KR102285724B1 - Composition for regenerating cartilage comprising polydeoxyribonucleotide and hyaluronic acid - Google Patents
Composition for regenerating cartilage comprising polydeoxyribonucleotide and hyaluronic acid Download PDFInfo
- Publication number
- KR102285724B1 KR102285724B1 KR1020180039522A KR20180039522A KR102285724B1 KR 102285724 B1 KR102285724 B1 KR 102285724B1 KR 1020180039522 A KR1020180039522 A KR 1020180039522A KR 20180039522 A KR20180039522 A KR 20180039522A KR 102285724 B1 KR102285724 B1 KR 102285724B1
- Authority
- KR
- South Korea
- Prior art keywords
- hyaluronic acid
- weight
- composition
- dna fragment
- fragment mixture
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Reproductive Health (AREA)
- Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Developmental Biology & Embryology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Rheumatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
본 발명은 연골 재생의 효과를 가진 조성물에 관한 것으로, 어류 정소로부터 분리된 DNA 단편 혼합물과 히알루론산을 포함하는 것을 특징으로 하여 관절 내 주입이 가능하도록 적절한 점성을 갖추며, 윤활기능과 동시에 세포 증식 효과에 따른 연골 재생을 극대화하여 골관절염을 치료할 수 있는 효과를 갖는다.The present invention relates to a composition having an effect of regenerating cartilage, comprising a mixture of DNA fragments isolated from fish testis and hyaluronic acid, and having an appropriate viscosity for intra-articular injection, lubricating function and cell proliferation effect It has the effect of maximizing the cartilage regeneration according to the osteoarthritis.
Description
본 발명은 연골 재생의 효과를 가진 조성물에 관한 것으로, 더욱 상세하게는 연골 재생 및 윤활기능에 따른 항통증 효과를 통한 골관절 치유 조성물에 관한 것이다.The present invention relates to a composition having an effect of cartilage regeneration, and more particularly, to a composition for healing osteoarthritis through an anti-pain effect according to cartilage regeneration and lubricating function.
퇴행성 관절염이라 불리는 골관절염(osteoarthritis)은 관절과 뼈의 구조적 변화와 더불어 관절 연골이 손상되어 심한 통증과 관절운동장애 등의 증상이나 증후를 나타내는 질환이다. 골관절염은 방사선 검사를 통해 연골 손실과 뼈의 변화 정도를 포함한 관절의 생리적 상태를 평가하는 질환이기에 명확한 정의를 내리기는 어렵지만, 일반적으로 “연골손실을 특징으로 활액낭, 관절, 관절 주위 뼈의 구조적 변화가 확인되는 상태”로 정의되고 있다.Osteoarthritis, also called degenerative arthritis, is a disease that exhibits symptoms or symptoms such as severe pain and joint movement disorders due to structural changes in joints and bones and damage to articular cartilage. Osteoarthritis is a disease that evaluates the physiological state of the joint, including cartilage loss and bone changes through radiographic examination, so it is difficult to define it clearly, but generally “characterized by cartilage loss, structural changes in the synovial sac, joint, and surrounding bones are It is defined as a “confirmed state”.
실제 퇴행성 관절염 환자의 무릎 관절을 평가한 결과 관절을 보호하고 있는 연골의 손상 또는 관절을 이루는 뼈와 인대 등에 손상이 일어나 비가역적으로 연골조직이 파괴되어 있음을 확인할 수 있다. As a result of evaluating the knee joint of an actual degenerative arthritis patient, it can be confirmed that the cartilage tissue is irreversibly destroyed due to damage to the cartilage protecting the joint or damage to the bones and ligaments constituting the joint.
현행 골관절염의 기본적인 치료 방법으로는, 초기에 있어 통증만 있는 경우에는 단순 진통제를 복용하여 통증을 없애는데 초점이 맞춰 있으나, 좀 더 진행된 후 통증이 계속된 경우에는 항염 효과가 강한 소염 진통제를 사용하게 된다. 그러나 이와 같은 소염 진통제는 코르티코스테로이드(corticosteroid) 유형의 소염 물질을 사용하게 되는데, 이러한 진통제의 일종인 하이드로코르티존(hydrocortisone) 및 베타메타손(betamethasone) 등을 장기간 사용하는 경우 위, 간, 신장의 기능을 저하 시키고, 연골 세포의 재생 능력을 저해시키는 등의 부작용이 있어 지속적인 관찰과 함께 경구투여가 아닌 히알루론산나트륨의 관절강 내 주사와 같은 요법 등이 사용되고 있다.Currently, the basic treatment method for osteoarthritis is to take simple analgesics to relieve the pain in the initial stage of pain only. . However, such an anti-inflammatory analgesic uses a corticosteroid-type anti-inflammatory substance. In the case of long-term use of these analgesics, such as hydrocortisone and betamethasone, the functions of the stomach, liver, and kidneys are impaired. There are side effects such as lowering the chondrocytes and inhibiting the regeneration ability of chondrocytes, so a therapy such as intra-articular injection of sodium hyaluronate rather than oral administration is being used with continuous observation.
