KR20020041712A - Process for Preparing Sustained Release Form of Micelle Employing Conjugate of Anticancer Drug and Biodegradable Polymer - Google Patents
Process for Preparing Sustained Release Form of Micelle Employing Conjugate of Anticancer Drug and Biodegradable Polymer Download PDFInfo
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Abstract
Description
본 발명은 생분해성 고분자와 항암제의 접합체를 이용한 서방형 미셀제제(micelle)의 제조방법에 관한 것이다. 좀 더 구체적으로, 본 발명은 생분해성 고분자를 항암제 등의 약물과 결합시킨 후, 수용액상에서 미셀구조를 형성시키는 서방형 미셀제제의 제조방법 및 전기 제조방법에 의하여 제조된 서방형 미셀제제에 관한 것이다.The present invention relates to a method for producing a sustained-release micelle using a conjugate of a biodegradable polymer and an anticancer agent. More specifically, the present invention relates to a sustained-release micelle preparation prepared by a method for preparing a sustained-release micelle preparation and an electrical preparation method of combining a biodegradable polymer with a drug such as an anticancer agent and then forming a micelle structure in an aqueous solution. .
일반적으로, 생체내로 약물을 전달하기 위한 전달체에 사용되는 고분자는 생체적으로 합성되거나 생체 분해성의 성질이 요구된다. 대표적으로, 이러한 조건을 만족시키는 고분자에는 폴리에스테르 결합으로 이루어진 지방족 폴리에스테르류가 있으며, 이는 미국식품의약품안전청(FDA)의 승인을 받아 오랫동안 약물전달용 담체 또는 수술용 봉합사 등으로 광범위하게 사용되고 있다. 전기 지방족 폴리에스테르의 구체적인 예로는, 폴리락트산(polylactic acid, PLA), 폴리글리콜산(polyglycolic acid, PGA), 폴리(D,L-락트산-co-글리콜산)(poly(D,L-lactic-co-glycolic acid, 이하 'PLGA'라 함), 폴리(카프로락톤), 폴리(발레로락톤), 폴리(하이드록시 부티레이트) 및 폴리(하이드록시 발러레이트) 등이 있다. 상술한 지방족 폴리에스테르 중에서도 특히, PLGA의 경우 락트산과 글리콜산 단량체의 비율을 조절하거나, 고분자 합성과정을 변형시킴으로써, 다양한 분해수명을 갖는 생분해성 고분자를 수득하는데 유용하게 사용된다.In general, polymers used in delivery vehicles for delivery of drugs in vivo are either synthesized in vivo or require biodegradable properties. Typically, polymers satisfying these conditions include aliphatic polyesters composed of polyester bonds, which have been widely used as drug delivery carriers or surgical sutures for a long time with approval from the US Food and Drug Administration (FDA). Specific examples of the electroaliphatic polyester include polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L-lactic-) co-glycolic acid, hereinafter referred to as “PLGA”), poly (caprolactone), poly (valerolactone), poly (hydroxy butyrate) and poly (hydroxy valerate), among the aliphatic polyesters described above. In particular, in the case of PLGA, by controlling the ratio of lactic acid and glycolic acid monomer, or by modifying the polymer synthesis process, it is useful to obtain a biodegradable polymer having various degradation life.
생체분해성 고분자는 생체내에서 일정시간이 지나면 자연적으로 분해되기 때문에 인체에 전혀 해를 미치지 않는다. 예를 들어, PLGA와 같은 고분자는 생체내에서 분해되어 락트산와 글리콜산으로 분해되어 인체에 아무런 해가 없다. 그러므로, 생체분해성 고분자를 이용하여 약물전달 담체를 만들게 되면, 여러가지 약물에 대해 지속적인 방출효과를 기대할 수 있다. 특히, 일정한 시간을 주기로 투약해야만 일정한 혈중농도를 유지하게 되어 약물의 효과가 나타나는 약물의 경우, 생체 분해성 고분자로 만든 약물전달 담체에 약물이 봉입되게 되면, 고분자의 담체의 분해에 따라 약물이 계속적으로 방출되므로, 이러한 서방형 제제가 다양한 약물에 응용되고 있다. 이러한 방출기작을 가진 담체에는 미립구(microsphere)와 나노입자(nanoparticle), 미셀(micelle) 등이 있는데, 미립구의 경우는 그 크기가 수십-수백 마이크로미터정도이며, 나노입자는 수백 나노미터정도이고, 미셀의 경우는 일백 나노미터 내외이다. 미립구는 피하근육 주사를 위한 약물전달체로 사용되며, 주로 단백질이나 기타 약물의 지속적인 방출효과를 얻기 위해 사용된다. 나노입자는 주로 혈관 주사를 목적으로 하는 제제이며, 약물의 지속적인 방출을 얻는 동시에 수동적인 암세포 지향을 위해 사용되는 약물전달 담체이며, 미셀 역시 유사한 용도로서 사용될 수 있다. 나노입자나 미셀을 이용하여 항암제를 투여하면, 수십에서 수백나노미터에 이르는 입자의 크기 때문에, 혈관벽내 세포연접이 느슨한 암세포조직에는 전달이 가능하지만, 상대적으로 혈관벽내 세포연접이 조밀한 일반세포조직에는 제대로 전달될 수 없으므로, 항암제의 비특이성을 물리적으로 극복할 수 있다.Biodegradable polymers do not harm the human body at all because they decompose naturally after a certain time in vivo. For example, polymers such as PLGA are degraded in vivo and decomposed into lactic acid and glycolic acid, which are harmless to the human body. Therefore, if a drug delivery carrier is made using a biodegradable polymer, a continuous release effect can be expected for various drugs. In particular, in the case of drugs in which a constant blood concentration is maintained only after a certain period of time to maintain the effect of the drug, when the drug is enclosed in a drug delivery carrier made of a biodegradable polymer, the drug continues to be decomposed according to the decomposition of the carrier of the polymer. Because of their release, these sustained release formulations are being applied to a variety of drugs. Carriers with such a release mechanism include microspheres, nanoparticles, and micelles, which are about tens to hundreds of micrometers in size, and nanoparticles about hundreds of nanometers in size. In the case of micelles, it is around 100 nanometers. Microspheres are used as drug carriers for subcutaneous muscle injection and are used primarily to achieve sustained release of proteins and other drugs. Nanoparticles are agents intended primarily for vascular injection, are drug delivery carriers used for passive cancer cell orientation while obtaining sustained release of drugs, and micelles can also be used for similar applications. When anticancer drugs are administered using nanoparticles or micelles, the size of particles ranging from tens to hundreds of nanometers enables them to be delivered to cancer cell tissues with loose cell junctions in the blood vessel walls, but relatively general cell tissues with relatively tight cell junctions in the blood vessel walls. Because it cannot be delivered properly, it can physically overcome the specificity of anticancer drugs.
