• Biotechnology and Bioprocess Engineering:BBE
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• 제7권6호
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• pp.340-346
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• 2002

The purpose of the present study was to examine the efficacy and mechanism of the PAB (para-amino benzamidine) affinity column chromatography, Viresolve NFP virus filtration, pasteurization (60$\^{C}$ heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine as regards the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and subjected to scale-down processes mimicking the manufacture of urokinase Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID$\_$50/), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing HAV with a log reduction factor of 3.24. HAV infectivity was rarely detected in the urokinase fraction, while most of the HAV infectivity was recovered in the unbound and wash fractions. HAV was completely removed during the Viresolve NFP filtration with a log reduction factor of $\geq$ 4.60. Pasteurization was also found to be an effective step in inactivating HAV where the titers were reduced from an initial titer of 7.18 log$\_$10/ TCID$\_$50/ to undetectable levels within 10 h of treatment. The log reduction factor achieved during pasteurization was $\geq$ 4.76. Lyophilization revealed the lowest efficacy for inactivating HAV with a log reduction factor of 1.48. The cumulative log reduction factor was $\geq$ 14.08. Accordingly, these results indicate that the production process for urokinase exhibited a sufficient HAV reducing capacity to achieve a high margin of virus safety.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.140-147
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• 2004

Urokinase is an enzyme with fibrinolytic activity (plasminogen activator) isolated from fresh urine of healthy men. Viral safety is an important prerequisite for clinical preparation of the protein from urine. In order to increase the viral safety of a high purity urokinase in regard to non-enveloped viruses, a virus removal process using a novel polyvinylidene fluoride membrane filter (Viresolve NFP) has been optimized. Urokinase was able to pass through the filter with recoveries of 95% in the production scale process. No substantial changes were observed in physical and biochemical characteristics of the filtered urokinase in comparison with those of the enzyme before filtration. A 47-mm disk membrane filter was used to simulate the process performance of the production scale cartridges and tested if it could remove several experimental model viruses for human pathogenic viruses, including porcine parvovirus (PPV), human hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), bovine viral diarrhoea virus (BVDV), and bovine herpes virus (BHV). Non-enveloped viruses (PPV, HAV, and EMCV) as well as enveloped viruses (BVDV and BHV) were completely removed during filtration. The log reduction factors achieved were $\geq$4.86 for PPV, $\geq$4.60 for HAV, $\geq$6.87 for EMCV, $\geq$4.60 for BVDV, and $\geq$5.44 for BHV. These results indicate that the virus filtration process successfully improved the viral safety of the final products.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.591-597
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• 2003

Human urokinase kringle domain, sharing homology with angiostatin kringles, has been shown to be an inhibitor of angiogenesis, which can be used for the treatment of cancer, rheumatoid arthritis, psoriasis, and retinopathy. Here, the expression of the kringle domain of urokinase (UK1) as a secreted protein in high levels is reported. UK1 was expressed in the methylotrophic yeast Pichia pastoris GS115 by fusion of the cDNA spanning from Ser47 to Lys135 to the secretion signal sequence of ${\alpha}-factor$ prepro-peptide. In a flask culture, the secreted UK1 reached about 1 g/l level after 120h of methanol induction and was purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the purified UK1 revealed that it was cleaved at the Ste13 signal cleavage site. The molecular mass of UK1 was determined to be 10,297.01 Da. It was also confirmed that the purified UK1 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, or epidermal growth factor, in a dose-dependent manner. These results suggest that a P. pastoris sytem can be employed to obtain large amounts of soluble and active UK1.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.615-618
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• 2000

The purpose of present study was to examine the efficacy of PAB (para-amino benzamidine) affinity column chromatography, pasteurization ($60^{\circ}C$ heat treatment for 10 h), and lyophilization steps, employed in the manufacture of urokinase from human urine, in the removal and/or inactivation of urine-born viruses. Bovine herpes virus (BHV) and Murine encephalomyocarditis virus (EMCV) were selected for this study. Samples from the relevant stages of the production process were spiked with the viruses and the amount of virus in each fraction was quantified by 50% tissue culture infectious dose ($TCID_{50}$). BHV and EMCV were effectively partitioned from urokinase during PAB chromatography with the log reduction factors of 6.71 and 5.27, respectively. Pasteurization was a robust and effective step in inactivating BHV and EMCV, of which titers were reduced from initial titers of $8.65\;log_{10}\;TCID_{50}$ and $7.81\;log_{10}\;TCID_{50}$, respectively, to undetectable levels within 1 hour of treatment. The log reduction factors achieved during lyophilization were 2.06 for BHV and 4.54 for EMCV. These results indicate that the production process for urokinase has sufficient virus reducing capacity to achieve a high margin of virus safety.

