40 times at 95° C. for 10 minutes, followed by 15 seconds at 95° C. and 1 minute at 60° C. In addition, the primers used for the quantitative RT-PCR are as follows; mouse GAPDH, forward: 5'- ACC CAG AAG ACT GTG GAT GG -3', reverse: 5'- CAC ATT GGG GGT AGG AAC AC -3'; mouse TNF- α, forward: 5'- AGC AAA CCA CCA AGT GGA GGA-3', reverse: 5'- GCT GGC ACC ACT AGT TGG TTG T -3'; mouse IL-1β, forward: 5'- TTG TGG CTG TGG AGA AGC TGT -3', reverse: 5'- AAC GTC ACA CAC CAG CAG GTT -3'; mouse IL-6, forward: 5'- TCC ATC CAG TTG CCT TCT TGG -3', reverse: 5'- CCA CGA TTT CCC AGA GAA CAT G -3'; mouse COX-2, forward: 5'- TGA GTA CCG CAA ACG CTT CT-3', reverse: 5'- CTC CCC AAA GAT AGC ATC TGG-3'; mouse iNOS, forward: 5'- GGC AAA CCC AAG GTC TAC GTT-3', reverse: 5'- TCG CTC AAG TTC AGC TTG GT-3'; rat GAPDH, forward: 5'- CTC ATG ACC ACA GTC CAT GC -3', reverse: 5'- TTC AGC TCT GGG ATG ACC TT -3'; rat TNF-α, forward: 5'- AGA TGT GGA ACT GGC AGA GG -3', reverse: 5'- CCC ATT TGG GAA CTT CTC CT -3'; rat IL-1β, forward: 5'- CAG CAG CAT CTC GAC AAG AG -3', reverse: 5'- CAT CAT CCC ACG AGT CAC AG -3'; rat IL-6, forward: 5'- CCG GAG AGG AGA CTT CAC AG -3', reverse: 5'- ACA GTG CAT CAT CGC TGT TC -3'; rat COX-2 forward: 5'- CAG TAT CAG AAC CGC ATT GCC -3', reverse: 5'- GAG CAA GTC CGT GTT CAA GGA-3'; rat TLR4 forward: 5'- TGA GCA GTC GTG CTG GTA TC -3', reverse: 5'- CAG GGC TTT TCT GAG TCG TC -3'. The mRNA expression level of each target gene was normalized to the expression level of GAPDH mRNA, and the value was calculated by the 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).
실험예 5. 면역조직화학염색Experimental Example 5. Immunohistochemical staining
래트를 소디움펜토바비탈(100mg/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 척추 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. Rats were anesthetized with sodium pentobarbital (100mg/kg, i.p.), and then heparinized PBS (pH 7.4) was perfused. Then, using a peristaltic pump, the rat was perfused with 4% paraformaldehyde (Merck, Darmstadt, Wetzlar, Germany) (in PBS) for 10 minutes at a rate of 20 ml/min. After the perfusion was completed, the rat spinal cord (L4-L6) section was immediately collected, immersed in a fixative solution overnight, and then immersed in a 10 to 30% sucrose solution. After 2 days, the spinal section was cut to a thickness of 30 μm using a cryostat (CM1950, Leica Biosystems, Germany), and the cut section was immersed in a storage buffer (30% glycerol, 30% ethylene glycol in PBS) to 4°C. Maintained.
면역염색을 위하여, 유지된 절편을 블로킹 버퍼(5% 정상 혈청, 0.3% Triton X-100 in PBS)와 실온에서 1시간 동안 반응시켰다. 그 후 절편과 1차 항체((iNOS, 1:100 [BD-610329; BD Transduction Laboratories, Franklin Lakes, NJ]; Iba1, 1:400 [019-19741; Wako, Osaka, Japan]; Arg1, 1:100 [sc-271430; Santa Cruz Biotechnology, Santa Cruz, CA])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.For immunostaining, the retained sections were reacted with blocking buffer (5% normal serum, 0.3% Triton X-100 in PBS) at room temperature for 1 hour. After that, fragments and primary antibodies ((iNOS, 1:100 [BD-610329; BD Transduction Laboratories, Franklin Lakes, NJ]; Iba1, 1:400 [019-19741; Wako, Osaka, Japan]; Arg1, 1: 100 [sc-271430; Santa Cruz Biotechnology, Santa Cruz, CA]) were incubated together and washed, and the sections were washed with Cy 3 or FITC (1:400, Jackson ImmunoResearch, West Grove, diluted in the same buffer as the primary antibody). PA) was incubated with secondary antibody conjugated and then washed to prepare the prepared section was counter-stained with DAPI (5 μg/ml) and mounted on a slide containing a mounting medium (Biomeda, Foster city, CA). Imaging of the stained section was performed using a confocal microscope (TCS SP8, Leica).
