KR20010000669A - Streptokinase-Human serum albumin conjugate as long-acting thrombolytic agent - Google Patents
Streptokinase-Human serum albumin conjugate as long-acting thrombolytic agent Download PDFInfo
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Abstract
Description
본 발명은 스트렙토키나아제(streptokinase, 이하 SK로 칭함)과 사람혈청알부민(HSA)의 포합체(이하 SK-HSA 포합체로 칭함)를 유효성분으로 함유하는 전신투여용 지속성 혈전 용해제에 관한 것으로, 구체적으로는 스트렙토키나제와 인간의 혈청 알부민의 몰비가 1:1 내지 10:1로 결합된 포합체를 유효성분으로 함유하는 전신투여용 지속성 혈전용해제에 관한 것이다. 본 발명의 SK-HSA 포합체를 함유하는 제제는 종래의 SK 제제와 비교하여 전신적으로 투여할 수 있으며 지속적인 혈전용해 효과를 기대할 수 있다.The present invention relates to a sustained thrombolytic agent for systemic administration containing a conjugate of streptokinase (hereinafter referred to as SK) and human serum albumin (HSA) (hereinafter referred to as SK-HSA conjugate) as an active ingredient. The present invention relates to a sustained thrombolytic agent for systemic administration containing a conjugate in which the molar ratio of streptokinase and human serum albumin is 1: 1 to 10: 1 as an active ingredient. Formulations containing SK-HSA conjugates of the present invention can be administered systemically compared to conventional SK formulations and can expect a sustained thrombolytic effect.
선진국에서는 전체 사망률의 35% 이상이 심혈관계 질환에 의한 것으로 집계되고 있다. 새로운 세기로 접어들면서 우리나라에서도 식생활 및 사회생활의 선진화, 서구화가 진행됨에 따라 선진국과 같이 심혈관계 질환으로 사망하는 인구의 비율이 점차 증가하는 추세에 있다. 이들의 질환의 치료에는 혈전용해제가 이용되는데 그 중에서도 대표적인 것들로 streptokinase, urokinase, tissue-type plasminogen activator, prourokinase 등이 있다. 이들 중에서 streptokinase는 가장 먼저 발견되어 널리 쓰이고 있는 혈전용해제 중의 하나이며, 또한 그 가격이 다른 혈전용해제와 비교하여 매우 저렴하므로 의약품으로서 유리한 점을 가지고 있다고 하겠다.In developed countries, more than 35% of all deaths are due to cardiovascular disease. As we enter the new century, as the dietary and social life advances and westernizes, the proportion of people who die from cardiovascular diseases like the developed countries is gradually increasing. Thrombolysis is used for the treatment of these diseases. Among them, streptokinase, urokinase, tissue-type plasminogen activator, and prourokinase are used. Among these, streptokinase is one of the first and widely used thrombolytics, and its price is very inexpensive compared to other thrombolytics.
SK는 β-hemolytic streptococci에 의해 생성되는 분자량 47000의 단백질이다. SK자신은 어떤 효소활성도 가지고 있지 않으나 혈중의 plasminogen과 결합하여 활성화되어 plasminogen의 Arg-561-Val-562의 부위를 절단함으로써 plasmin을 유리시킨다. 유리된 plasmin은 혈전에 작용하여 혈전을 용해시킴으로써 혈전용해 효과를 나타내게 된다. SK는 매우 강력한 혈전용해활성을 가지고 있으나 사람에게 SK를 정맥주사후 분포상의 반감기는 약11~17분, 분포후상에서의 반감기는 약83분으로 보고되어 있다.(A.D. Fletcher, N. Alkjaersig and S. Sherry, The clearance of heterologous protein from the circulation of normal and immunized man, J. Clin. Invest., 37, 1306-1315, 1959)SK is a protein of molecular weight 47000 produced by β-hemolytic streptococci. SK itself does not have any enzymatic activity, but it binds to plasminogen in the blood and is activated to release the plasmin by cleaving the site of Arg-561-Val-562 of the plasminogen. The free plasmin acts on the thrombus to dissolve the thrombus, resulting in a thrombolytic effect. SK has a very strong thrombolytic activity, but the half-life in the distribution of SK after intravenous injection is about 11 to 17 minutes, and the half-life in post-distribution is reported to be about 83 minutes (AD Fletcher, N. Alkjaersig and S). Sherry, The clearance of heterologous protein from the circulation of normal and immunized man, J. Clin.Invest., 37, 1306-1315, 1959.
이상과 같은 단점을 보완하기 위해 아니솔화한 플라스미노겐 스트렙토키나제 활성화 복합체(anisolyated streptokinase activator complex, Anitreplase, Smith-Klein Beecham Inc.)가 개발되었으나 반감기의 연장효과는 70~120분 정도에 지나지 않았다.To ameliorate these shortcomings, the anisolated plasminogen streptokinase activator complex (Anitoreplase, Smith-Klein Beecham Inc.) was developed, but the half-life extension effect was only 70-120 minutes.