한편, 기존의 많은 약물전달 담체의 문제점 중의 하나는 약물이 지속적으로 방출되지 않는다는 점이다. 즉, 종래의 약물전달 담체의 경우에 초기에는 담체의 표면에 있는 약물이 확산의 형태로 방출되어 초기 방출량은 상당히 높은 반면, 시간이 지날수록 방출량이 점차 줄어들기 때문에 혈중농도를 일정하게 유지시키는데 문제가 발생하게 된다. 그 외에도, 담체 내에 다량의 약물을 봉입해야 하는 바, 다량의 약물이 사용됨으로써 약물전달 담체의 제제화 과정이나 기타 조제과정에서 약물 소실율이 높기 때문에, 담체를 제조한 후 담체내에 존재하는 약물의 양을 조사해보면, 50퍼센트 이상의 약물이 소실된 경우가 많다는 것은 이미 주지된 사실이다. 따라서, 전세계의 많은 연구진이 약물의 봉입률을 높이기 위해서 노력하고 있다.On the other hand, one of the problems of many existing drug delivery carriers is that the drug is not continuously released. That is, in the case of a conventional drug delivery carrier, the drug on the surface of the carrier is initially released in the form of diffusion so that the initial release amount is quite high, while the release amount gradually decreases over time, thereby maintaining a constant blood concentration. Will occur. In addition, since a large amount of drug must be encapsulated in a carrier, since a large amount of drug is used, the drug loss rate is high during the formulation or other preparation of the drug delivery carrier. Investigations have already shown that more than 50 percent of drugs are lost. Therefore, many researchers around the world are working to increase the drug's inclusion rate.
한편, 항암치료를 위하여 현재까지 독소루비신(doxorubicin), 아드리아마이신(adriamycin), 시스플라틴(cisplatin), 택솔(taxol), 5-플루오로우라실(5-fluorouracil) 등의 약물을 사용한 화학 치료요법이 광범위하게 사용되고 있다. 그러나, 전기 항암제는 과도한 부작용으로 인하여 다량을 투여할 수 없으며, 치료가능한 정도의 양만 투여해도 환자에게 심한 고통을 가져다 주고 있는 실정이다. 이러한 부작용이 나타나는 원인은 항암제의 비선택성 때문인데, 항암제가 암세포에만 작용하지 않고 일반세포에도 작용함으로써 암세포만을 사멸시키지 못하고, 정상적인 세포의 성장도 억제, 괴사시키기 때문에 환자는 심한 고통을 느끼게 되는 것이다.Meanwhile, chemotherapy using drugs such as doxorubicin, adriamycin, cisplatin, cisplatin, taxol, and 5-fluorouracil has been widely used for chemotherapy. It is used. However, the anticancer drugs cannot be administered in large amounts due to excessive side effects, and even if the amount is curable, the situation is causing severe pain to the patient. The cause of these side effects is due to the non-selectivity of the anticancer drugs, the anticancer drugs do not act only on cancer cells, but also on normal cells to kill only cancer cells, and also inhibits and grows the normal cells, the patient will feel severe pain.
따라서, 암세포만 선택적으로 사멸시킬 수 있다면 보다 많은 양의 항암제를 투여할 수 있기 때문에 암환자를 치료하는 효과적인 수단이 될 수 있을 것으로 예상되고 있다. 이를 위하여, 지금까지 암세포만을 목표로 하기 위한 방법으로 암세포에만 특별히 존재하는 항원에 대한 수용체를 약물에 부착하여 항암제가 암세포에만 부착되도록 하는 방법이 주로 이용되고 있다(참조: T. Minko, et al., J. Control. Rel., 54:223-233, 1998; A. Colin de Verdiere, et al., Cancer Chemother. Pharmacol., 33:504-508, 1994).Therefore, if only cancer cells can be selectively killed, it is expected to be an effective means of treating cancer patients since a greater amount of anticancer drugs can be administered. To this end, a method for targeting only cancer cells has been mainly used to attach a receptor for an antigen specifically present only on cancer cells to a drug so that the anticancer agent is attached only to cancer cells (T. Minko, et al. J. Control. Rel., 54: 223-233, 1998; A. Colin de Verdiere, et al., Cancer Chemother. Pharmacol., 33: 504-508, 1994).