히알루론산은 관절 활액 중에 기 존재하는 완전히 생체 적합한 성분으로써, 이상 반응의 발현 빈도가 매우 낮은 장점에도 불구하고, 진통 효과 발현이 매우 느리고, 관절강 내 주입시 국소 부위의 통증으로 인해 환자의 치료 만족도가 낮으며, 연골 재생 효과가 낮은 단점이 있어왔다.Hyaluronic acid is a completely biocompatible component already present in the joint synovial fluid, and despite the advantage of having a very low frequency of adverse reactions, the onset of analgesic effect is very slow, and the patient's satisfaction with the treatment is high due to local pain during intra-articular injection. low, and there has been a disadvantage in that the cartilage regeneration effect is low.
따라서 본 발명이 해결하고자 하는 과제는 상기한 한계점을 극복하기 위한 것으로, 관절의 세포 증식 효능에 따른 연골 재생 효과 및 윤활기능에 따른 골관절 치유의 효과가 뛰어난 DNA 단편 혼합물 및 히알루론산을 포함하는 골관절염 치료 조성물을 제공하는 것이다.Therefore, the problem to be solved by the present invention is to overcome the above-described limitations, and treatment of osteoarthritis containing a DNA fragment mixture and hyaluronic acid, which is excellent in the effect of cartilage regeneration according to the cell proliferation effect of the joint and osteoarthritis healing effect according to the lubricating function to provide a composition.
상기 기술적 과제를 달성하기 위하여,In order to achieve the above technical task,
어류 정소로부터 분리된 DNA 단편 혼합물과 히알루론산을 포함하는 것을 특징으로 하는 연골 재생용 조성물을 제공한다.It provides a composition for cartilage regeneration comprising a mixture of DNA fragments isolated from fish testis and hyaluronic acid.
상술한 바와 같은 본 발명에 따른 연골 재생용 조성물에 있어서, 상기 DNA 단편 혼합물의 분자량은 300~3,000kDa이고, 상기 히알루론산의 분자량은 100~3,000kDa인 것이 바람직하다.In the composition for cartilage regeneration according to the present invention as described above, the molecular weight of the DNA fragment mixture is 300 to 3,000 kDa, and the molecular weight of the hyaluronic acid is preferably 100 to 3,000 kDa.
상술한 바와 같은 본 발명에 따른 연골 재생용 조성물에 있어서, 상기 DNA 단편 혼합물의 농도는 0.2~6%이고, 상기 히알루론산의 농도는 0.1~3%인 것이 바람직하다.In the composition for cartilage regeneration according to the present invention as described above, the concentration of the DNA fragment mixture is 0.2 to 6%, and the concentration of the hyaluronic acid is preferably 0.1 to 3%.
상술한 바와 같은 본 발명에 따른 연골 재생용 조성물에 있어서, 상기 DNA 단편 혼합물과 히알루론산의 중량비는 2:1인 것이 바람직하다.In the composition for cartilage regeneration according to the present invention as described above, the weight ratio of the DNA fragment mixture and hyaluronic acid is preferably 2:1.
상술한 바와 같은 본 발명에 따른 연골 재생용 조성물에 있어서, 상기 어류는 연어과 어류인 것이 바람직하다.In the composition for cartilage regeneration according to the present invention as described above, the fish is preferably salmonid fish.
본 발명에 따른 DNA 단편 혼합물(PDRN)과 히알루론산(HA) 혼합 조성물은 관절 내 주입이 가능하도록 적절한 점성을 갖추며, 윤활기능과 동시에 세포 증식 효과에 따른 연골 재생을 극대화하여 골관절염을 치료할 수 있는 효과를 갖는다.The DNA fragment mixture (PDRN) and hyaluronic acid (HA) mixture composition according to the present invention has an appropriate viscosity to enable intra-articular injection, and can treat osteoarthritis by maximizing cartilage regeneration according to the lubricating function and cell proliferation effect. has
이하, 본 발명에 대하여 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다.Hereinafter, the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention.
본 발명에서의 히알루론산은 히알루론산 또는 그의 약학적으로 허용되는 염 또는 그의 수화물 또는 이의 혼합물로써, 히알루론산의 약학적으로 허용되는 염은, 예를 들어, 히알루론산 나트륨, 히알루론산 마그네슘, 히알루론산 아연, 히알루론산 코발트 등과 같은 무기염과, 히알루론산 테트라부틸암모늄 등과 같은 유기염일 수 있으며, 이들 염이 한개 이상이 조합되어 사용될 수 있다. 본 발명의 히알루론산의 분자량은 100~3,000kDa인 것이 바람직하다.Hyaluronic acid in the present invention is hyaluronic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof or a mixture thereof, and a pharmaceutically acceptable salt of hyaluronic acid is, for example, sodium hyaluronate, magnesium hyaluronate, hyaluronic acid. It may be an inorganic salt such as zinc or cobalt hyaluronate, and an organic salt such as tetrabutylammonium hyaluronate, and one or more of these salts may be used in combination. The molecular weight of the hyaluronic acid of the present invention is preferably 100 to 3,000 kDa.