한국공개특허공보(공개번호 98-67002, 1998. 10.15)에서는 이와 같은 단점을 보완하여 혈중체류성 스트렙토키나제를 개발하기 위해 스트렙토키나제를 리포좀에 봉입하는 발명을 보고하고 있다. 혈중체류성을 이루기 위한 방법으로는 두가지를 생각할 수 있는데 첫번째는 제제로부터의 방출을 지속적으로 진행되게 함으로써 지속성을 얻는 방법이 있고, 두번째는 약물 자체를 수식하여 약물의 체내로부터의 소실을 느리게 만들어 지속성을 얻는 방법이 있다.Korean Laid-Open Patent Publication (Publication No. 98-67002, Oct. 15, 1998) reports the invention of encapsulating streptokinase in liposomes to develop blood-retentive streptokinase by supplementing these shortcomings. There are two ways to achieve blood retention. First, there is a method of obtaining sustainability by continually progressing the release from the agent. Second, the drug itself is modified to slow the loss of the drug from the body. There is a way to get it.
상기 한국공개특허공보 제 98-67002호에 보고된 스트렙토키나제를 리포좀에 봉입하는 방법은 첫번째 방법에 속하는 것으로 리포좀으로부터 스트렙토키나제의 방출이 지속적으로 일어나게 함으로써 지속성 혈전용해제로 개발한 것이다. 이와 같이 스트렙토키나제의 제제로부터의 방출을 조절하는 방법은 스트렙토키나제 자체의 소실과정을 수식한 것은 아니므로 원하는 혈전 용해 효과를 얻기위해 투여하는 총 투여량은 기존의 스트렙토키나제 제제와 비교하였을 때 오히려 더 많은 양을 필요로 하게 된다. 왜냐하면 리포좀에 스트렙토키나제를 봉입할 때, 그 봉입율이 100%에 미치지 못하기 때문이다. 결과적으로 혈중 지속성은 가능하게 하였으나 경제적인 측면에서 오히려 더 많은 스트렙토키나제를 필요로 한다는 단점을 가지고 있다. 또한, 리포좀에 봉입한 스트렙토키나제 제제는 전신투여를 하였을 때, 그 혈전용해효과가 보장되어 있지 않다. 왜냐하면 리포좀과 같이 큰 vesicle내에 스트렙토키나제를 봉입하여 놓았기 때문에 스트렙토키나제의 체내에서의 움직임이 리포좀의 체내분포에 의해 지배되기 때문이다. 즉, 간의 endoplasmic reticulum과 같은 조직에 리포좀이 포획됨으로써 실제로 혈전이 생성되어 있는 목적부위까지 리포좀에 봉입되어 있는 스트렙토키나제가 도달하지 못하는 경우도 일어날 수 있기 때문이다. 따라서, 이와같은 리포좀 제제는 혈전이 생겨 있는 부위 근방에 피하주사와 같은 방법으로 국소 투여함으로써 혈전용해 효과를 기대할 수 있지만, 전신적으로 투여할 수 있는 제제는 되지 못한다.The method of encapsulating streptokinase reported in Korean Patent Application Laid-Open No. 98-67002 into liposomes belongs to the first method and is developed as a sustained thrombolytic agent by continuously releasing streptokinase from liposomes. This method of controlling the release of streptokinase from the formulation does not modify the process of streptokinase itself, so the total dosage administered to achieve the desired thrombolytic effect is rather higher than that of conventional streptokinase. You need a lot. This is because when the streptokinase is encapsulated in liposomes, the encapsulation rate is less than 100%. As a result, blood persistence is possible, but it has the disadvantage of requiring more streptokinase in terms of economy. In addition, the thrombolytic effect of streptokinase preparations encapsulated in liposomes is not guaranteed when administered systemically. Because streptokinase is encapsulated in large vesicles such as liposomes, the movement of streptokinase in the body is controlled by the distribution of liposomes in the body. That is, when liposomes are captured in tissues such as liver endoplasmic reticulum, streptokinase encapsulated in liposomes may not reach the target site where thrombus is actually generated. Therefore, such liposome preparations can be expected to have a thrombolytic effect by topical administration in the vicinity of the site of thrombus by a subcutaneous injection, but they cannot be administered systemically.