그러나, 이러한 항암제도 반복적으로 투여하게 되면, 암세포가 세포내로 침투한 항암제를 세포 밖으로 다시 내보내는 펌프가 작동되어 일정 농도 이상의 항암제가 세포질 내에서 존재할 수 없게 되어, 계속적인 항암제의 투여에도 항암효과를 나타낼 수 없다. 따라서, 이러한 문제를 해결하기 위한 노력이 계속되고 있으나, 서방형의 암세포 지향성 약물제제에 대한 주목할만한 결과가 아직까지 나타나지 않고 있는 실정이다.However, if the anticancer agent is repeatedly administered, a pump for reactivating the anticancer agent penetrated into the cell to the outside of the cell is activated, so that a certain concentration of the anticancer agent cannot exist in the cytoplasm, and thus the anticancer agent may be effective even after continuous administration of the anticancer agent. Can not. Therefore, efforts to solve these problems continue, but the results of the sustained-release cancer cell-oriented drug formulations have not yet appeared.
이에, 본 발명자들은 종래의 항암제의 문제점인 비특이성으로 인한 심한 부작용을 줄이며 약물의 지속적인 방출이 가능한 혈관 주사용 제제를 만들기 위해 예의 연구 노력한 결과, 항암제를 수용액상에서 미셀의 구조를 가질 수 있는 생체 분해성 고분자와 화학적으로 결합시켜 생체 분해성과 암세포 지향성을 가진 항암제 담체인 서방형 미셀제제를 제조할 경우, 약물의 함유율이 높아지며 약물의 방출 속도를 조절할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made diligent research efforts to reduce the serious side effects due to non-specificity, which is a problem of conventional anticancer drugs, and to make vascular injection preparations capable of sustained release of drugs. When chemically combined with a polymer to produce a sustained-release micelle formulation, which is an anticancer carrier having biodegradability and cancer cell orientation, it was confirmed that the drug content is increased and the release rate of the drug can be controlled, thereby completing the present invention.
결국, 본 발명의 주된 목적은 고분자와 약물의 접합체를 이용한 서방형 미셀제제를 제조하는 방법을 제공하는 것이다.After all, the main object of the present invention is to provide a method for producing a sustained-release micelle formulation using a conjugate of a polymer and a drug.
본 발명의 다른 목적은 전기 방법에 의하여 제조된 서방형 미셀제제를 제공하는 것이다.Another object of the present invention is to provide a sustained release micelle preparation prepared by the electric method.
도 1은 본 발명에 의한 서방형 미셀제제의 제조방법을 모식적으로 나타낸 그림이다.BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows typically the manufacturing method of a sustained-release micelle preparation by this invention.
도 2는 독소루비신이 결합된 서방형 미셀단위체의 제조방법을 도식적으로 나타낸 그림이다.2 is a diagram schematically illustrating a method for preparing a sustained-release micelle unit in which doxorubicin is bound.
도 3은 하이드라진을 이용한 독소루비신이 결합된 서방형 미셀단위체의 제조방법을 도식적으로 나타낸 그림이다.3 is a diagram schematically illustrating a method for preparing a sustained-release micelle unit in which doxorubicin is bound using hydrazine.
도 4는 시간에 따른 독소루비신의 누적 방출량을 나타낸 그래프이다.4 is a graph showing the cumulative release amount of doxorubicin over time.
도 5는 독소루비신과 본 발명의 독소루비신이 결합된 서방형 미셀제제의 항암효과를 비교한 그래프이다.Figure 5 is a graph comparing the anticancer effect of doxorubicin and the sustained-release micelles combined doxorubicin of the present invention.
본 발명의 서방형 미셀제제의 제조방법은 생분해성 폴리에스테르계 고분자와 폴리에틸렌글리콜(PEG)계 고분자를 옥토산주석산염(stannous octoate)의 존재하에 진공상태에서 160 내지 200℃의 온도에서 2 내지 6시간 동안 공중합반응시켜서, 한쪽에는 소수성 부분을 가지고, 다른 한쪽에는 친수성 부분을 가지며, 소수성 부분의 말단에 하이드록시기를 가지는 블록 공중합체를 수득하는 공정; 전기 블록 공중합체를 유기용매에 용해시키고, 상온에서 피리딘과 질소의 존재하에 링커 화합물과 반응시켜서, 블록 공중합체의 하이드록시기에 링커 화합물을 결합시키는 공정; 링커 화합물이 결합된 블록 공중합체를 히드라진과 반응시키거나 또는 반응시키지 않은 후, 약물과 공유결합시켜서, 약물과 블록 공중합체가 결합된 형태의 미셀단위체를 수득하는 공정; 및, 전기 수득한 미셀단위체를 수용액상에서 분산시켜, 서방형 미셀제제를 제조하는 공정을 포함한다(참조: 도 1).The preparation method of the sustained-release micelle preparation of the present invention is a biodegradable polyester-based polymer and polyethylene glycol (PEG) -based polymer in the presence of stannous octoate in a vacuum state of 2 to 6 at a temperature of 160 to 200 ℃ Copolymerizing for a time period to obtain a block copolymer having a hydrophobic portion on one side, a hydrophilic portion on the other, and a hydroxyl group at the end of the hydrophobic portion; Dissolving the electric block copolymer in an organic solvent and reacting with a linker compound in the presence of pyridine and nitrogen at room temperature to bond the linker compound to the hydroxy group of the block copolymer; Reacting or not reacting the block copolymer in which the linker compound is bound with hydrazine and then covalently bonding the drug to obtain a micelle unit in a form in which the drug and the block copolymer are bound; And dispersing the previously obtained micelle unit in an aqueous solution to prepare a sustained-release micelle preparation (see FIG. 1).
이하, 본 발명의 서방형 미셀제제를 제조하는 방법을 공정별로 나누어 설명하고자 한다.Hereinafter, the method for preparing the sustained-release micelle formulation of the present invention will be described by dividing by process.