본 발명에서, 히알루론산 또는 그의 약학적으로 허용되는 염 또는 그의 수화물은, 본 발명의 조성물의 총 중량에 대하여 히알루론산 또는 그의 약학적으로 허용되는 염으로서 0.1~3중량%로 포함되는 것이 바람직하다. 히알루론산 또는 그의 염의 농도가 0.1중량% 미만인 경우에는 골관절염 치료 효과가 미미할 수 있고, 3중량% 초과인 경우에는 조성물의 급격한 점성 증가로 인하여 프리필드 시린지 또는 앰플 용기 충전에 어려움이 있고, 또한 환자의 환부 투여 시 어려움을 야기할 수 있다.In the present invention, hyaluronic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof is preferably included in an amount of 0.1 to 3% by weight as hyaluronic acid or a pharmaceutically acceptable salt thereof, based on the total weight of the composition of the present invention. . If the concentration of hyaluronic acid or its salt is less than 0.1% by weight, the osteoarthritis treatment effect may be insignificant, and if it is more than 3% by weight, it is difficult to fill the prefilled syringe or ampoule container due to the rapid increase in viscosity of the composition, and also It may cause difficulties when administering to the affected area.
또한, 본 발명에 따른 DNA 단편 혼합물(Polydeoxyribonucleotide, PDRN)의 분자량은 300~3,000kDa이며, 0.2~6중량%로 포함되는 것이 바람직하다. 0.2중량% 미만에서는 효과가 미미하며, 6중량% 초과의 농도로 제조할 경우에는 점성 증가로 인하여 골관절염 치료의 효능이 떨어지게 된다.In addition, the molecular weight of the DNA fragment mixture (Polydeoxyribonucleotide, PDRN) according to the present invention is 300 ~ 3,000 kDa, preferably contained in 0.2 ~ 6% by weight. If it is less than 0.2% by weight, the effect is insignificant, and when it is prepared at a concentration of more than 6% by weight, the efficacy of treating osteoarthritis is reduced due to increased viscosity.
또한, 본 발명에서 DNA 단편 혼합물과 히알루론산의 중량비는 2:1인 것이 바람직하다. 이 비율을 벗어나는 경우 골관절염의 치료 효과가 상대적으로 낮아진다.In addition, in the present invention, the weight ratio of the DNA fragment mixture and hyaluronic acid is preferably 2:1. If it is outside this ratio, the therapeutic effect of osteoarthritis is relatively low.
본 발명에 따른 조성물은 액상으로서 주사제형으로 제제화하는 것이 바람직하며, 골관절염 치료를 위하여 관절강 내 주사로 투여되는 것이 바람직하다.The composition according to the present invention is preferably formulated as an injection as a liquid, and is preferably administered by intra-articular injection for the treatment of osteoarthritis.
본 발명에 따른 주사제형의 조제예는 히알루론산 0.5g, 폴리데옥시리보뉴클레오티드 1g, 염화나트륨 0.9g, 인산수소나트륨 0.2g, 인산수소이나트륨 2.5g을 주사용수 100ml에 완전히 용해시킨 다음 제균 여과 하여 제제화 할 수 있다.The preparation example of the injection formulation according to the present invention is formulated by completely dissolving 0.5 g of hyaluronic acid, 1 g of polydeoxyribonucleotide, 0.9 g of sodium chloride, 0.2 g of sodium hydrogen phosphate, and 2.5 g of disodium hydrogen phosphate in 100 ml of water for injection, followed by sterilization and filtration. can do.
본 발명의 조성물의 pH는 NaH2PO4, Na2HPO4를 사용하여 6.0 내지 8.0로 조절하는 것이 바람직하다. 6.0 미만 또는 8.0을 초과하는 pH에서는 히알루론산이 불안정하고, 관절 내 상기 액상 조성물 투여 시 국소자극을 통한 통증, 부종, 염증 등의 부작용이 나타날 수 있다.The pH of the composition of the present invention is preferably adjusted to 6.0 to 8.0 using NaH 2 PO 4 , Na 2 HPO 4 . At a pH of less than 6.0 or exceeding 8.0, hyaluronic acid is unstable, and side effects such as pain, edema, and inflammation may occur through local stimulation when the liquid composition is administered in a joint.
또한, 본 발명의 약학적 조성물에는, 항류머티즘제, 비-스테로이드성 항염증성 약물(NSAID), 진통제, 마취제, 국소 마취제, 면역 억제제, 성장 호르몬, 호르몬 대체 약물, 방사성 약제 등의 약물을 더 포함할 수 있다.In addition, the pharmaceutical composition of the present invention further includes drugs such as antirheumatic agents, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, local anesthetics, immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, etc. can do.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention. it will be self-evident
<< 준비예preparation example 1. 송어 정소로부터 DNA 단편 혼합물의 제조> 1. Preparation of DNA fragment mixture from trout testis>
1)분쇄1) Crushing
무지개송어(바다에서 양식된 송어로 바다송어라칭함)의 정소 10㎏을 정제수 150L와 NaCl 37.4 ㎏을 넣어 분쇄기로 분쇄하였다.10 kg of testes of rainbow trout (trout farmed in the sea, called sea trout) were ground with 150 L of purified water and 37.4 kg of NaCl with a grinder.