본 발명은 상기와 같은 종래기술의 문제점을 해결하기 위한 것으로, 본 발명에서는 종래의 짧은 혈장중 반감기와 국소적 투여에 의해서만 이용되어 지고 있는 SK 제제의 투여방법 및 지속성을 개선시킨 제제를 개발하기 위하여, SK에 사람 혈청 알부민(HSA)을 포합시킨 SK-HSA포합체를 제조함으로써 신장에서의 사구체여과에 의한 배설속도를 감소시키고, 그 결과 체내에 더 오래 머물수 있는 지속성 혈전용해제를 개발하고 동시에 전신적으로 투여하여도 혈전용해효과를 얻을 수 있도록 투여방법을 개선시킨 제제를 개발하였다.The present invention is to solve the problems of the prior art as described above, in the present invention to develop an improved method of administration and sustainability of the SK formulation that is used only by conventional short plasma half-life and topical administration , SK-HSA conjugate containing human serum albumin (HSA) in SK reduces the rate of excretion caused by glomerular filtration in the kidney, and as a result, develops a sustained thrombolytic agent that can stay in the body longer and systemically In order to obtain a thrombolytic effect even when administered, a formulation with improved administration method was developed.
도 1은 흰쥐의 혈전모델에서 생리식염수의 정맥주사 후 혈류의 경시적 변화를 나타낸 것이다.Figure 1 shows the changes over time after the intravenous injection of physiological saline in the blood clot model of the rat.
도 2는 흰쥐의 혈전모델에서 SK의 정맥주사후 혈류의 경시적 변화를 나타낸 것이다.Figure 2 shows the changes over time after the intravenous injection of SK in the blood clot model of the rat.
도 3은 흰쥐의 혈전모델에서 SK-HSA 포합체의 정맥주사후 혈류의 경시적 변화를 나타낸 것이다.Figure 3 shows the changes over time after the intravenous injection of SK-HSA conjugate in the blood clot model of the rat.
본 발명은 SK와 HSA의 결합몰비가 1:1 ~ 10:1인 SK와 HSA의 포합체를 유효성분으로 함유하는 SK의 전신투여용 지속성 혈전용해제를 제공한다. 상기 제제는 정맥, 근육주사 또는 피하주사제로 제형화되는 것이 바람직하나 상기 투여방법에 국한되는 것은 아니다. 또한, 본 발명은 완충액에 용해시킨 SK에 HSA를 가교제(cross linker)인 N-succinimidyl 3-[2-pyridyldithio]propionate (SPDP)를 이용하여 SK:HSA = 1:1 ~ 10:1의 몰비로 가하여 포합반응시키고 투석, 농축, 겔 여과, 전기영동에 의해 생성된 포합체를 분리하여 SK와 HSA의 포합체를 제조하는 방법을 제공한다. SK와 HSA의 포합반응을 위해 사용되는 cross linker는 SPDP에 국한되지 않는다.The present invention provides a sustained thrombolytic agent for systemic administration of SK containing a conjugate of SK and HSA having a molar ratio of SK to HSA of 1: 1 to 10: 1 as an active ingredient. The preparation is preferably formulated as an intravenous, intramuscular or subcutaneous injection, but is not limited to the method of administration. In addition, the present invention provides a molar ratio of SK: HSA = 1: 1 to 10: 1 using N-succinimidyl 3- [2-pyridyldithio] propionate (SPDP), a cross linker of HSA, in SK dissolved in buffer. The present invention provides a method of preparing a conjugate of SK and HSA by adding and reacting and separating the conjugate produced by dialysis, concentration, gel filtration, and electrophoresis. The cross linker used for the combined reaction of SK and HSA is not limited to SPDP.
이하, 본 발명을 더욱 상세히 설명하면 아래와 같다.Hereinafter, the present invention will be described in more detail.
본 발명에서는 SK의 전신투여용 지속성 제제를 제공하기 위하여 SK가 혈관내에 비교적 장시간 체류하도록 하기 위해 SK에 내인성 고분자인 HSA를 결합시켜 SK의 체내로부터의 소실속도를 감소시키는 방법을 사용하였다. 왜냐하면 분자량이 약 69000인 HSA에 분자량이 약 47000인 SK를 여러가지 몰비로 결합시킴으로써 신장에서 사구체여과를 받는 임계분자량 (약 50000)보다 분자량이 더 큰 포합체를 제조하여 사구체여과속도를 저하시킬 수 있기 때문이다.In the present invention, in order to provide a long-acting formulation for systemic administration of SK, in order to allow SK to stay in the blood vessel for a relatively long time, a method of reducing the loss rate of SK from the body by combining HSA, an endogenous polymer, is used. Because by combining various molar ratios of SK having a molecular weight of about 47000 to HSA having a molecular weight of about 69000, a conjugate having a higher molecular weight than a critical molecular weight (about 50000) subjected to glomerular filtration in the kidney can be produced to decrease glomerular filtration rate. Because.