제 1공정: 블록공중합체의 수득 First Step : Obtaining Block Copolymer
생분해성 폴리에스테르계 고분자와 폴리에틸렌글리콜(PEG)계 고분자를 옥토산주석산염(stannous octoate)의 존재하에 진공상태에서 160 내지 200℃의 온도에서 2 내지 6시간 동안 공중합반응시켜서, 한쪽에는 소수성 부분을 가지고, 다른 한쪽에는 친수성 부분을 가지며, 소수성 부분의 말단에 하이드록시기를 가지는 블록 공중합체를 수득한다: 이때, 폴리에스테르계 고분자는 폴리락트산(PLA), 폴리글리콜산(PGA), 폴리(D,L-락트산-co-그리콜산, PLGA), 폴리(카프로락톤), 폴리(발레로락톤), 폴리(하이드록시 부티레이트) 또는 폴리(하이드록시 발러레이트)를 사용할 수 있으나, PLGA를 사용함이 바람직하고, 글리콜산과 락트산을 50:50(몰비)으로 반응시켜 제조한 PLGA를 사용함이 가장 바람직하다. 폴리에틸렌글리콜계 고분자는 메톡시폴리에틸렌글리콜(methoxypolyethyleneglycol, mPEG)을 사용함이 바람직하다.The biodegradable polyester polymer and the polyethylene glycol (PEG) polymer are copolymerized in the presence of stannous octoate at a temperature of 160 to 200 ° C. for 2 to 6 hours in a vacuum state, and a hydrophobic portion is formed on one side. A block copolymer having a hydrophilic moiety on the other side and a hydroxyl group at the end of the hydrophobic moiety: wherein the polyester-based polymer is polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid, PLGA), poly (caprolactone), poly (valerolactone), poly (hydroxy butyrate) or poly (hydroxy valerate) may be used, but PLGA is preferred. Most preferably, PLGA prepared by reacting glycolic acid and lactic acid at 50:50 (molar ratio) is used. The polyethylene glycol polymer is preferably methoxy polyethylene glycol (mPEG).
제 2공정: 블록 공중합체의 기능기의 활성화에 의한 링커의 접합 2nd step : conjugation of linker by activation of functional group of block copolymer
전기 블록 공중합체를 유기용매에 용해시키고, 상온에서 피리딘과 질소의 존재하에 링커 화합물과 반응시켜서, 블록 공중합체의 하이드록시기에 링커 화합물을 결합시킨다: 이때, 유기용매는 특별히 제한되는 것은 아니나, 메틸렌클로라이드를 사용함이 바람직하고, 링커 화합물로는 p-니트로페닐클로로포름산염, 카르보닐디이미다졸(CDI), N,N'-디숙신이미딜 카르보네이트(DSC) 또는 이들의 혼합물을 사용할수 있으나, p-니트로페닐클로로포름산염을 사용함이 바람직하며, 반응시 블록 공중합체, 링커 화합물 및 피리딘의 혼합비율은 1:2:2 내지 1:2:6(몰비)이고, 반응시간은 2 내지 6시간이다.The electric block copolymer is dissolved in an organic solvent and reacted with a linker compound in the presence of pyridine and nitrogen at room temperature to bond the linker compound to the hydroxy group of the block copolymer: wherein the organic solvent is not particularly limited, It is preferable to use methylene chloride, and as the linker compound, p-nitrophenylchloroformate, carbonyldiimidazole (CDI), N, N'-disuccinimidyl carbonate (DSC) or a mixture thereof can be used. However, it is preferable to use p-nitrophenylchloroformate, and the mixing ratio of the block copolymer, the linker compound and the pyridine during the reaction is 1: 2: 2 to 1: 2: 6 (molar ratio), and the reaction time is 2 to 6 It's time.
제 3공정: 약물ㆍ생분해성 고분자의 중합체 수득 Step 3 : obtaining a polymer of drug and biodegradable polymer
링커 화합물이 결합된 블록 공중합체를 히드라진과 반응시키거나 또는 반응시키지 않은 후, 약물과 공유결합시켜서, 약물과 블록 공중합체가 결합된 형태의 미셀단위체를 수득한다: 이때, 히드라진과 반응시킨 전기 블록 공중합체는 블록 공중합체의 링커 화합물이 약물의 케톤기와 결합하여 미셀단위체를 형성하고, 히드라진과 반응시키지 않은 전기 블록 공중합체는 블록 공중합체의 링커 화합물이 약물의 아민기와 결합하여 미셀단위체를 형성한다. 또한, 약물은 특별히 제한 되지 않으나, 독소루비신, 아드리아마이신, 시스플라틴, 택솔, 5-플루오로우라실 등의 항암제를 사용함이 바람직하다.The block copolymer in which the linker compound is bound is reacted with or not reacted with the hydrazine and then covalently bonded with the drug to obtain a micelle unit in a form in which the drug and the block copolymer are bound: an electric block reacted with the hydrazine. In the copolymer, the linker compound of the block copolymer combines with the ketone group of the drug to form a micelle, and in the electric block copolymer that is not reacted with hydrazine, the linker compound of the block copolymer combines with the amine group of the drug to form a micelle unit. . In addition, the drug is not particularly limited, but anticancer agents such as doxorubicin, adriamycin, cisplatin, taxol, and 5-fluorouracil are preferably used.
제 4공정: 서방형 미셀제제의 제조 Fourth step : preparation of sustained-release micelle preparation
전기 수득한 미셀단위체를 수용액상에서 분산시켜, 서방형 미셀제제를 제조한다: 도 1에서 보듯이, 일정 농도 이상의 미셀단위체를 수용액상에서 분산시키게 되면, 열역학적 평형성에 의하여 자연적으로 미셀 구조를 형성하게 된다. 이처럼형성된 서방형 미셀제제는 생체 조건에서 가수분해 및 효소의 작용으로 인하여 약물이 방출될 수 있으며, 방출된 약물은 블록 공중합체와 결합되지 않은 약물과 동일한 효과를 가지게 된다.The obtained micellar unit is dispersed in an aqueous solution to prepare a sustained-release micelle: As shown in FIG. 1, when the micelle unit of a certain concentration or more is dispersed in an aqueous solution, a micelle structure is naturally formed by thermodynamic equilibrium. The sustained-release micelle formulation thus formed can be released due to hydrolysis and the action of enzymes in vivo, and the released drug has the same effect as the drug which is not bound to the block copolymer.