2)분자저감2) Molecular reduction
분쇄된 정소에 증류수 150L을 추가로 가하고, 소디움도데실설페이트(이하 SDS라 칭함) 1.2㎏을 투입한 다음 100~105℃에서 4~5시간 동안 반응하여 분자저감하였다.150 L of distilled water was additionally added to the pulverized testis, 1.2 kg of sodium dodecyl sulfate (hereinafter referred to as SDS) was added, and the reaction was performed at 100 to 105° C. for 4 to 5 hours to reduce the molecular weight.
3)정제과정3) Purification process
분자저감 후 실온까지 냉각하여 15,000rpm에서 원심분리하여 정소 찌꺼기를 제거하고 여과하였다. 에탄올에 여과액을 서서히 첨가하여 흰색 고체를 수득하였다.After molecular reduction, it was cooled to room temperature and centrifuged at 15,000 rpm to remove testis residues and filtered. The filtrate was slowly added to ethanol to obtain a white solid.
4)탈수, 건조과정4) Dehydration and drying process
흰색고체를 70%(v/v)에탄올로 세척한 다음 무수에탄올로 탈수 후 여과, 건조 후 800g의 DNA 단편 혼합물(PDRN, 순도 99.5%)을 얻었다. 평균 분자량은 겔 이미지 분석 시스템으로 측정한 결과, 저분자량인 300~3,000kDa에 주로 분포하였다. The white solid was washed with 70% (v/v) ethanol, then dehydrated with absolute ethanol, filtered and dried to obtain 800 g of a DNA fragment mixture (PDRN, purity 99.5%). As a result of measuring the average molecular weight by a gel image analysis system, it was mainly distributed in the low molecular weight range of 300 to 3,000 kDa.
상기 과정을 통해 얻어진 DNA 단편 혼합물(PDRN)을 0.9%의 염화나트륨 용액 첨가하여 혼합한 뒤 120℃에서 15분간 습윤멸균한 다음 냉각시켜 하기의 실험예에 사용되었다.The DNA fragment mixture (PDRN) obtained through the above process was mixed by adding 0.9% sodium chloride solution, followed by wet sterilization at 120° C. for 15 minutes, followed by cooling, and used in the following experimental examples.
<실시예 1 내지 5 및 비교예 1 내지 6. DNA 단편 혼합물 및 히알루론산 조성물의 제조><Examples 1 to 5 and Comparative Examples 1 to 6. Preparation of DNA fragment mixture and hyaluronic acid composition>
상기 준비예 1에서 제조된 DNA 단편 혼합물(PDRN)과 100~3,000kDa 분자량의히알루론산(HA, 시그마알드리치에서 구입)을 하기 표 1과 같은 농도 및 중량비가 되도록 0.9% 염화나트륨이 첨가된 용액에 혼합한 뒤 실린지 필터로 멸균하여 액상의 조성물을 제조하였다.The DNA fragment mixture (PDRN) prepared in Preparation Example 1 and hyaluronic acid (HA, purchased from Sigma-Aldrich) having a molecular weight of 100 to 3,000 kDa were mixed in a solution with 0.9% sodium chloride added so as to have the same concentration and weight ratio as shown in Table 1 below. After sterilization with a syringe filter, a liquid composition was prepared.
(PDRN, 중량%)DNA fragment mixture
(PDRN, wt%)
(HA, 중량%)hyaluronic acid
(HA, wt%)
<실험예 1. DNA 단편 혼합물과 히알루론산 혼합물의 연골세포 성장 효과><Experimental Example 1. Chondrocyte growth effect of DNA fragment mixture and hyaluronic acid mixture>
1-1. 세포 수 측정1-1. Cell counting
사람 연골 세포(CHON-001, ATCC CRL-2846)를 10% FBS(fetal bovine serum)와 1% 페니실린/스트렙토마이신이 포함된 DMEM(Dulbeco’s Modified Eagle’s Medium)에서 배양하였고, 배양된 세포에 트립신/EDTA를 처리하여 바닥으로부터 세포를 떼어낸 후 헤마사이토미터(hemacytometer)를 이용하여 세포 수를 측정하였다. 상기 세포를 3×103 cells/200ul로 96-well plate에 접종하고 24시간 안정화시켰다. 이후 각 well의 배지를 제거하고 실시예 1 내지 5 및 비교예 1 내지 6의 조성물이 포함된 DMEM 배지를 세포에 넣어주었다. 그 후 5% CO2가 포함된 37℃ 인큐베이터에서 72시간 동안 배양하였다. 이때, 음성 대조군으로는 PDRN과 히알루론산이 모두 첨가되지 않은 0.9% 염화나트륨이 첨가된 DMEM 배지를 사용하였다. 이후, 세포수 측정을 위해서 상기 세포에 트립신/EDTA를 처리하여 바닥으로부터 세포를 떼어낸 후 헤마사이토미터(hemacytometer)를 이용하여 세포 수를 측정하였다(표 2).Human chondrocytes (CHON-001, ATCC CRL-2846) were cultured in DMEM (Dulbeco's Modified Eagle's Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and trypsin/EDTA was added to the cultured cells. After removing the cells from the bottom by treatment, the number of cells was measured using a hemacytometer. The cells were inoculated in a 96-well plate at 3×10 3 cells/200ul and stabilized for 24 hours. After removing the medium from each well, DMEM medium containing the compositions of Examples 1 to 5 and Comparative Examples 1 to 6 was put into the cells. Thereafter, 5% CO 2 was cultured in an incubator at 37° C. for 72 hours. At this time, as a negative control, a DMEM medium supplemented with 0.9% sodium chloride to which neither PDRN nor hyaluronic acid was added was used. Thereafter, to measure the number of cells, the cells were treated with trypsin/EDTA to remove the cells from the bottom, and then the number of cells was measured using a hemacytometer (Table 2).