DAB(diaminobenzidine) 염색을 위하여, 척추 조직을 0.3 % H2O2(in PBS)에 10분 동안 노출시킨 후 블로킹 버퍼(5% 정상 혈청, 0.3% Triton X-100 in PBS)로로 실온에서 1시간 동안 배양하였다. 내인성 퍼옥시다제의 활성이 차단된 조직을 1차 항체(Iba-1, 1 : 400, Wako)와 4℃에서 밤새도록 배양한 후, 바이오티닐화된(biotinylated) 2차 항체와 스트렙타비딘 퍼옥시다아제 복합체(streptavidin peroxidase complex, Vector Laboratories, Inc., Burlingame, CA)를 처리하였다. 제작된 표본을 DAB-퍼옥시다아제 기질 용액(PBS 중 0.05% DAB/0.015%H2O2)으로 시각화하고 폴리마운트(Polysciences, Warrington, PA)를 사용하여 유리 슬라이드 상에 올려놓았다. 상기 슬라이드를 명시야 현미경(ECLIPSE E600 POL, Nikon, Tokyo, Japan)으로 관찰 및 이미징을 수행하였다. 이미지 J 소프트웨어(NIH, Bethesda, MD)를 사용하여, 얻어진 이미지로부터 척수 등쪽뿔에서 Iba1 면역 반응을 측정 하였다.For DAB (diaminobenzidine) staining, the spinal tissue was exposed to 0.3% H 2 O 2 (in PBS) for 10 minutes, and then 1 hour at room temperature with blocking buffer (5% normal serum, 0.3% Triton X-100 in PBS). During incubation. After incubating the tissues in which the activity of endogenous peroxidase is blocked with primary antibody (Iba-1, 1: 400, Wako) overnight at 4°C, biotinylated secondary antibody and streptavidin fur The oxidase complex (streptavidin peroxidase complex, Vector Laboratories, Inc., Burlingame, CA) was treated. The prepared samples were visualized with a DAB-peroxidase substrate solution (0.015% DAB/0.015%H 2 O 2 in PBS) and placed on a glass slide using Polysciences (Warrington, PA). The slide was observed and imaged with a bright field microscope (ECLIPSE E600 POL, Nikon, Tokyo, Japan). Using Image J software (NIH, Bethesda, MD), the Iba1 immune response was measured in the spinal dorsal horn from the obtained images.
실험예 6. 활성산소종(reactive oxygen species, ROS) 검출 분석Experimental Example 6. Reactive oxygen species (ROS) detection analysis
척수의 과산화물 음이온(Superoxide anion)의 농도는 Zhang Y, et al.의 방법(Zhang Y, Yu XJ, Chen WS, Gao HL, Liu KL, Shi XL, et al. Exercise training attenuates renovascular hypertension partly via RAS- ROS- glutamate pathway in the hypothalamic paraventricular nucleus. Sci Rep. 2016; 6: 37467.)으로 검출하였다. 구체적으로, 척추 절편과 DHE(1μM) 용액을 실온에서 5분 동안 반응시켰다. 반응이 완료된 척추 절편을 슬라이드 글라스에 올린 후 공촛점 현미경(Leica Biosystems, Germany)을 이용하여 신호를 검출하였으며, 형광 강도는 Image J 프로그램(NIH, Bethesda, MD, USA)을 통해 정량화하였다.The concentration of superoxide anion in the spinal cord was determined by Zhang Y, et al.'s method (Zhang Y, Yu XJ, Chen WS, Gao HL, Liu KL, Shi XL, et al. Exercise training attenuates renovascular hypertension partly via RAS- ROS-glutamate pathway in the hypothalamic paraventricular nucleus.Sci Rep. 2016; 6: 37467.). Specifically, the spinal section and DHE (1 μM) solution were reacted at room temperature for 5 minutes. After the reaction was completed spinal section was placed on a slide glass, the signal was detected using a confocal microscope (Leica Biosystems, Germany), and the fluorescence intensity was quantified through the Image J program (NIH, Bethesda, MD, USA).