전기 방법에 의하여 제조된 본 발명의 서방형 미셀제제는 다음과 같은 특성을 갖는다: 첫째, 약물이 생분해성 고분자의 중합체에 약물이 화학적으로 결합함으로써 물리적으로 약물이 고분자에 봉입된 종래의 미셀에 비하여, 약물의 봉입 효율이 증가된다; 둘째, 약물이 미셀 형성이 가능한 고분자에 연결됨으로써 수용액상에서 자발적으로 미셀의 구조가 형성이 된다; 셋째, 생분해성 고분자의 분해속도에 의존하여 약물이 서서히 방출되는 특징을 갖게 된다.The sustained-release micelle preparation of the present invention prepared by the above method has the following characteristics: First, compared with conventional micelles in which the drug is physically encapsulated in the polymer by the drug chemically bonding to the polymer of the biodegradable polymer. , The encapsulation efficiency of the drug is increased; Second, the drug is spontaneously formed in an aqueous solution by being linked to a polymer capable of forming micelles; Third, the drug is slowly released depending on the rate of degradation of the biodegradable polymer.
한편, 본 발명에 의하여 제조된 서방형 미셀제제의 생체내 약동학(pharmacokinetics)은 다음과 같다: 미셀 구조는 신장배출(renal exclusion)을 회피하게 할 뿐만 아니라, 수동확산(passive diffusion)에 의하여 표적부위로 약물의 혈관투과성(vascular permeability)을 높이게 된다. 아울러, 세포내이입(endocytosis)의 증가 및 약제다제내성(multi-drug resistance, MDR) 효과의 감소에 의하여 생체내에서 미셀구조의 약물의 섭취가 증가하게 된다. 또한, 약물에 접합된 PLGA 골격이 화학적으로 분해되면서 수용성 약물-PLGA 올리고머 분획상에서 약물이 서서히 방출되게 된다.Meanwhile, the in vivo pharmacokinetics of the sustained-release micelle prepared according to the present invention are as follows: micelle structure not only avoids renal exclusion, but also target sites by passive diffusion. This increases the vascular permeability of the drug. In addition, ingestion of micelle-structured drugs is increased in vivo by increasing endocytosis and decreasing the effect of multi-drug resistance (MDR). In addition, the chemical degradation of the PLGA backbone conjugated to the drug causes the drug to be released slowly on the water-soluble drug-PLGA oligomer fraction.
본 발명의 서방형 미셀제제의 제조방법으로 항암제로 알려진 독소루비신을 포함하는 서방형 미셀제제를 제조하고, 이를 종래의 미셀과 비교하였을 경우, 단순히 고분자와 항암제를 물리적으로 혼합하여 제조한 미셀에 비하여 다량의 항암제를 서방형 미셀제제내에 봉입할 수 있음을 확인할 수 있었다. 아울러, 본 발명의 서방형 미셀제제가 종래의 항암제에 비하여, 지속적인 항암제의 방출에 의하여 효과적인 항암 효과를 나타냄을 알 수 있었다.As a method for preparing a sustained-release micelle formulation of the present invention, a sustained-release micelle formulation containing doxorubicin known as an anticancer agent is prepared, and compared with a conventional micelle, a large amount of micelle prepared by physically mixing a polymer and an anticancer agent. It was confirmed that the anticancer agent of can be enclosed in a sustained release micelle formulation. In addition, it was found that the sustained-release micelle formulation of the present invention exhibited an effective anticancer effect by sustained release of the anticancer agent, compared to the conventional anticancer agent.
이하, 실시예를 통하여 본 발명을 보다 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 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예 1: mPEG가 접합된 폴리에스테르계 고분자 mPEG-PLGA의 수득 Example 1 : Obtaining mPEG-bonded polyester polymer mPEG-PLGA
폴리(D,L-락트산-co-글리콜산)(PLGA) 26g을 분자량 700인 메톡시폴리에틸렌글리콜(mPEG) 2g, 분자량 3350인 mPEG 8g 및 분자량 8000인 mPEG 16g과 각각 플라스크에서 혼합하고, 질소 기체 존재하에서 140℃로 가열하여 완전히 융해시켰다. 이어, 진공하에서 전체 중량의 0.05%에 해당하는 옥토산주석산염(stannous octoate)를 첨가한 후, 180℃에서 3시간 동안 반응시켰다. 이어, 반응물을 메틸렌클로라이드(methylenechloride)와 혼합하고, 차갑게 냉각된디에틸에테르(diethylether)에 적가하여 침전시킨 물질을 거름종이로 여과하고, 감압조건하에서 건조시켜 mPEG-PLGA를 수득하였다. 그런 다음, 겔투과 크로마토그래피(gel permeation chromatography, GPC) 및 자기공명분석기를 사용하여, 합성된 mPEG-PLGA의 분자량을 측정하고, 시차 주사 열량계(differential scanning calorimetry, DSC)를 사용하여 이들의 결정질화 온도를 측정하였다(참조: 표 1).26 g of poly (D, L-lactic acid-co-glycolic acid) (PLGA) were mixed in a flask with 2 g of methoxy polyethylene glycol (mPEG) having a molecular weight of 700, 8 g of mPEG having a molecular weight of 3350, and 16 g of mPEG having a molecular weight of 8000, respectively, in a nitrogen gas In the presence, it was heated to 140 ° C. to completely dissolve it. Subsequently, stannous octoate corresponding to 0.05% of the total weight was added under vacuum, followed by reaction at 180 ° C. for 3 hours. Subsequently, the reaction mixture was mixed with methylene chloride, added dropwise to cold cooled diethylether, and the precipitated material was filtered through a filter paper and dried under reduced pressure to obtain mPEG-PLGA. The molecular weights of the synthesized mPEG-PLGA were then measured using gel permeation chromatography (GPC) and magnetic resonance spectroscopy, and their crystallization using differential scanning calorimetry (DSC). The temperature was measured (see Table 1).