(5.2±1)×104
(5.2±1)×10 4
그 결과, 비교예 1 내지 6에서 확인할 수 있듯이, 히알루론산(HA)을 단독 처리 했을 때 보다 DNA 단편 혼합물(PDRN)을 단독 처리 했을 때 세포 성장률이 더 높았다. As a result, as can be seen in Comparative Examples 1 to 6, the cell growth rate was higher when the DNA fragment mixture (PDRN) was treated alone than when the hyaluronic acid (HA) was treated alone.
또한, 실시예와 비교예에서 확인할 수 있듯이, DNA 단편 혼합물(PDRN)과 히알루론산(HA) 혼합 조성물에서는 단독 처리하였을 때의 세포 성장률보다 우수한 세포증식 효능을 보였으며 특히, DNA 단편 혼합물(PDRN)과 히알루론산(HA)의 중량비가 2:1이 될때 가장 우수한 세포증식 효능을 보였다.In addition, as can be seen in Examples and Comparative Examples, the DNA fragment mixture (PDRN) and hyaluronic acid (HA) mixture composition showed better cell proliferation efficacy than the cell growth rate when treated alone. In particular, the DNA fragment mixture (PDRN) When the weight ratio of hyaluronic acid (HA) to 2:1 was 2:1, the best cell proliferation efficacy was shown.
1-2. CCK-8 assay1-2. CCK-8 assay
실험예 1-1에서 실시한 동일한 방법으로 사람 연골세포를 배양한 다음, 실시예 1 내지 5 및 비교예 1 내지 6의 조성물을 세포에 처리하였다. 그 후, 5% CO2가 포함된 37℃ 인큐베이터에서 72시간 동안 배양하였다.Human chondrocytes were cultured in the same manner as in Experimental Example 1-1, and then the cells were treated with the compositions of Examples 1 to 5 and Comparative Examples 1 to 6. Then, incubated for 72 hours in a 37 ℃ incubator containing 5% CO 2 .
세포 증식률은 CCK-8 (Dojindo Molecular Technologies, Japan) 분석법을 이용하였다. 72시간 배양 세포에 CCK-8 시약을 첨가하여 2시간 동안 37℃, 5% CO2 배양기에서 배양하였다. ELISA reader (SPECTRAmax® M2 Microplate Spectrophotometer; Molecular Devices, USA)를 사용하여 450nm에서 흡광도를 측정하였으며, 음성 대조군의 흡광도를 기준으로 세포 증식률을 산출하였다.Cell proliferation rate was measured using CCK-8 (Dojindo Molecular Technologies, Japan) assay. CCK-8 reagent was added to 72-hour cultured cells and cultured in an incubator at 37° C., 5% CO 2 for 2 hours. Absorbance was measured at 450 nm using an ELISA reader (SPECTRAmax® M2 Microplate Spectrophotometer; Molecular Devices, USA), and the cell proliferation rate was calculated based on the absorbance of the negative control group.
100±1.1
100±1.1
표 3의 결과에서도, DNA 단편 혼합물(PDRN)과 히알루론산(HA) 혼합 조성물에서는 단독 처리하였을 때의 세포 성장률보다 우수한 세포 증식 효능을 보였다.Also in the results of Table 3, the DNA fragment mixture (PDRN) and the hyaluronic acid (HA) mixture composition showed superior cell proliferation efficacy than the cell growth rate when treated alone.
또한, DNA 단편 혼합물(PDRN)과 히알루론산의 중량비가 2:1이 될때 가장 우수한 세포증식 효능을 보였다.In addition, when the weight ratio of the DNA fragment mixture (PDRN) and hyaluronic acid was 2:1, the best cell proliferation efficacy was shown.
<< 실시예Example 6 내지 9. 분자량에 따른 DNA 단편 혼합물 및 히알루론산 조성물의 제조> 6 to 9. Preparation of DNA fragment mixture and hyaluronic acid composition according to molecular weight>
상기와 같이 세포증식 효능이 가장 우수한 DNA 단편 혼합물(PDRN)과 히알루론산(HA)의 중량비가 2:1이 될 때(실시예 3), 각 분자량에 따른 세포증식 효능을 알아보고자 실시예 6 내지 9의 조성물을 제조하였다.When the weight ratio of the DNA fragment mixture (PDRN) and hyaluronic acid (HA) having the best cell proliferation efficacy as described above is 2:1 (Example 3), Examples 6 to A composition of 9 was prepared.