실험예 7. MIA(monoiodoactate)로 유도된 무릎 관절염(knee osteoarthritic, OA) 통증 모델 및 행동 검사Experimental Example 7. MIA (monoiodoactate)-induced knee osteoarthritic (OA) pain model and behavior test
무릎 관절염 통증 모델은 “Thakur M, Rahman W, Hobbs C, Dickenson AH, Bennett DL. Characterisation of a peripheral neuropathic component of the rat monoiodoacetate model of osteoarthritis. PLoS One. 2012; 7: e33730.”에 기재된 동물 모델을 이용하였다. 구체적으로, 아이소플루레인(2% in oxygen mixture; Hana Pharm, 대한민국)을 이용하여 래트를 마취시킨 후 MIA(식염수 25μl에 MIA가 2mg 포함된 것)를 26.5G 주사 바늘이 달린 해밀턴 주사기를 사용하여 왼쪽 슬개건을 통해 왼쪽 뒷다리의 무릎 관절의 관절 내 공간에 주사하였다. 대조군은 MIA 대신 PBS 25μl를 주사하였다. MIA 주사 후 7일째에 무릎 관절염 통증 모델의 무릎 관절염 통증이 확립되었다. 상기 무릎 관절염 통증 모델의 척수강에 PAT4(25nmol)를 주입하였으며, PAT4의 진통 감소 효과를 확인하기 위하여 본 프레이 필라멘트 행동 검사를 실시하였다.The knee arthritis pain model is described in “Thakur M, Rahman W, Hobbs C, Dickenson AH, Bennett DL. Characterization of a peripheral neuropathic component of the rat monoiodoacetate model of osteoarthritis. PLoS One. 2012; 7: The animal model described in “e33730.” was used. Specifically, after anesthetizing the rat with isoflurane (2% in oxygen mixture; Hana Pharm, Korea), MIA (25 μl of saline containing 2 mg of MIA) was used using a Hamilton syringe with a 26.5G injection needle. It was injected into the intraarticular space of the knee joint of the left hind limb through the left patellar tendon. The control group was injected with 25 μl of PBS instead of MIA. Knee arthritis pain in the knee arthritis pain model was established 7 days after MIA injection. PAT4 (25 nmol) was injected into the spinal cavity of the knee arthritis pain model, and a von Frey filament behavior test was performed to confirm the analgesic reduction effect of PAT4.
실시예 1. PAT4 제작Example 1. PAT4 production
신경병증성 통증의 치료를 위해 TLR4에 특이적으로 결합하는 펩타이드를 제작하였으며, 제작된 펩타이드 중 TLR4와 결합 친화도가 높은 PAT4(peptide antagonist of Toll-like receptor 4)를 선별하였다. 상기 PAT4는 서열번호 1의 아미노산 서열로 표시된다.For the treatment of neuropathic pain, a peptide that specifically binds to TLR4 was produced, and PAT4 (peptide antagonist of Toll-like receptor 4) having a high binding affinity to TLR4 was selected among the produced peptides. The PAT4 is represented by the amino acid sequence of SEQ ID NO: 1.
실시예 2. 마우스 미세아교세포주에서 PAT4의 전염증 매개인자의 발현 억제 효과 확인Example 2. Inhibition of expression of pro-inflammatory mediators of PAT4 in mouse microglia cell lines
마우스 미세아교세포주인 BV2 세포를 이용하여 상기 실시예 1에서 제작된 PAT4의 전염증 매개인자의 발현 억제 효과를 확인하였다. 구체적으로, BV2 세포를 PAT4(10mM)로 30분 동안 전처리하였다. 전처리된 BV2 세포를 LPS(lipopolysaccharide)(1000ng/ml)와 함께 3시간 동안 추가 배양하였다. 음성 대조군은 PAT4 대신 PBS를 처리한 BV2 세포이며, 양성 대조군은 PBS로 전처리한 후 LPS로 자극한 BV2 세포이다. 배양 종료 후 전염증 매개인자인 TNF-α, IL-1β, IL-6, COX-2 및 iNOS의 발현을 정량적 RT-PCR(실험예 4)을 통해 확인하였으며, 그 결과는 도 1에 나타내었다.The effect of inhibiting the expression of the pro-inflammatory mediator of PAT4 prepared in Example 1 was confirmed using BV2 cells, which are mouse microglia cell lines. Specifically, BV2 cells were pretreated with PAT4 (10 mM) for 30 minutes. The pretreated BV2 cells were further incubated with LPS (lipopolysaccharide) (1000 ng/ml) for 3 hours. The negative control was BV2 cells treated with PBS instead of PAT4, and the positive control was BV2 cells pretreated with PBS and then stimulated with LPS. After completion of the culture, the expression of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS was confirmed through quantitative RT-PCR (Experimental Example 4), and the results are shown in FIG. .