• 대한핵의학회지
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• 제25권1호
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• pp.95-100
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• 1991

Computerized scintigraphv using $^{99m}Tc-DTPA$ was performed to 37 isolated rabbit kidneys after preservation for 48 hours in perfusates differing in their compositions, i. e., Group 1 (N 9) in Collins' solution, Group 2 (N 10) in Collins' plus trifluoperazine, Group 3 (N 9) in Collins' plus urokinase and Group 4 (N 9) in Collins pius urokinase plus verapamil. Satisfactory images, and statistically analyzable quantitative indices such as perfusion score, filtration rate and cortical uptake ratio (CUR) were obtained by the evaluations of first-pass perfusion, equilibration slopes and postequilibration images. Significant improvements in CUR were observed by adding trifluoperazine (Group 2) and urokinase (group 3) as compared to Collins' only group (Group 1), p<0.05 for each, and all of the three indices (perfusion score, filtration rate and CUR) were also significantly (p=0.0092 p<0.05 and p<0.05) improved by adding urokinase plus verapamil (Group 4). We concluded that the computerized scintigraphy with Tc-99m DTPA provide valuable quantitative indices for evaluation of preserved kidney funcitions and suggest its possible clinical applicability in cadaver kidney transplantation considering the safety and easiness of the prodedure.

• Tuberculosis and Respiratory Diseases
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• 제55권3호
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• pp.297-302
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• 2003

저자들은 좌주기관지내 피덩이로 인해 폐허탈과 심한 호흡부전을 보인 환자에서 기관지내 국소 우로키나아제의 사용으로 피덩이를 효과적으로 제거한 예를 경험하였기에 보고하는 바이다.

• Journal of Korean Neurosurgical Society
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• 제38권4호
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• pp.281-286
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• 2005

Objective : The authors report our experience of urokinase thrombolysis in treating patients harboring nonaneurysmal spontanesous intraventricular hemorrhage[IVH] and evaluated complications, safety and feasibility of this procedure retrospectively. Methods : Fifty-three patients with nonaneurysmal IVH>15mL without underlying structural etiology or coagulopathy were recruited. The patients with Glasgow Coma Scale[GCS]<5 were excluded. A catheter was directed into the IVH. Hematoma aspiration was followed by instillation of urokinase at the ear level of drainage bag under intracranial pressure monitoring system. This was repeated every 6hours until half of its initial volume. For analysis of prognostic factors, we classified the patients into two groups by Glasgow outcome scale[GOS]; good [$GOS\;{\ge}3$] and bad [GOS<3] prognosis group, and performed comparative analysis between two groups. Results : Mean age was 60.2years. The baseline hematoma size ranged 16 to 72mL. IVH volume reduction was done by an average of 74.2%. As complications, there were 3cases of rebleeding and 2cases of ventriculitis. No intracranial adverse effects were observed during thrombolytic theraphy. At 6months after the procedure, 29patients had achieved a good recovery, 15remained vegetative. 9patients died in hospital. The main good prognostic factors were young age, small IVH volume, and high GCS. Conclusion : The results of this study suggest that this relatively easy and safe method of treatment will improve the prognosis. However, further clinical studies also must assess optimal thrombolytic dosage, frequency, and timing of urokinase instillation for safety and effectiveness and must include controlled comparisons of mortality, disability outcome, quality of life, time until convalescence, and cost of care in treated and untreated patients.

• Tuberculosis and Respiratory Diseases
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• 제44권3호
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• pp.621-628
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• 1997