본 발명에 따른 HSA와 SK의 포합체는 1:1 ~ 1:10의 몰비를 갖는 것이 바람직하다. HSA의 양이 지나치게 증가할 경우, SK의 효소활성을 저하시킬 우려가 있기 때문이다.It is preferable that the conjugate of HSA and SK according to the present invention has a molar ratio of 1: 1 to 1:10. If the amount of HSA is excessively increased, there is a concern that the enzyme activity of SK may be lowered.
본 발명은 SK와 HSA의 포합체를 약제학적 분야에서 통상적으로 허용하는 담체와 배합하여 약제학적 분야에서 통상적인 제제로 제형화시킬 수 있다. 특히 주사용 용액, 현탁액 또는 주사용 건조분말로 제조하여 사용할 수 있으며, 주사용 건조분말의 형태로 제형화시키는 것이 바람직하다. 그러나 이상에서 언급한 제형에 국한되는 것은 아니다. 본 발명에서 사용되는 담체로는 약제학적 분야에서 통상적인 것으로, 예를 들면 보존제, 무통화제, 가용화제 또는 안정화제 등이 포함될 수 있다. 이렇게 제조된 약제학적 제제는 예를 들면 정맥, 근육, 또는 피하로 투여될 수 있으며, 이 경우 투여량이 바로 흡수량에 근사하므로 투여량을 줄일 수 있다는 장점이 있다. 본 발명 제제에서 혈전용해를 기대할 수 있는 투여량은 SK-HSA 포합체로서 약 1000~200,000 IU/kg이며, 바람직하게는 10,000~30,000 IU/kg이다.The present invention can be formulated into a formulation conventional in the pharmaceutical art by combining the conjugate of SK and HSA with a carrier that is commonly acceptable in the pharmaceutical art. In particular, it can be prepared and used as an injectable solution, suspension or dry powder for injection, and is preferably formulated in the form of a dry powder for injection. However, it is not limited to the formulation mentioned above. Carriers used in the present invention are conventional in the pharmaceutical field, and may include, for example, preservatives, analgesics, solubilizers or stabilizers. The pharmaceutical preparations thus prepared may be administered, for example, intravenously, intramuscularly, or subcutaneously. In this case, the dosage may be directly approximated by the absorption, thereby reducing the dosage. The dose that can be expected to be thrombolytic in the formulation of the present invention is about 1000-200,000 IU / kg, preferably 10,000-30,000 IU / kg, as the SK-HSA conjugate.
이하, 본 발명의 구성 및 작용을 바람직한 실시예 및 실험예에 의거 더욱 상세히 설명하고자 하나, 본 발명의 이해를 돕기위한 것일 뿐 본 발명의 범위를 제한하고자 하는 것은 아니다.Hereinafter, the configuration and operation of the present invention will be described in more detail based on the preferred embodiments and experimental examples, but are not intended to limit the scope of the present invention only to help the understanding of the present invention.
실시예. 스트렙토키나아제와 사람 혈청알부민과의 포합체의 제조Example. Preparation of conjugates of streptokinase and human serum albumin
스트렙토키나아제 (streptokinase, 이하 SK라 칭함)의 0.1M 인산완충액(39.78mg/ml) 40㎕ (SK 1.6mg)을 960㎕의 0.1M 인산완충액(phosphate buffer)을 함유하는 0.1M NaCl (pH 7.4)에 가하고(a) 5mg의 N-succinimidyl 3-[2-pyridyldithio]propionate (SPDP, Sigma Co., USA)를 1ml의 N,N-dimethyl formamide (DMF, Sigma Co., USA)에 녹인 후 5㎕의 SPDP용액을 (a)용액에 부드럽게 흔들며 가하였다. 실온에서 30분간 방치한 후 반응액 전체를 sephadex-G25 (Sigma Co., USA) column에 가하고 0.1M 인산완충액(phosphate buffer)을 함유하는 0.1M NaCl (pH 7.4)를 용리전개완충액(elution buffer)으로 하여 각 fraction당 1ml의 용적이 되도록 수집하였다. 각 fraction 10㎕에 단백측정용 시액 (BIO-RAD Laboratories, USA) 5배 희석액을 200㎕가한 후 microplate reader (Molecular Device, USA)를 이용하여 650nm에서 흡광도를 측정하고 단백 peak를 합하여 냉장보관 하였다 (A). 별도로 5mg의 human serum albumin (이하 HSA라 칭함, Sigma Co., USA)을 1ml의 0.1M 인산완충액(phosphate buffer)을 함유하는 0.1M NaCl(pH 7.4)에 녹이고(b), DMF에 녹인 SPDP 용액 30㎕를 취하여 부드럽게 흔들면서 가하였다. 30분동안 실온에서 반응시킨 후 반응액 전체를 sephadex-G25 column에 가하고 0.1M sodium acetate buffer를 함유하는 0.1M NaCl을 용리전개완충액(elution buffer)으로 하여 각fraction당 1ml의 용적이 되도록 수집하였다. 각 fraction 10㎕에 단백측정용 시액 5배 희석액을 200㎕가한 후 microplate reader를 이용하여 650nm에서 흡광도를 측정하였다. 