도 1에 나타낸 바와 같이, PAT4를 처리한 LPS로 자극된 BV2 세포는 전염증 매개인자인 TNF-α, IL-1β, IL-6, COX-2 및 iNOS의 발현이 음성 대조군 수준으로 감소하는 것을 확인하였다. 상기 결과는 PAT4가 미세아교세포에서 항염증 효과를 나타낸다는 것을 의미한다.As shown in Figure 1, BV2 cells stimulated with LPS treated with PAT4 showed that the expression of pro-inflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS decreased to the level of negative control. Confirmed. The above results indicate that PAT4 exhibits anti-inflammatory effects in microglia.
실시예 3. 신경병증성 통증 동물 모델에서 PAT4의 타겟 세포 확인Example 3. Identification of target cells of PAT4 in neuropathic pain animal model
SNL 수술 후 신경병증성 통증 동물 모델의 미세아교세포에서 TLR4의 발현이 급증하기 때문에, 실험예 1의 신경병증성 통증 동물 모델에서 PAT4가 미세아교세포를 표적하는지 확인하였다. 구체적으로, SNL 수술 후 3일 째에 FITC가 결합된 PAT4를 신경병증성 통증 동물 모델의 척수강 내에 투여하였다(실험예 3). 투여 4일 후 L5 척추 절편을 제조하고, 미세아교세포 마커인 lba1에 대한 항체와 함께 배양하여 면역염색하였다(실험예 5). 면역염색 결과는 도 2에 나타내었다.Since the expression of TLR4 rapidly increased in microglia of the neuropathic pain animal model after SNL surgery, it was confirmed whether PAT4 targets microglia in the neuropathic pain animal model of Experimental Example 1. Specifically, 3 days after SNL surgery, FITC-conjugated PAT4 was administered into the spinal cavity of the neuropathic pain animal model (Experimental Example 3). Four days after administration, an L5 spinal section was prepared, and immunostained by incubating with an antibody against lba1, a microglia marker (Experimental Example 5). The immunostaining results are shown in FIG. 2.
도 2에 나타낸 바와 같이, 척수 등쪽뿔(lpsi)의 PAT4/Iba1 양성 세포의 수는 배측(contra)보다 3배 이상 많은 것을 확인하였다.As shown in FIG. 2, it was confirmed that the number of PAT4/Iba1 positive cells in the dorsal horn of the spinal cord (lpsi) was three times greater than that of the contra.
실시예 4. 신경병증성 통증 동물 모델에서Example 4. In an animal model of neuropathic pain PAT4의 진통 감소 및 미세아교세포 활성 억제 효과 확인Confirmation of the effect of PAT4 on reducing analgesia and inhibiting microglia activity
실험예 1의 신경병증성 통증 동물 모델을 이용해서 PAT4가 SNL 수술에 의해 유발된 통증을 감소시키는지 여부를 조사하였다. 구체적으로, SNL 수술 3일 째인 래트 중 본 프레이 필라멘트에 민감한 래트(<2g)의 척수강 내에 PAT4(25nmol) 20μl를 투여하였다(실험예 3). 음성 대조군은 SNL 수술 3일 째인 신경병증성 통증 동물 모델에 PAT4 대신 PBS 20μl을 투여하였고, 양성 대조군은 신경병증성 통증 치료제인 가바펜틴 5mg을 투여하였다. 상기 실험예 2의 통증 행동 시험을 통해 동물 모델의 기계적 역치를 측정하였으며 그 결과는 도 3A에 나타내었다.Using the neuropathic pain animal model of Experimental Example 1, it was investigated whether PAT4 reduces pain induced by SNL surgery. Specifically, 20 μl of PAT4 (25 nmol) was administered into the spinal cavity of rats (<2 g) sensitive to von Frey filaments among rats on the 3rd day of SNL surgery (Experimental Example 3). The negative control group received 20 μl of PBS instead of PAT4 to the neuropathic pain animal model on the 3rd day of SNL surgery, and the positive control group received 5 mg of gabapentin, a neuropathic pain treatment. The mechanical threshold of the animal model was measured through the pain behavior test of Experimental Example 2, and the results are shown in FIG. 3A.