연구배경 : 흉막의 염증성 변화가 생기면 흉막의 혈관 및 임파관의 투과성의 증가로 인한 혈장단백질의 유입으로 인해 흉막 비후와 격막의 형성으로 인하여 소방이 형성된다. 이러한 소방이 형성된 흉막질환에 있어 흉막강내 섬유소용해 치료의 하나로 유로키나제 치료가 효과적인 것으로 보고되고 있으나, 이에 대한 치료효과 예측을 위한 지표에 대한 연구는 미미한 실정이다. 이에 저자들은 소방이 형성된 흉막질환에서 일반적으로 시행되는 생화학적 지표들과 초음파검사상의 격막의 정도를 이용하여 유로카나제 치료효과에 대한 예측인자에 대해 알아보고자 하였다. 방 법 : 1993년 4월부터 1996년 4월까지 인하병원에서 소방이 형성된 흉막삼출액 소견을 보인 35명의 환자를 대상으로 흉부 초음파검사와 흉막삼출액에 대한 생화학적 검사를 시행하였고, 1개의 경피적 도관을 삽입하여 배액량이 100ml/day 이하로 감소되면 유로키나제를 주입하였고 배액량이 50ml/day 이하로 측정되면 유로키나제 주입을 중단하였다. 흉부단순촬영 추적검사에서 50% 이상의 폐실질의 확장을 보이면 치료효과군으로, 50% 미만의 확장을 보이면 치료실패군으로 분류하여, 각군들에 대한 초기 흉막 삼출액의 당, 단백질, LDH, ADA, pH, 백혈구 수와 초음파 상의 격막 정도를 무에코형, 선상 격막형, 벌집형으로 구분하여 비교하였다. 결 과 : 1) 대상환자 35명중 치료효과군은 27명 (77.1%)이었고, 치료실패군은 8명(22.9%)였으며, 성과 연령에 있어서의 차이는 없었다. 2) 치료효과군과 치료실패군 사이의 증상발현 기간에 있어서 치료실패군에서 유의하게 길었으며, 유로키나제 주입치료 기간중의 배액량도 적음을 알 수 있었다. 치료효과군에서 $89.7{\pm}35.9mg/dl$, 치료실패군에서 $41.2{\pm}47.1mg/dl$로 치료실패군에서 의미있게 낮은 결과를 보였으며, LDH는 치료효과군에서 $878.7{\pm}654.3IU/L$, 치료실패군에서 $2711.1{\pm}973.1IU/L$로 치료실패군에서 의미있게 높은 결과를 보였고(P<0.05), 단백질, pH, ADA, 백혈구수에 있어서는 의미있는 차이가 없었다. 결 론 : 이상의 결과로 소방이 형성된 흉막질환에서 경피적 도관 삽입을 통한 유로키나제 주입치료의 효과를 판정할 수 있는 지표로 증상발현 기간이 길수록, 흉막삼출액의 당이 낮을수록, LDH 의 수치가 높을수록, 초음파 검사상 격막의 정도가 심할수록 치료가 어려운 것을 나타내는 예측인자임을 추정할수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.162-165
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• 2003

In order to study the secretion of the human urokinase-type plasminogen activator, u-PA, from the yeast yarrowia lipolytica, three kinds of integrative expression vector were constructed. These vectors differed only in their secretion control legions, pre-, pre-dip-(dipeptide Stretch) or pre-dip-pro sequences of the alkaline extracellular protease, which were joined inflame to the human u-PA cDNA. The recombinant Y. lipolytica Strains, transformed with the expression vectors, secreted the hyperglycosylated u-PA. A fibrin plate assay of the culture supernatants showed that the hyperglycosylated u-PA proteins could catalyze fibrinolysis, and that the pre-dip sequence was the most efficient secretory signal for the secretion of the u-PA from Y. lipolyica. This result suggests that Y. lipolytica can be developed as a potential host for the production of recombinant human u-PA.

• Archives of Reconstructive Microsurgery
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• 제4권1호
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• pp.1-8
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• 1995

장시간 허혈상태의 토끼 이개를 실험대상으로 하여 유로키나제와 헤파린을 병용 또는 단독으로 사용시와 또한 약물을 사용하지 않았을 때, 이들이 모세혈관의 개존성과 아울러 미세수술후의 조직 생존율에 미칠 수 있는 효과를 보기 위하여 허혈상태의 토끼 이개를 미세수술로 접합한 후 모세혈관으로의 혈류를 측정하기 위하여 레이저 초음파 혈류측정기(Laser doppler flowmetry)를 이용하였으며 방사선 구슬들(Cobalt-57 with plastic material with average diameter 15 micron)을 주입한 결과 유로키나제 조합에서 통계적으로 유효한 성적을 얻었다. 광학현미경 소견은 유로키나제와 헤파린을 병용한 조합에서 모세혈관내 내피세포의 배열이 유지되어 있었으며 헤파린을 사용한 조합에서도 유사한 소견을 보였으나 약물을 사용하지 않은 조합에서는 국소적인 내피세포의 배열이 결핍되어 있었다. 전자현미경 소견에서 유로키나제와 헤파린을 병용한 조합에서 내피세포가 혈관내벽에 배열되어 있었고 또 불규칙한 세포질이 돌출되어 있었다.