분자량(kDa)DNA fragment mixture (PDRN)
Molecular Weight (kDa)
분자량(kDa)Hyaluronic acid (HA)
Molecular Weight (kDa)
2-1. 실시예 6 내지 9의 세포 수 측정2-1. Cell number measurement of Examples 6 to 9
상기 1-1의 세포수 측정에 따라 실시예 6 내지 9의 세포 증식 효능을 측정한 결과, 하기 표 5에서와 같이 실시예 3과 비교하여 DNA 단편혼합물(PDRN)과 히알루론산(HA)의 분자량이 커지거나 작아지면 세포 증식 효능이 떨어짐을 알 수 있었다.As a result of measuring the cell proliferation efficacy of Examples 6 to 9 according to the cell number measurement of 1-1, the molecular weight of the DNA fragment mixture (PDRN) and hyaluronic acid (HA) compared to Example 3 as shown in Table 5 below It was found that the cell proliferation efficacy was reduced when the cells became larger or smaller.
이것으로 실시예 3과 같이 DNA 단편혼합물(PDRN)의 분자량이 300~3,000kDa, 히알루론산(HA)의 분자량이 100~3,000kDa이며 중량비가 2:1일 때가 세포 증식 효능이 가장 우수한 최적의 분자량 즉, 최적의 점도임을 확인하였다.As in Example 3, the molecular weight of the DNA fragment mixture (PDRN) is 300-3,000 kDa, the molecular weight of hyaluronic acid (HA) is 100-3,000 kDa, and the optimal molecular weight is the best when the weight ratio is 2: 1 That is, it was confirmed that the viscosity was optimal.
(5.2±1)×104
(5.2±1)×10 4
<< 실험예Experimental example 2. 모노-요오드아세테이트(Mono- 2. Mono-iodine acetate (Mono- iodoacetateiodoacetate ) 유발 ) cause 골관염osteoarthritis 모델에on the model 서의 항통증 효력평가>Evaluation of anti-pain efficacy
약 8주령의 Male SD rat 각 조성물 당 5마리씩 75마리를 구입한 후, 온도 23±2℃, 상대습도 40-60%이 유지되는 청정구역에서 1주일 동안 안정화하였다. 각 Male SD rat의 오른쪽 다리에 Mono-idoacetate(MIA)를 50μL(60 mg/mL)를 투여하여 골관절염을 유발하였다. MIA 투여 3일 후 오른쪽 다리의 통증정도를 체중 밸런스를 측정함으로서 평가하였다. 측정 후 실시예 1 내지 7 및 비교예 1 내지 8의 조성물 당 5마리씩 총 75마리에 50uL를 실린지로 관절강 내에 1회 투여하였다. 투여 1, 3, 7 일 후 관절의 통증정도를 평가하고, 표 6에 나타내었다.After purchasing 75 male SD rats of about 8 weeks of age, 5 for each composition, they were stabilized for 1 week in a clean area where a temperature of 23±2℃ and a relative humidity of 40-60% was maintained. Osteoarthritis was induced by administering 50 μL (60 mg/mL) of Mono-idoacetate (MIA) to the right leg of each male SD rat. After 3 days of MIA administration, the pain level in the right leg was evaluated by measuring the weight balance. After the measurement, 50 uL of each of the compositions of Examples 1 to 7 and Comparative Examples 1 to 8 was administered to a total of 75 mice once intra-articularly by syringe. After 1, 3, and 7 days of administration, the pain level of the joint was evaluated, and it is shown in Table 6.
통증의 정도를 평가하는 방법은 가장 일반적으로 사용되는 weight distribution(%)을 이용하여 오른쪽 다리 체중부하율(%)을 측정하였으며, 이를 바탕으로 체중 부하 회복율(%)을 계산하였다. 체중부하율은 Incapacitance meter tester를 이용하여 각 뒷다리에 가해진 세기를 10초에 걸쳐 평균을 산출하였다. 체중부하율 및 체중 부하 회복율은 각각 하기 수학식 1 및 2와 같다.To evaluate the severity of pain, the weight-bearing rate (%) of the right leg was measured using the most commonly used weight distribution (%), and the weight-bearing recovery rate (%) was calculated based on this. The weight-bearing rate was calculated as the average of the strength applied to each hind leg over 10 seconds using an incapacitance meter tester. The weight-bearing rate and the weight-bearing recovery rate are as shown in Equations 1 and 2, respectively.