도 3A에 나타낸 바와 같이, PAT4를 투여한 신경병증성 통증 동물 모델은 기계적 역치가 음성 대조군(PBS)보다 현저히 증가하는 것을 확인하였다. 또한 신경병증성 통증의 치료제로 사용되는 가바펜틴은 진통 감소 효과가 약 1일 정도 유지되는 반면, PAT4는 21일 이상 진통 감소 효과가 지속되는 것을 확인하였다.As shown in FIG. 3A, it was confirmed that the neuropathic pain animal model administered with PAT4 significantly increased the mechanical threshold compared to the negative control (PBS). In addition, it was confirmed that gabapentin, which is used as a therapeutic agent for neuropathic pain, maintains an analgesic reduction effect for about 1 day, while PAT4 has an analgesic reduction effect for more than 21 days.
신경병증성 통증 동물 모델에서 PAT4의 미세아교세포 활성 억제 효과를 조사하였다. 구체적으로, SNL 수술 3일 째인 신경병증성 통증 동물 모델의 척수강 내에 PAT4(25nmol) 20μl를 투여하였다(실험예 3). PAT4 투여 4일 후 L5 척추 절편을 제작하고, 항 lba1 항체와 배양하였다(실험예 5). 음성 대조군은 SNL 수술을 하지 않은 개체이며, 양성 대조군은 SNL 수술 3일째인 신경병증성 통증 동물 모델에 PAT4 대신 PBS 20μl을 투여하였다. 항 Iba1 항체의 결합 강도는 NIH Image J 소프트웨어를 사용하여 측정하였으며, 그 결과는 도 3B에 나타내었다.The inhibitory effect of PAT4 on microglia activity in an animal model of neuropathic pain was investigated. Specifically, 20 μl of PAT4 (25 nmol) was administered into the spinal cavity of the neuropathic pain animal model on the 3rd day of SNL surgery (Experimental Example 3). Four days after PAT4 administration, an L5 spinal section was prepared and incubated with an anti-lba1 antibody (Experimental Example 5). The negative control group was an individual who did not undergo SNL surgery, and the positive control group was administered 20 μl of PBS instead of PAT4 to the neuropathic pain animal model on the 3rd day of SNL surgery. The binding strength of the anti-Iba1 antibody was measured using NIH Image J software, and the results are shown in FIG. 3B.
도 3B에 나타낸 바와 같이, PAT4 처리군의 Iba1 면역 염색의 강도는 PBS로 처리한 대조군에 비해 67% 감소하였으며, 이는 PAT4가 미세아교세포의 활성을 감소시킨다는 것을 의미한다.As shown in FIG. 3B, the intensity of Iba1 immunostaining of the PAT4 treatment group was reduced by 67% compared to the control group treated with PBS, which means that PAT4 decreases the activity of microglia.
상기 결과는 PAT4가 척수에서 미세아교세포를 표적하고, 미세아교세포의 활성을 감소시킴으로써 SNL 수술로 유도된 신경병증성 통증을 감소시킨다는 의미한다.The above results indicate that PAT4 targets microglia in the spinal cord and decreases the activity of microglia, thereby reducing neuropathic pain induced by SNL surgery.
실시예 5. 신경병증성 통증 동물 모델에서 PAT4의 신경병증성 통증 감소 효과 확인Example 5. Confirmation of Neuropathic Pain Reduction Effect of PAT4 in Neuropathic Pain Animal Model
5-1. PAT4의 전염증 매개인자의 발현 억제 효과 확인5-1. Confirmation of the inhibitory effect of PAT4 on the expression of pro-inflammatory mediators
신경병증성 통증 동물모델에서 상기 실시예 2에서 제작된 PAT4의 전염증 매개 인자의 발현 역제 효과를 확인하였다. 구체적으로, 실험예 1에서 제조된 신경병증성 통증 동물 모델의 척수강에 PAT4(25nmol) 20μl를 주입하였다. PAT4 투여 4일 후 L5 척추 절편(0.7cm)로부터 전체 RNA를 분리하였다. 음성 대조군은 SNL 수술을 하지 않은 동물이며, 양성 대조군은 신경병증성 통증 동물 모델에 PBS 20μl을 투여하였다. 전염증 매개인자인 TNF-α, IL-1β, IL-6, COX-2 및 iNOS의 발현을 정량적 RT-PCR(실험예 4)을 통해 확인하였으며, 그 결과는 도 4A에 나타내었다.In the neuropathic pain animal model, the inverse effect of the expression of the pro-inflammatory mediating factor of PAT4 prepared in Example 2 was confirmed. Specifically, 20 μl of PAT4 (25 nmol) was injected into the spinal cavity of the neuropathic pain animal model prepared in Experimental Example 1. Total RNA was isolated from the L5 spinal section (0.7 cm) 4 days after PAT4 administration. The negative control group was an animal that did not undergo SNL surgery, and the positive control group was administered
도 4A에 나타낸 바와 같이, PAT4를 투여한 신경병증성 통증 동물 모델에서 전염증 매개인자인 TNF-α, IL-1β, IL-6, COX-2 및 iNOS의 발현이 양성 대조군(SNL+PBS)보다 현저히 감소한 것을 확인하였다.As shown in Figure 4A, the expression of pro-inflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS in the neuropathic pain animal model administered with PAT4 was positive control (SNL+PBS) It was confirmed that it was significantly reduced.