단백 peak에 dithiothreitol (Sigma Co., USA)의 최종 농도가 0.025M이 되도록 가하고 실온에서 40분간 방치한 후 반응액을 sephadex-G25에 loading하고 0.1M 인산완충액(phosphate buffer)을 함유하는 0.1M NaCl(pH 7.4)를 용리전개완충액(elution buffer)으로 하여 fraction당 1ml이 되도록 수집한 후 단백정량을 하여 단백 peak를 (B)라 하였다. (A)와 (B)의 몰비율이 1:1이 되도록 두 용액을 섞은 다음 실온에서 overnight로 방치하였다. Overnight방치가 끝난 반응액을 sephacryl-200HR column(Pharmacia, Sweden)에 1.5ml씩 가하여 0.1M phosphate buffer (pH 7.4)를 elution buffer로 1 fraction의 용적이 1ml가 되도록 수집하였다. 각 fraction 10㎕에 단백측정용 시액 5배 희석액을 200㎕가하여 단백정량을 하고 peak를 분리하였다. Sephacryl column으로부터 용출되어 나온 단백분획을 수집하여 농축시킨 후, 농축된 샘플을 다시 model-491 prep cell (Bio-Rad, CA, USA)을 이용하여 분리, 정제하여 SK-HSA 포합체를 얻었다. 이때 SK 1.6mg을 반응시켜 0.5mg의 SK-HSA포합체를 얻었다.40 μl (SK 1.6 mg) of 0.1 M phosphate buffer (39.78 mg / ml) of streptokinase (hereinafter referred to as SK) was added to 0.1 M NaCl (pH 7.4) containing 960 μl of 0.1 M phosphate buffer. (A) 5 mg of N-succinimidyl 3- [2-pyridyldithio] propionate (SPDP, Sigma Co., USA) was dissolved in 1 ml of N, N-dimethyl formamide (DMF, Sigma Co., USA) and 5 μl SPDP solution of was gently added to (a) solution. After standing at room temperature for 30 minutes, the whole reaction solution was added to a sephadex-G25 (Sigma Co., USA) column, and 0.1M NaCl (pH 7.4) containing 0.1M phosphate buffer was added to the elution buffer. In order to collect a volume of 1ml per fraction. 200 μl of a 5-fold dilution of the protein measuring solution (BIO-RAD Laboratories, USA) was added to 10 μl of each fraction, and then the absorbance was measured at 650 nm using a microplate reader (Molecular Device, USA). A). Separately, 5 mg of human serum albumin (hereinafter referred to as HSA, Sigma Co., USA) was dissolved in 0.1 M NaCl (pH 7.4) containing 1 ml of 0.1 M phosphate buffer (b) and dissolved in DMF. 30 μl was taken and gently shaken. After reacting at room temperature for 30 minutes, the entire reaction solution was added to a sephadex-G25 column, and 0.1M NaCl containing 0.1M sodium acetate buffer was collected as an elution buffer to a volume of 1 ml per fraction. 200 μl of a 5-fold dilution of the protein solution was added to 10 μl of each fraction, and the absorbance was measured at 650 nm using a microplate reader. To the protein peak, dithiothreitol (Sigma Co., USA) was added at a final concentration of 0.025M and allowed to stand at room temperature for 40 minutes.The reaction solution was then loaded into sephadex-G25 and 0.1M NaCl containing 0.1M phosphate buffer. (pH 7.4) was collected to be 1 ml per fraction using elution buffer and the protein peak was defined as (B). The two solutions were mixed so that the molar ratio of (A) and (B) was 1: 1, and then left overnight at room temperature. Overnight reaction solution was added to the sephacryl-200HR column (Pharmacia, Sweden) by 1.5ml each and 0.1M phosphate buffer (pH 7.4) was collected as elution buffer so that the volume of 1 fraction was 1ml. 200 μl of a 5-fold dilution of the protein solution was added to 10 μl of each fraction to determine the protein and isolate the peaks. After collecting and concentrating the protein fraction eluted from the Sephacryl column, the concentrated sample was separated and purified again using model-491 prep cell (Bio-Rad, CA, USA) to obtain SK-HSA conjugate. At this time, 1.6 mg of SK was reacted to obtain 0.5 mg of SK-HSA conjugate.