[수학식 1] [Equation 1]
체중부하율 (%) ={오른쪽 다리 체중부하율 / (오른쪽 다리 체중부하율 + 왼쪽 다리 체중부하율)}×100Weight-bearing ratio (%) = {Right leg weight-bearing ratio / (Right leg weight-bearing ratio + left leg weight-bearing ratio)}×100
[수학식 2][Equation 2]
체중 부하 회복율 (%) = {(약물투여 후 오른쪽 다리 체중부하율 - MIA 투여 3일 후 오른쪽 다리 체중부하율) / MIA 투여 3일 후 오른쪽 다리 체중부하율}×100 Weight bearing recovery rate (%) = {(Right leg weight bearing rate after drug administration - Right leg weight bearing rate 3 days after MIA administration) / Right leg weight bearing rate 3 days after MIA administration}×100
상기 실험 결과의 비교예 1 내지 6에서 알 수 있는 바와 같이, 히알루론산(HA)을 단독 처리 했을 때 보다 DNA 단편 혼합물(PDRN)을 단독 처리 했을 때 체중 부하 회복율이 더 높았다. As can be seen from Comparative Examples 1 to 6 of the experimental results, the recovery rate of weight bearing was higher when the DNA fragment mixture (PDRN) was treated alone than when the hyaluronic acid (HA) was treated alone.
또한, 실시예 1 내지 5과 비교예 1 내지 6에서 확인할 수 있듯이, DNA 단편 혼합물(PDRN)과 히알루론산(HA) 혼합 조성물에서는 단독 처리하였을 때 보다 우수한 체중 부하 회복 효능을 보였으며 특히, DNA 단편 혼합물(PDRN)과 히알루론산(HA)의 중량비가 2:1이 될 때 가장 우수한 체중 부하 회복 효능을 보였다.In addition, as can be seen in Examples 1 to 5 and Comparative Examples 1 to 6, the DNA fragment mixture (PDRN) and hyaluronic acid (HA) mixture composition showed better weight-bearing recovery efficacy than when treated alone, and in particular, DNA fragments When the weight ratio of the mixture (PDRN) and hyaluronic acid (HA) was 2:1, the best weight bearing recovery effect was shown.
게다가 실시예 6 내지 9에서와 같이 실시예3 보다 분자량이 크거나 낮아질 때, 즉 점도가 높아지거나 낮아지면 체중 부하 회복 효능이 떨어짐을 보여, 실시예 3과 같이 DNA 단편혼합물(PDRN)의 분자량이 300~3,000kDa, 히알루론산(HA)의 분자량이 100~3,000kDa이며 중량비가 2:1일때가 항통증 효능이 가장 우수한 최적의 분자량 즉, 최적의 점도임을 확인하였다.In addition, as in Examples 6 to 9, when the molecular weight is higher or lower than in Example 3, that is, when the viscosity is increased or lowered, the weight-bearing recovery efficacy is lowered. As in Example 3, the molecular weight of the DNA fragment mixture (PDRN) is When the molecular weight of 300-3,000 kDa, hyaluronic acid (HA) was 100-3,000 kDa, and the weight ratio was 2:1, it was confirmed that the optimal molecular weight with the best anti-pain effect, that is, the optimal viscosity.
따라서 본 발명은 세포증식 효능의 극대화에 따른 연골 재생과 윤활기능으로 골관절염 치료에 효과가 있는 최적의 중량비와 분자량을 갖춘 DNA 단편 혼합물(PDRN)과 히알루론산(HA) 혼합 조성물을 제공한다.Accordingly, the present invention provides a DNA fragment mixture (PDRN) and hyaluronic acid (HA) mixture composition having an optimal weight ratio and molecular weight that is effective in treating osteoarthritis with cartilage regeneration and lubricating function according to maximization of cell proliferation efficacy.
Claims (5)
2% by weight of a DNA fragment mixture isolated from fish testis having a molecular weight of 300 to 3,000 kDa as an active ingredient, and 1% by weight of hyaluronic acid having a molecular weight of 100 to 3,000 kDa Contains the DNA fragment mixture and hyaluronic acid in a weight ratio of 2:1 Cartilage regeneration composition, characterized in that.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020180039522A KR102285724B1 (en) | 2018-04-05 | 2018-04-05 | Composition for regenerating cartilage comprising polydeoxyribonucleotide and hyaluronic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020180039522A KR102285724B1 (en) | 2018-04-05 | 2018-04-05 | Composition for regenerating cartilage comprising polydeoxyribonucleotide and hyaluronic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20190116636A KR20190116636A (en) | 2019-10-15 |
| KR102285724B1 true KR102285724B1 (en) | 2021-08-03 |
Family
ID=68209233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020180039522A KR102285724B1 (en) | 2018-04-05 | 2018-04-05 | Composition for regenerating cartilage comprising polydeoxyribonucleotide and hyaluronic acid |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102285724B1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT202100001496A1 (en) * | 2021-01-26 | 2022-07-26 | I R A St Ricerche Applicate S P A | PROCESS FOR THE PRODUCTION OF A POLYNUCLEOTIDE-BASED GEL AND ITS USE IN THE TREATMENT OF OSTEOARTICULAR PATHOLOGIES |
| KR102503635B1 (en) * | 2021-12-29 | 2023-02-24 | 주식회사 비알팜 | Composition comprising fragmented dna mixture and protamine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010049898A1 (en) * | 2008-10-30 | 2010-05-06 | Medevice S.P.A. | Injectable polydeoxyribonucleotide composition for the treatment of osteoarticular diseases |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101739409B1 (en) * | 2011-10-21 | 2017-05-25 | 주식회사 파마리서치프로덕트 | Composition comprising mixture of DNA fragments separated from fish's testis for regenerating cartilage |