5-2. PAT4의 미세아교세포 활성 억제 효과 확인5-2. Confirmation of the inhibitory effect of PAT4 on microglia activity
실험예 1에서 제조된 신경병증성 통증 동물 모델의 척수강에 PAT4(25nmol) 20μl를 주입하였다. PAT4 투여 4일 후 L5 척추 절편을 제작하였다. 상기 L5 척추 절편을 미세아교세포 마커인 lba1에 대한 항체와 함께 배양하여 면역염색 하였다(실험예 5). 음성 대조군은 SNL 수술을 하지 않은 동물이며, 양성 대조군은 신경병증성 통증 동물 모델에 PBS 20μl을 투여하였다. 면역염색 결과는 도 4B에 나타내었다.PAT4 (25 nmol) 20 μl was injected into the spinal cavity of the neuropathic pain animal model prepared in Experimental Example 1. Four days after PAT4 administration, an L5 spinal section was prepared. The L5 spine section was incubated with an antibody against lba1, a microglia marker, and immunostained (Experimental Example 5). The negative control group was an animal that did not undergo SNL surgery, and the positive control group was administered
도 4B에 나타낸 바와 같이, PAT4를 투여한 신경병증성 통증 동물 모델에서 iNOS 및 iNOS/lba1 양성 세포 수가 양성 대조군(PBS)에 비해 각각 39% 및 46% 감소한 것을 확인하였다.As shown in FIG. 4B, it was confirmed that the number of iNOS and iNOS/lba1 positive cells decreased by 39% and 46%, respectively, compared to the positive control (PBS) in the neuropathic pain animal model administered with PAT4.
5-3. PAT4의 활성산소종 생성 억제 효과 확인5-3. Confirmation of the inhibitory effect of PAT4 on the production of reactive oxygen species
활성산소종(ROS)은 SNL 수술에 의한 미세아교세포의 활성을 향상시키므로, 본 실시예에서는 PAT4 투여에 따른 활성산소종 생성 억제여부를 확인하였다. 구체적으로, 실험예 1에서 제조된 신경병증성 통증 동물 모델의 척수강에 PAT4(25nmol) 20μl를 주입하였다. PAT4 투여 4일 후 L5 척추 절편을 제작하였다. L5 척추 절편을 이용하여 상기 실험예 6의 활성산소종 검출 분석을 수행하였다. 대조군은 신경병증성 통증 동물 모델에 PBS 20μl을 투여하였다. 활성산소종 검출 분석 결과는 도 5에 나타내었다.Since reactive oxygen species (ROS) improves the activity of microglia by SNL surgery, in this example, it was confirmed whether or not the production of reactive oxygen species was inhibited by administration of PAT4. Specifically, 20 μl of PAT4 (25 nmol) was injected into the spinal cavity of the neuropathic pain animal model prepared in Experimental Example 1. Four days after PAT4 administration, an L5 spinal section was prepared. The detection analysis of reactive oxygen species in Experimental Example 6 was performed using an L5 spine section. The control group was administered 20 μl of PBS to the neuropathic pain animal model. The results of the analysis for detecting reactive oxygen species are shown in FIG. 5.
도 5에 나타낸 바와 같이, PAT4를 투여한 신경병증성 통증 동물 모델에서 활성산소종이 대조군에 비해 약 55% 감소된 것을 확인하였다.As shown in FIG. 5, it was confirmed that in the neuropathic pain animal model administered with PAT4, reactive oxygen species decreased by about 55% compared to the control group.
상기 결과는 PAT4가 신경병증성 통증 동물 모델에서 전염증 매개인자의 발현 감소, 미세아교세포의 활성 억제 및 활성산소종 생성 억제를 통해 신경병증성 통증을 감소시킨다는 것을 의미한다.The above results indicate that PAT4 reduces neuropathic pain in an animal model of neuropathic pain by reducing the expression of pro-inflammatory mediators, inhibiting the activity of microglia, and inhibiting the production of reactive oxygen species.