상기 표 1에서 보듯이, 각각 다른 분자량을 가지는 3가지의 PLGA-PEG 중합체가 합성되었고, GPC에 의한 분자량(Mw)은 모두 20,000이상이며, DSC에 의한 결정화 온도는 PEG의 분자량이 증가할수록 감소하는 결과를 보였다.As shown in Table 1, three PLGA-PEG polymers each having a different molecular weight were synthesized, the molecular weight (Mw) by GPC are all 20,000 or more, and the crystallization temperature by DSC decreases as the molecular weight of PEG increases. The results were shown.
실시예 2: mPEG-PLGA와 독소루비신이 결합된 서방형 미셀제제의 제조 Example 2 Preparation of Sustained-release micelles Containing mPEG-PLGA and Doxorubicin
전기 수득한 mPEG-PLGA 3g과 링커 화합물인 p-니트로페닐플로로포름산염 80㎎을 30㎖의 메틸렌클로라이드에 용해시키고, 63㎎의 피리딘(pyridine)을 첨가하여 질소기체하에 3시간 동안 반응시켰다. 이어, 반응물을 차가운 디에틸에테르에 적가하여 침전시키고, 침전물을 거름종이에 여과시킨 후, 감압조건에서 건조시켰다. 건조된 반응물 0.25g을 디메틸포름아마이드(dimethylformamide) 7.5㎖에 용해시키고, 10㎎의 독소루비신(doxorubicin-HCl, Sigma Chem. Co., U.S.A) 및 6.75㎎의 트리에틸아민(triethylamine)을 첨가한 후, 실온에서 질소기체하에 24시간동안 반응시켜서, 독소루비신이 결합된 미셀단위체를 수득하였다(참조: 도 2). 전기 미셀단위체를 3차 증류수 300㎖에 분산시켜 독소루비신이 결합된 서방형 미셀제제를 형성하고, 별도의 3차 증류수 3ℓ를 사용하여, 5회에 걸쳐 투석함으로써, 미셀단위체와 결합되지 않은 독소루비신을 제거하였다. 이어, 초원심 분리하여 독소루비신이 결합된 서방형 미셀제제를 강제 침전시킨 후, 상등액을 제거하고 동결건조시켜서, 독소루비신이 결합된 미셀단위체의 형태로 보존하였다.3 g of mPEG-PLGA obtained above and 80 mg of p-nitrophenylfloroformate, a linker compound, were dissolved in 30 ml of methylene chloride, and 63 mg of pyridine was added to react for 3 hours under nitrogen gas. The reaction was then precipitated by dropwise addition to cold diethyl ether, and the precipitate was filtered through a filter paper and dried under reduced pressure. 0.25 g of the dried reactant was dissolved in 7.5 ml of dimethylformamide, 10 mg of doxorubicin-HCl, Sigma Chem. Co., USA and 6.75 mg of triethylamine were added. The reaction was carried out under nitrogen gas at room temperature for 24 hours to obtain doxorubicin-bound micelle unit (see FIG. 2). Disperse the micelle unit in 300 ml of tertiary distilled water to form a sustained-release micelle formulated with doxorubicin, and dialysate five times with another 3 liters of tertiary distilled water to remove doxorubicin not bound to the micelle unit. It was. Subsequently, ultracentrifugation separated the forced release of doxorubicin-linked sustained-release micelles, and the supernatant was removed and lyophilized to preserve doxorubicin-bound micelles.
전기 합성반응에서, mPEG-PLGA에 대한 독소루비신의 접합 효율은 합성된 미셀단위체를 DMSO에 용해시킨 후, 분광계로 독소루비신의 양을 정량하여, 최초에 첨가한 독소루비신 양과의 비율로 결정하였다(참조: 표 2).In the electrosynthesis reaction, the conjugation efficiency of doxorubicin to mPEG-PLGA was determined by dissolving the synthesized micelle unit in DMSO and quantifying the amount of doxorubicin with a spectrometer, and then determining the ratio with the amount of doxorubicin initially added (see Table: 2).
상기 표 2에서 보듯이, mPEG-PLGA에 대한 독소루비신의 접합효율은 34 내지38%(w/w)이며, 미셀단위체에서 독소루비신은 2.2%(w/w)를 차지함을 알 수 있었다.As shown in Table 2, the conjugated efficiency of doxorubicin to mPEG-PLGA was 34 to 38% (w / w), doxorubicin occupies 2.2% (w / w) in the micelle unit.