| KR102580370B1 (en) * | 2015-04-27 | 2023-09-20 | 주식회사 파마리서치 | Adhesion barrier containing natural-polymer with biocompatibility and DNA fragment mixture, and process for producing the same |
| KR101916193B1 (en) * | 2016-08-25 | 2018-11-07 | 주식회사 파마리서치프로덕트 | Injectable composition comprising nucleic acid and chitosan for articular cavity |
-
2018
- 2018-04-05 KR KR1020180039522A patent/KR102285724B1/en active IP Right Review Request
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010049898A1 (en) * | 2008-10-30 | 2010-05-06 | Medevice S.P.A. | Injectable polydeoxyribonucleotide composition for the treatment of osteoarticular diseases |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20190116636A (en) | 2019-10-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100989109B1 (en) | Use of a mixture of sodium hyaluronate and chondroitin sulfate for the treatment of osteoarthritis | |
| Grecomoro et al. | Therapeutic synergism between hyaluronic acid and dexamethasone in the intra-articular treatment of osteoarthritis of the knee: a preliminary open study | |
| EP0433817B1 (en) | Combined anti-inflammatory agent | |
| US3966908A (en) | Method of treating degenerative joint afflictions | |
| JP6782636B2 (en) | Aqueous capsaicinoid formulations and manufacturing and usage | |
| US20210145850A1 (en) | Composition, for osteoarthritis treatment, comprising hydrophilized sulfasalazine and hyaluronic acid and method for preparing same | |
| EP2090307A1 (en) | Pharmaceutical composition for the treatment or prevention of osteoarticular diseases | |
| KR102285724B1 (en) | Composition for regenerating cartilage comprising polydeoxyribonucleotide and hyaluronic acid | |
| CA2100657A1 (en) | Method for treatment of acute and chronic painful arthropathic conditions in human and other mammals | |
| EP2497468A1 (en) | Polyacrylamide hydrogel for use in the treatment and/or prevention of joint swelling and/or bone oedema in a mammal suffering from arthritis | |
| KR101831168B1 (en) | A composition for treating osteoarthritis comprising hyaluronic acid and magnesium | |
| TWI448292B (en) | Pharmaceutical compositions containing bradykinin antagonists and hyaluronic acid, and uses thereof | |
| US20220280555A1 (en) | Composition Comprising Hyaluronic Acid and Pluronic for Preventing or Treating Articular and Cartilage Injury | |
| ES2616004T3 (en) | Prophylactic agent and / or therapeutic agent and / or exacerbating suppressing agent for human knee osteortritis | |
| KR101637883B1 (en) | Polyethylene glycol hydrogel injection | |
| AU2018258100B2 (en) | Variants of human BMP7 protein | |
| JP2018536642A (en) | Cross-linked serum albumin containing hyaluronic acid for the treatment of facet joint diseases | |
| KR20220152904A (en) | Composition for preventing or treating arthritis containing dna fraction and chondroitin sulfate | |
| BRPI0902144A2 (en) | process for preparing a solid oral administration pharmaceutical composition containing the active ingredients glycosamine and meloxicam and use of the association between glycosamine and meloxicam | |
| CA3223710A1 (en) | Pharmaceutical compositions comprising glp-1r agonists | |
| WO2010109736A1 (en) | Cartilage production promoter and prophylactic or therapeutic agent for diseases associated with cartilage damage | |
| KR20210076580A (en) | Composition for preventing and treating arthritis comprising niclosamide | |
| JP5629940B2 (en) | Combination preparations for the treatment of joint diseases | |
| KR20220003551A (en) | Pharmaceutical composition comprising a statin and a hyaluronic acid derivative | |
| Ghazal et al. | EffECT Of INTRa-aRTICUlaR CORTICOsTEROID INjECTIONs ON TEmpOROmaNDIbUlaR jOINT sTRUCTURE (ExpERImENTal sTUDy) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E601 | Decision to refuse application | ||
| J201 | Request for trial against refusal decision | ||
| J301 | Trial decision |
Free format text: TRIAL NUMBER: 2020101001441; TRIAL DECISION FOR APPEAL AGAINST DECISION TO DECLINE REFUSAL REQUESTED 20200528 Effective date: 20210624 |
|
| GRNO | Decision to grant (after opposition) | ||
| GRNT | Written decision to grant | ||
| Z072 | Maintenance of patent after cancellation proceedings: certified copy of decision transmitted [new post grant opposition system as of 20170301] | ||
| Z131 | Decision taken on request for patent cancellation [new post grant opposition system as of 20170301] | ||
| Z072 | Maintenance of patent after cancellation proceedings: certified copy of decision transmitted [new post grant opposition system as of 20170301] |