실험예 1. 흰쥐의 혈전모델에서 생리식염수의 정맥주사 후 혈류의 경시적 변화 (도 1)Experimental Example 1. Changes of blood flow over time after iv saline in rat thrombus model (FIG. 1)
나트륨 펜토바비탈 (sodium pentobarbital)로 마취한 Sparague-Dawley계 웅성 흰쥐의 대퇴정맥에 PE-50튜빙으로 삽관한 후, 대퇴동맥을 노출시키고 FeCl3를 흰쥐의 대퇴동맥에 떨어뜨려 혈전을 생성시킨 후, 약 5분 경과후에 생리식염수를 정맥주사한 후 대퇴동맥의 혈류를 laser doppler flow meter를 이용하여 경시적으로 관찰한 결과를 나타내었다(도 1). FeCl3를 대퇴동맥에 가함으로써 혈류가 급격히 저하하는 것을 관찰할 수 있으며(혈전생성을 의미함), 혈전 생성 직후에 생리식염수를 대퇴정맥을 통하여 주사한 후에도 대퇴동맥의 혈류는 감소한 그대로 유지되고 있음을 나타낸다. 도 1에는 3마리의 흰쥐의 실험예를 나타내었다.After inserting PE-50 tubing into the femoral vein of Sparague-Dawley male rats anesthetized with sodium pentobarbital, exposing the femoral artery and dropping FeCl 3 into the femoral artery of rats to generate blood clots. After about 5 minutes, the saline was injected intravenously and the blood flow of the femoral artery was observed with a laser doppler flow meter over time (FIG. 1). FeCl 3 was added to the femoral artery and the blood flow rapidly decreased (meaning thrombus formation), and the blood flow in the femoral artery was maintained intact even after injection of physiological saline through the femoral vein immediately after thrombus formation. Indicates. 1 shows an experimental example of three rats.
실험예 2. 흰쥐의 혈전모델에서 SK의 정맥주사 후 혈류의 경시적 변화 (도 2)Experimental Example 2 Changes of blood flow over time after intravenous injection of SK in the thrombus model of rats (FIG. 2)
나트륨 펜토바비탈 (sodium pentobarbital)로 마취한 Sparague-Dawley계 웅성 흰쥐의 대퇴정맥에 PE-50튜빙으로 삽관한 후, 대퇴동맥을 노출시키고 FeCl3를 흰쥐의 대퇴동맥에 떨어뜨려 혈전을 생성시킨 후, 약5분 경과후에 SK(약 15000 units per rat)를 정맥주사한 후 대퇴동맥의 혈류를 laser doppler flow meter를 이용하여 경시적으로 관찰한 결과를 나타내었다. FeCl3를 대퇴동맥에 가함으로써 혈류가 급격히 저하하는 것을 관찰할 수 있으며 (혈전생성을 의미함), 혈전 생성 직후에 SK를 대퇴정맥을 통하여 주사한 후에도 대퇴동맥의 혈류는 감소한 그대로 유지되고 있음을 나타낸다(도 2). 즉 혈전이 생성되어 있는 부위인 대퇴동맥으로부터 가장 멀리 떨어져 있는 부위에 SK를 투여한 경우 (전신적 투여에 해당함)에 SK는 혈전용해활성을 나타내지 못하였다. 도 2에서는 2마리의 흰쥐의 실험예를 나타내었다.After inserting PE-50 tubing into the femoral vein of Sparague-Dawley male rats anesthetized with sodium pentobarbital, exposing the femoral artery and dropping FeCl 3 into the femoral artery of rats to generate blood clots. After 5 minutes of intravenous injection of SK (approximately 15000 units per rat), the blood flow of the femoral artery was observed with a laser doppler flow meter. It can be observed that blood flow is rapidly decreased by adding FeCl 3 to the femoral artery (meaning thrombus formation), and the blood flow of the femoral artery remains intact even after SK is injected through the femoral vein immediately after thrombus formation. (FIG. 2). In other words, when SK was administered to the most distant part of the femoral artery, the site where thrombus was formed (corresponding to the systemic administration), SK did not show thrombolytic activity. 2 shows an experimental example of two rats.
앞에서도 언급한 바와 같이 SK의 혈장중 반감기는 매우 짧기 때문에 SK를 전신적으로 정맥주사에 의해 투여하는 경우 대부분의 SK가 조직분포 등에 의해 혈중으로부터 신속하게 사라지므로 혈중의 SK농도가 격감하여 대퇴동맥에 생성되어 있는 혈전을 용해시키기에는 충분하지 못한 농도로 감소되어 있기 때문인 것으로 판단된다. 바로 이러한 이유 때문에 SK를 사용할 때 혈전생성부위에 국소적으로 SK를 장시간에 걸쳐 IV infusion에 의해 투여하여야만 효과를 볼 수 있는 것이다.As mentioned earlier, the half-life of SK in plasma is very short, so when SK is administered systemically by intravenous injection, most SK disappears rapidly from the blood due to tissue distribution and so on. This may be because the concentration is reduced to a concentration not sufficient to dissolve the generated blood clot. It is for this reason that SK should be administered by IV infusion for a long time locally to thrombus generation site.