실시예 3: 히드라존 링커가 결합된 mPEG-PLGA의 수득 Example 3 Obtaining mPEG-PLGA with Hydrazone Linker
mPEG-PLGA 3g과 링커 화합물인 p-니트로페닐클로로포름산염 80㎎을 30㎖의 메틸렌클로라이드에 용해시키고, 피리딘 63㎎을 첨가한 후, 상온에서 질소기체하에 3시간동안 반응시킨 다음, 냉각된 디에틸에테르에 적가하여 침전시켰다. 이어, 침전물을 여과 및 건조시키고, 다시 메틸렌클로라이드에 용해시킨 후, 교반하에 하이드라진(hydrazine, NH2-NH2) 2㎎이 용해된 메틸렌클로라이드 용액을 한방울씩 천천히 적가하며, 계속 교반시키면서 3시간동안 상온에서 반응시켰다. 그런 다음, 반응물을 냉각된 디에틸에테르에 적가하여 침전한 후, 침전물을 여과하고 건조시켜서, 히드라존 링커가 결합된 mPEG-PLGA를 수득하였다(참조: 도 3).3 g of mPEG-PLGA and 80 mg of p-nitrophenylchloroformate, a linker compound, were dissolved in 30 ml of methylene chloride, 63 mg of pyridine was added, followed by reaction under nitrogen gas at room temperature for 3 hours, followed by cooled diethyl. Precipitation was added dropwise to ether. The precipitate was then filtered and dried, dissolved in methylene chloride again, and then slowly added dropwise to the methylene chloride solution in which 2 mg of hydrazine (hydrazine, NH 2 -NH 2 ) was dissolved dropwise, under stirring, for 3 hours while continuing to stir. The reaction was carried out at room temperature. The reaction was then precipitated by dropwise addition to cooled diethyl ether, and then the precipitate was filtered and dried to give mPEG-PLGA bound hydrazone linker (see Figure 3).
실시예 4: 히드라존 링커가 결합된 mPEG-PLGA와 독소루비신이 결합된 서방형 미셀제제의 제조 Example 4 Preparation of Sustained Release Micelle Formulated with mPEG-PLGA Conjugated with Hydrazone Linker and Doxorubicin
실시예 3에서 수득한 히드라존 링커가 결합된 mPEG-PLGA 0.25g과 10㎎의 독소루비신을 7.5㎖의 디메틸포름아마이드에 용해시키고, 상온에서 질소기체하에 24시간동안 반응시켜서 독소루비신이 결합된 미셀단위체를 수득한 다음, 전기 미셀단위체를 3차 증류수 300㎖에 분산시켜 독소루비신이 결합된 서방형 미셀제제를 형성하고, 별도의 3차 증류수 3ℓ를 사용하여, 5회에 걸쳐 투석함으로써, 미셀단위체와 결합하지 않은 독소루비신을 제거하였다. 이어, 초원심 분리하여 독소루비신이 결합된 서방형 미셀제제를 강제 침전시킨 후, 상등액을 제거하고 동결건조시켜서, 독소루비신이 결합된 미셀단위체의 형태로 보존하였다(참조: 도 3).0.25 g of mPEG-PLGA conjugated with Hydrazone linker and 10 mg of doxorubicin obtained in Example 3 were dissolved in 7.5 ml of dimethylformamide, and reacted for 24 hours under nitrogen gas at room temperature to form doxorubicin-coupled micelle unit. The obtained micellar unit was dispersed in 300 ml of tertiary distilled water to form a sustained-release micelle containing doxorubicin-bound, and dialyzed five times using another 3 liters of tertiary distilled water, thereby not binding to the micelle unit. Doxorubicin was removed. Subsequently, ultracentrifugation separated the doxorubicin-linked sustained-release micelles and precipitated them. Then, the supernatant was removed and lyophilized to preserve doxorubicin-coupled micelles (see FIG. 3).
실시예 2에서와 동일한 방법으로, 독소루비신의 접합 효율을 측정하였다(참조: 표 3)In the same manner as in Example 2, the conjugation efficiency of doxorubicin was measured (see Table 3).
상기 표 3에서 보듯이, mPEG-PLGA에 대한 독소루비신의 접합효율은 26 내지 29%(w/w)이며, 독소루비신 미셀단위체에서 독소루비신은 1.7%(w/w)를 차지함을 알 수 있었다.As shown in Table 3, the conjugated efficiency of doxorubicin to mPEG-PLGA was 26 to 29% (w / w), it can be seen that doxorubicin occupies 1.7% (w / w) in the doxorubicin micelle unit.
실시예 5: 서방형 미셀제제의 제조 Example 5 Preparation of Sustained-release Micelles
전기 실시예 2 및 4에서 보존된 각각의 독소루비신이 결합된 미셀단위체 100㎎을 10㎖의 아세톤에 용해시키고, 이를 100㎖의 3차 증류수 첨가하면서 6시간동안 교반하여 아세톤을 증발시켜서 독소루비신이 결합된 서방형 미셀제제를 제조하였다. 각 미셀단위체에 포함된 mPEG의 분자량에 따라, 전기 제조된 독소루비신이 결합된 서방형 미셀제제의 크기를 DLS(dynamic light scattering) 방법으로 측정하였다(참조: 표 4).100 mg of each of the doxorubicin-bound micelle units preserved in Examples 2 and 4 was dissolved in 10 ml of acetone, which was stirred for 6 hours while adding 100 ml of tertiary distilled water to evaporate acetone to bind doxorubicin. A sustained release micelle formulation was prepared. According to the molecular weight of mPEG contained in each micelle unit, the size of the sustained-release micelles combined with the doxorubicin prepared previously was measured by DLS (dynamic light scattering) method (see Table 4).
표 4에서 보듯이, 독소루비신이 결합된 서방형 미셀제제의 크기는 mPEG의 분자량과는 독립적임을 알 수 있었다.As shown in Table 4, the size of the sustained-release micelles in which doxorubicin is bound was independent of the molecular weight of mPEG.