실시예 6. 무릎 관절염 통증 모델에서 PAT4의 통증 감소 효과 확인Example 6. Confirmation of pain reduction effect of PAT4 in knee arthritis pain model
신경병증성 통증과 생리학적으로 관련이 있는 MIA로 유도된 무릎 관절염 통증 모델을 이용하여, PAT4의 통증 감소 효과를 상기 실험예 7의 방법으로 확인하였다. 구체적으로, MIA를 주사한지 7일째인 MIA로 유도된 무릎관절염 통증 동물 모델을 PBS(20μl, 25 nmol)를 척수강 내에 투여하였다. PAT4를 투여한 동물 모델의 기계적 역치는 주사 후 1 내지 24일(MIA 주사 후 7 내지 31일)에 조사하였다. 대조군으로는 PAT4 대신 PBS 20μl를 투여하였다. 기계적 역치를 조사한 결과는 도 6에 나타내었다.Using the MIA-induced knee arthritis pain model that is physiologically related to neuropathic pain, the pain reduction effect of PAT4 was confirmed by the method of Experimental Example 7. Specifically, PBS (20 μl, 25 nmol) was administered into the spinal cavity in an animal model of knee arthritis pain induced by MIA on the 7th day after MIA injection. The mechanical threshold of the animal model administered with PAT4 was investigated 1 to 24 days after injection (7 to 31 days after MIA injection). As a control group, 20 μl of PBS was administered instead of PAT4. The results of examining the mechanical threshold are shown in FIG. 6.
도 6에 나타낸 바와 같이, PAT4를 투여한 무릎 관절염 통증 모델은 대조군에 비해 기계적 역치가 현저히 증가한 것을 확인하였다. 또한 PAT4는 통증 감소 효과가 24일 이상 유지되는 것을 확인하였다. 상기 결과는 PAT4가 MIA에 의해 유발된 무릎 관절염 통증을 장기적으로 감소시킨다는 것을 의미하며, 무릎 관절염 통증을 포함하는 신경병증성 통증의 치료에 이용할 수 있다는 것을 시사한다.As shown in FIG. 6, it was confirmed that the knee arthritis pain model administered with PAT4 significantly increased the mechanical threshold compared to the control group. In addition, it was confirmed that the pain reduction effect of PAT4 was maintained for more than 24 days. The above results indicate that PAT4 reduces knee arthritis pain caused by MIA in the long term, and suggests that it can be used for the treatment of neuropathic pain including knee arthritis pain.
종합적으로 본 발명자들은 PAT4가 미세아교세포를 타겟하여 전염증 매개인자의 발현을 억제, 활성산소종 생성 억제, 미세아교세포의 활성을 억제 및 기계적 역치를 증가시킴으로써, 만성통증을 감소시킨다는 것을 확인하였다. 이는 PAT4가 만성통증의 치료제로서 이용될 수 있음을 의미하는 바, 본 발명의 PAT4는 만성통증의 예방, 개선 및 치료 분야에서 다양하게 활용될 수 있다.Overall, the present inventors have confirmed that PAT4 targets microglia, inhibits the expression of pro-inflammatory mediators, inhibits the production of reactive oxygen species, inhibits the activity of microglia, and increases the mechanical threshold, thereby reducing chronic pain. . This means that PAT4 can be used as a therapeutic agent for chronic pain, and PAT4 of the present invention can be variously used in the field of prevention, improvement and treatment of chronic pain.
이하, 제제예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 제제예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 제제예에 의해 제한되는 것으로 해석되지 않는다.Hereinafter, the present invention will be described in more detail through formulation examples. Formulation examples are for illustrative purposes only, and the scope of the present invention is not construed as being limited by formulation examples.
제제예 1. 만성통증의 예방 또는 치료용 약학적 조성물의 제조Formulation Example 1. Preparation of a pharmaceutical composition for preventing or treating chronic pain
1-1. 산제의 제조1-1. Preparation of powder
PAT4 20 mg
유당 100 mg100 mg lactose
탈크 10 mg10 mg of talc
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
1-2. 정제의 제조1-2. Manufacture of tablets
PAT4 10 mg
옥수수전분 100 mg100 mg corn starch
유당 100 mg100 mg lactose
스테아린산 마그네슘 2 mg2 mg of magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.