실험예 3. 흰쥐 혈전모델에서 SK-HSA포합체의 정맥주사후 혈류의 경시적 변화 (도 3)Experimental Example 3. Changes of blood flow over time after intravenous injection of SK-HSA conjugate in rat thrombus model (FIG. 3)
나트륨 펜토바비탈 (sodium pentobarbital)로 마취한 Sparague-Dawley계 웅성 흰쥐의 대퇴정맥에 PE-50튜빙으로 삽관한 후, 대퇴동맥을 노출시키고 FeCl3를 흰쥐의 대퇴동맥에 떨어뜨려 혈전을 생성시킨 후, 약 5분 경과후에 SK-HSA포합체 (6000 units per rat)를 정맥주사한 후 대퇴동맥의 혈류를 laser doppler flow meter를 이용하여 경시적으로 관찰한 결과를 나타내었다. FeCl3를 대퇴동맥에 가함으로써 혈류가 급격히 저하하는 것을 관찰할 수 있으며(혈전생성을 의미함), 혈전 생성 직후에 SK-HSA포합체를 대퇴정맥을 통하여 주사한 후에 대퇴동맥의 혈류가 다시 흐르기 시작하는 것을 관찰할 수 있었다(도 3). 즉, 전신적으로 SK-HSA포합체를 단 한번 주사하여 준 후에 혈전이 생성되었던 대퇴동맥의 혈류가 약 5시간 이후까지도 흐르는 것이 관찰되었다. 본 발명에서는 흰쥐모델(E.M.Lyle, S.D. Lewis, E.D. Lehman, S.J. Gardell, S.L. Motzel and J.J. Lynch Jr, Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis, Thromb. Haemost., 79, 656-662, 1998.)을 이용하여 SK의 혈전용해효과를 평가하였다. SK는 비교적 새롭게 생성된 혈전을 용해하는데 있어서 강력한 효과를 보인다. 따라서, 본 발명에서는 FeCl3를 흰쥐의 대퇴동맥에 떨어뜨려 혈전을 생성시킨 후, 약 5분 경과 후에 약물을 정맥주사 함으로써 비교적 새롭게 형성된 혈전을 용해하는 모델을 이용하여 SK-HSA 포합체의 혈전용해활성을 평가하였다. 이 모델에 의해 평가한 결과에 따르면, SK-HSA 포합체는 단1회 전신투여에 의해서도 현저한 혈전용해효과를 장시간 나타내는 것이 관찰되었으므로 기존의 SK제제가 국소적으로 정맥주입에 의해 장시간 투여 되어야 하는 것과 비교할 때, 월등히 뛰어난 혈전용해제로 사용할 수 있을 것으로 생각된다.After inserting PE-50 tubing into the femoral vein of Sparague-Dawley male rats anesthetized with sodium pentobarbital, exposing the femoral artery and dropping FeCl 3 into the femoral artery of rats to generate blood clots. After about 5 minutes, SK-HSA conjugate (6000 units per rat) was injected intravenously and the blood flow in the femoral artery was observed with a laser doppler flow meter. FeCl 3 was added to the femoral artery to see a sharp drop in blood flow (meaning thrombus formation), and the flow of the femoral artery flowed again after injection of the SK-HSA complex through the femoral vein immediately after thrombus formation. Starting could be observed (FIG. 3). That is, it was observed that the blood flow in the femoral artery, in which thrombus was generated, flowed up to about 5 hours after the systemic injection of the SK-HSA complex only once. In the present invention (EMLyle, SD Lewis, ED Lehman, SJ Gardell, SL Motzel and JJ Lynch Jr, Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis, Thromb. Haemost., 79, 656- 662, 1998.) to evaluate the thrombolytic effect of SK. SK has a strong effect on dissolving relatively newly generated thrombi. Therefore, in the present invention, after dropping FeCl 3 into the femoral artery of rats to generate a blood clot, the thrombolysis of the SK-HSA conjugate using a model of dissolving a relatively newly formed blood clot by intravenous injection of the drug about 5 minutes later. Activity was evaluated. According to the results evaluated by this model, it was observed that SK-HSA conjugate exhibits a prominent thrombolytic effect even after a single systemic administration for a long time. Therefore, the SK-HSA conjugate should be administered locally by intravenous injection for a long time. In comparison, it can be used as an excellent thrombolytic agent.