실시예 6: 독소루비신이 결합된 서방형 미셀제제에서 독소루비신의 방출량 측정 Example 6 Determination of Doxorubicin Release Rate in Sustained-release Micelle Containing Doxorubicin
실시예 5에서 제조된 독소루비신이 결합된 미셀제제인 DOX-PLGA-mPEG8000을 1mg/ml의 농도로 포함하는 인산완충용액(pH 6.5) 20ml를 투석막(dialysis bag, 분자량제한: 10,000)에 주입하고, 이를 동일한 인산완충용액 60ml에 넣고 교반하여,37℃에서 16일간 방치하면서, 투석막을 통하여 외부 완충용액으로 방출되어, 외부 완충용액에 함유된 독소루비신의 양을 분광계를 이용하여 480nm에서 측정하였다(참조: 도 4). 도 4는 시간에 따른 독소루비신의 누적 방출량을 나타낸 그래프이다. 도 4에서 보듯이, 독소루비신이 결합된 서방형 미셀제제는 생분해성 고분자로 구성되어 있어, 자연상태에서 서서히 분해되므로, 서서히 분해되는 독소루비신이 결합된 미셀제제로부터 독소루비신이 장기간에 걸쳐 서서히 방출됨을 알 수 있었다.20 ml of a phosphate buffer solution (pH 6.5) containing DOX-PLGA-mPEG8000, a doxorubicin-bound micelle prepared in Example 5, at a concentration of 1 mg / ml was injected into a dialysis membrane (molecular weight limit: 10,000), The solution was added to 60 ml of the same phosphate buffer solution, stirred, and left for 16 days at 37 ° C., released into an external buffer solution through a dialysis membrane, and the amount of doxorubicin contained in the external buffer solution was measured at 480 nm using a spectrometer. 4). 4 is a graph showing the cumulative release amount of doxorubicin over time. As shown in Figure 4, doxorubicin-bound sustained-release micelles are composed of a biodegradable polymer, so slowly decomposes in nature, it can be seen that doxorubicin is slowly released over a long period of time from the micelles that are slowly decomposed doxorubicin there was.
실시예 7: 암세포에 대한 독소루비신이 결합된 서방형 미셀제제의 작용 Example 7 Action of Sustained Release Micelle Formulated with Doxorubicin on Cancer Cells
독소루비신이 결합된 서방형 미셀제제가 독소루비신과 비교하여 효과적으로 암세포에 침투되는지를 알아보기 위하여, 실시예 2에서 제조된 독소루비신이 결합된 서방형 미셀제제를 간암세포(HepG2 cell line, KCLB(한국세포주은행))와 함께 배양시킨 후, 흐름세포측정기(flow cytometry)와 현미경을 사용하여 암세포 독성을 조사하였다(참조: 도 5). 도 5는 독소루비신과 본 발명의 독소루비신이 결합된 서방형 미셀제제의 항암효과를 비교한 그래프이다. 도 5에서 보듯이, 독소루비신이 결합된 서방형 미셀제제는 독소루비신에 비해 향상된 항암효과를 지님을 확인할 수 있었으며, 이러한 효과는 독소루비신이 결합된 서방형 미셀제제가 세포내이입(endocytosis)을 통하여 암조직의 세포내로 침투(transport)되어 독소루비신보다 세포내로 축적된 양이 많기 때문인 것으로 여겨진다.To determine whether doxorubicin-coupled sustained-release micelles are effectively penetrated into cancer cells as compared to doxorubicin, the doxorubicin-coupled sustained-release micelles prepared in Example 2 may be treated with hepatocarcinoma cells (HepG2 cell line, KCLB). After incubation with)), cancer cytotoxicity was examined using flow cytometry and microscope (see FIG. 5). Figure 5 is a graph comparing the anticancer effect of doxorubicin and the sustained-release micelles combined doxorubicin of the present invention. As shown in Figure 5, it was confirmed that doxorubicin-linked sustained-release micelles have an improved anti-cancer effect compared to doxorubicin, this effect is doxorubicin-coupled sustained-release micelles through endocytosis cancer tissue It is believed that this is due to the greater amount accumulated in cells than doxorubicin that has been transported into cells.
이상에서 상세히 설명하고 입증하였듯이, 본 발명은 생분해성 고분자를 항암제 등의 약물과 결합시킨 후, 수용액상에서 미셀구조를 형성시키는 서방형 미셀제제의 제조방법 및 전기 제조방법에 의하여 제조된 서방형 미셀제제를 제공한다. 본 발명의 서방형 미셀제제의 제조방법은 생분해성 폴리에스테르계 고분자와 폴리에틸렌글리콜(PEG)계 고분자를 공중합반응시켜서, 블록 공중합체를 수득하는 공정; 전기 블록 공중합체를 링커 화합물과 반응시켜서, 블록 공중합체의 하이드록시기에 링커 화합물을 결합시키는 공정; 링커 화합물이 결합된 블록 공중합체를 약물과 결합시켜서, 약물과 블록 공중합체가 결합된 형태의 미셀단위체를 수득하는 공정; 및, 전기 수득한 미셀단위체를 수용액상에서 분산시켜, 서방형 미셀제제를 제조하는 공정을 포함한다. 본 발명에 의하여 제조된 서방형 미셀제제를 통하여 약물의 투여량을 증진시킬 수 있고, 약물의 방출 속도를 조절할 수 있었는 바, 전기 서방형 미셀제제를 항암치료에 효과적으로 활용할 수 있을 것이다.As described and demonstrated in detail above, the present invention is a sustained-release micelle prepared by the method of preparing a sustained-release micelle prepared by the biodegradable polymer and a drug such as an anticancer agent, to form a micelle structure in an aqueous solution and the electrical preparation method To provide. The method for producing a sustained-release micelle preparation of the present invention comprises the steps of copolymerizing a biodegradable polyester polymer and a polyethylene glycol (PEG) polymer to obtain a block copolymer; Reacting the electric block copolymer with a linker compound to bind the linker compound to the hydroxy group of the block copolymer; Combining the block copolymer in which the linker compound is bound with the drug to obtain a micelle unit in a form in which the drug and the block copolymer are bound; And dispersing the previously obtained micelle unit in an aqueous solution to prepare a sustained-release micelle formulation. Through the sustained-release micelles prepared according to the present invention can improve the dosage of the drug, it was possible to control the rate of release of the drug, the electrical sustained-release micelles will be effectively used in anti-cancer treatment.
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