1-3. 캡슐제의 제조1-3. Preparation of capsules
PAT4 10 mg
결정성 셀룰로오스 3 mg3 mg of crystalline cellulose
락토오스 14.8 mg14.8 mg lactose
마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.
1-4. 주사제의 제조1-4. Preparation of injections
PAT4 10 mg
만니톨 180 mgMannitol 180 mg
주사용 멸균 증류수 2974 mg2974 mg of sterile distilled water for injection
인산이나트륨 이수화물 26 mgDisodium phosphate dihydrate 26 mg
통상의 주사제의 제조방법에 따라 1 앰플당(2 ml) 상기의 성분 함량으로 제조한다.It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.
1-5. 액제의 제조1-5. Preparation of liquid
PAT4 20 mg
이성화당 10 g10 g of isomerized sugar
만니톨 5 g5 g of mannitol
정제수 적량Purified water appropriate amount
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100ml로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.Add and dissolve each component in purified water according to the usual preparation method of liquid, add lemon zest, mix the above ingredients, add purified water and add purified water to adjust the total to 100 ml, then fill in a brown bottle for sterilization. To prepare a solution.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. Above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
<110> The Industry & Academic Cooperation in Chungnam National University (IAC)
<120> Composition for prevention, improving or treatment of chronic
pain comprising PAT4
<130> 1-69P
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> PAT4
<400> 1
Ala Met Ala Leu Asp Cys Phe Arg Trp Gly Trp Arg Met Trp Cys Ser
1 5 10 15
<110> The Industry & Academic Cooperation in Chungnam National University (IAC)
<120> Composition for prevention, improving or treatment of chronic
pain comprising PAT4
<130> 1-69P
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> PAT4
<400> 1
Ala Met Ala Leu Asp Cys Phe Arg Trp Gly Trp Arg Met
Claims (7)
A pharmaceutical composition for preventing or treating chronic pain diseases, comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
상기 만성통증 질환은 신경병증성 통증, 관절염에 의한 만성통증, 복합부위 통증 증후군, 대상포진 후 신경통 및 척추 수술 후 통증 증후군으로 이루어진 군에서 선택되는 1종 이상의 질환인, 만성통증 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The chronic pain disease is one or more diseases selected from the group consisting of neuropathic pain, chronic pain due to arthritis, complex site pain syndrome, neuralgia after shingles, and pain syndrome after spinal surgery, preventing or treating chronic pain diseases Pharmaceutical composition for use.
상기 신경병증성 통증은 말초 신경 손상 또는 중추 신경 손상에 의해 유도된 것인, 만성통증 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 2,
The neuropathic pain is induced by peripheral nerve injury or central nerve injury, a pharmaceutical composition for preventing or treating chronic pain disease.
상기 신경병증성 통증은 말초신경계 이상 또는 손상, 척수신경 손상을 포함한 다발성경화증, 척수부상, 통각과민, 감각과민, 신경병증, 당뇨성 신경장애, 신경염, 신경통, 작열통, 이질통, 포진후신경통, 요추 신경근압박, 암과 관련된 통증, 알코올 중독, 비정형 안면통증, 포진성 신경통, 중풍후통증, HIV 연관 신경병증, 골관절염 및 환상사지통증으로 이루어진 군에서 선택되는 1종 이상의 질환인, 만성통증 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 2,
The neuropathic pain is peripheral nervous system abnormality or damage, multiple sclerosis including spinal nerve injury, spinal cord injury, hyperalgesia, hypersensitivity, neuropathy, diabetic neuropathy, neuritis, neuralgia, burning pain, allodynia, postherpetic neuralgia, lumbar spine Prevention of chronic pain disease, one or more diseases selected from the group consisting of neuromuscular compression, cancer-related pain, alcoholism, atypical facial pain, herpes neuralgia, post-stroke pain, HIV-related neuropathy, osteoarthritis and phantom limb pain Or a therapeutic pharmaceutical composition.
상기 PAT4는 미세아교세포(microglia cell), 뉴런 세포(neuronal cell) 및 성상세포(astrocytic cell)로 이루어진 군에서 선택되는 1종 이상을 타겟하는, 만성통증 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The PAT4 is a pharmaceutical composition for preventing or treating chronic pain diseases targeting at least one selected from the group consisting of microglia cells, neuronal cells and astrocytic cells.
A food composition for preventing or improving chronic pain diseases, comprising PAT4 represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
A method for treating chronic pain disease comprising a; administering the pharmaceutical composition according to any one of claims 1 to 5 to an individual other than a human.
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