제제예Formulation example
실시예로부터 수득한 SK-HSA포합체 5,000 ~ 1,000,000 단위SK-HSA conjugate obtained from the Example 5,000 ~ 1,000,000 units
염화나트륨 5.84mgSodium Chloride 5.84mg
구연산나트륨 5.80mgSodium citrate 5.80mg
무수구연산 0.057mgCitric anhydride 0.057mg
주사용증류수 적량Distilled water for injection
계 1mlTotal 1ml
상기한 조성에 따라 실시예로부터 수득된 SK-HSA 포합체를 주사용 증류수에 용해시켜 주사액을 제조하였다.According to the composition described above, the SK-HSA conjugate obtained from the Example was dissolved in distilled water for injection to prepare an injection solution.
이상 설명하고 실시예 및 실험예를 통하여 알 수 있는 바와 같이, 본 발명은 스트렙토키나아제(streptokinase, 이하 SK로 칭함)과 사람혈청알부민(HSA)와의 포합체 (이하 SK-HSA 포합체로 칭함)를 유효성분으로 함유하는 전신투여용 지속성 혈전 용해제에 관한 것으로, 구체적으로는 스트렙토키나제와 인간의 혈청 알부민의 몰비가 1:1 내지 10:1로 결합된 포합체를 유효성분으로 함유하는 전신투여용 지속성 혈전용해제에 관한 것이다. 본 발명의 SK-HSA 포합체를 함유하는 제제는 종래의 SK 제제와 비교하여, 전신적으로 투여할 수 있으며 지속적인 혈전용해 효과를 기대할 수 있다.As described above and as can be seen through Examples and Experimental Examples, the present invention is a conjugate of streptokinase (hereinafter referred to as SK) and human serum albumin (HSA) (hereinafter referred to as SK-HSA conjugate). The present invention relates to a sustained thrombolytic agent for systemic administration containing as an active ingredient, specifically, to a systemic administration containing a conjugate in which the molar ratio of streptokinase and human serum albumin is 1: 1 to 10: 1. Relates to thrombolytic release. The formulation containing the SK-HSA conjugate of the present invention can be administered systemically compared to the conventional SK formulation and can expect a sustained thrombolytic effect.
Claims (3)
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| KR1020000059994A KR20010000669A (en) | 2000-10-12 | 2000-10-12 | Streptokinase-Human serum albumin conjugate as long-acting thrombolytic agent |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4808405A (en) * | 1979-11-05 | 1989-02-28 | Beecham Group P.L.C. | P-anisoyl streptokinase/plasminogen complex |
| JPH0965879A (en) * | 1995-08-30 | 1997-03-11 | Ensuiko Sugar Refining Co Ltd | New thrombolytic enzyme and its production |
| KR970020109A (en) * | 1995-10-18 | 1997-05-28 | 강재헌 | Covalent conjugate of streptokinase and polysaccharide and preparation method thereof |
| KR19980067002A (en) * | 1997-01-30 | 1998-10-15 | 김종국 | Plasmolytic thrombolytics obtained by encapsulating streptokinase in liposomes and a method of manufacturing the same |
| US6087332A (en) * | 1997-12-23 | 2000-07-11 | Galler; Lawrence Isaac | Streptokinase derivatives with high affinity for activated platelets and methods of their production and use in thrombolytic therapy |
| KR20010099583A (en) * | 2000-04-18 | 2001-11-09 | 최호일 | Preparation Method of sustained release dosage forms of protein or peptide drugs and the drugs prepared by that method |
| KR101325912B1 (en) * | 2011-01-12 | 2013-11-07 | 경희대학교 산학협력단 | Polydiacetylene based biosenser |
-
2000
- 2000-10-12 KR KR1020000059994A patent/KR20010000669A/en not_active Application Discontinuation
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4808405A (en) * | 1979-11-05 | 1989-02-28 | Beecham Group P.L.C. | P-anisoyl streptokinase/plasminogen complex |
| JPH0965879A (en) * | 1995-08-30 | 1997-03-11 | Ensuiko Sugar Refining Co Ltd | New thrombolytic enzyme and its production |
| KR970020109A (en) * | 1995-10-18 | 1997-05-28 | 강재헌 | Covalent conjugate of streptokinase and polysaccharide and preparation method thereof |
| KR19980067002A (en) * | 1997-01-30 | 1998-10-15 | 김종국 | Plasmolytic thrombolytics obtained by encapsulating streptokinase in liposomes and a method of manufacturing the same |
| US6087332A (en) * | 1997-12-23 | 2000-07-11 | Galler; Lawrence Isaac | Streptokinase derivatives with high affinity for activated platelets and methods of their production and use in thrombolytic therapy |
| KR20010099583A (en) * | 2000-04-18 | 2001-11-09 | 최호일 | Preparation Method of sustained release dosage forms of protein or peptide drugs and the drugs prepared by that method |
| KR101325912B1 (en) * | 2011-01-12 | 2013-11-07 | 경희대학교 산학협력단 | Polydiacetylene based biosenser |
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