Vibrio vulnipcus 에 대한 경구용백신 CJ-50002 의 일반약리작용

박완제(Wan Je Park),김영훈(Young Hoon Kim),정성목(Seong Mok Jeong),이영수(Young Soo Lee),신재규(Jae Kyu Shin),최재묵(Jae Mook Choi),이나경(Na Gyong Lee),이윤하(Youn Ha Lee) 한국응용약물학회 1999 Biomolecules & Therapeutics(구 응용약물학회지) Vol.7 No.1

CJ-50002 is an oral vaccine against V. vulnificus infection composed of whole cell lysate of V. vulnificus. The general pharmacological properties of CJ-50002 were evaluated in various animals and in vitro system. CJ-50002 at oral doses of 0.2, 2 and 20 mg/kg had no effect on general behavior in mice, chemoand electro-convulsions in mice, writhing syndrome induced by acetic acid in mice, body temperature in rats, charcoal meal propulsion in mice and urine and electrolytes excretion in rats. However, oral administration of CJ-50002 at dose of 20 mg/kg prolonged the hexobarbital-inuced sleeping inducing time in mice. In anesthetized dogs, CJ-50002 showed no effect on blood pressure, heart rate and ECG but decreased the respiratory rate and femoral blood flow at dose of 20 mg/kg. p.o. CJ-50002 had no effect on the contractile response of the isolated guinea pig ileum to various spasmogen at concentrations of 0.2, 2 and 20 ㎍/ml, respectively. Since these pharmacological effects of CJ-50002 were observed at dose much greater than those in clinical use (approximately 0.16 mg/kg, p.o.), it is likely that this vaccine may be relatively free of undesirable effects in clinical practice.

Purification and Properties of Malic Enzyme from the Uropygial Gland of Peking Duck.

박완제,김유삼,Park, Wan-Je,Kim, Yu-Sam 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.1

Uropygial gland에 있는 malic enzyme을 2',5'-ADP agarose affinity chromatography, DEAE-Sephacel ion exchange chromatography에 의해서 183배 정제하였으며 specific activity는 174 unit/mg protein이었다. 이렇게 정제된 효소는 SDS-polyacrylamide gel electrophoresis를 하였을 때 95% 이상의 순도를 나타냈으며, subunit의 분자크기는 약 60,000dalton이었다. 최적 pH는 7.5이었으며 이 효소의 활성을 나타내는데는 $Mn^{++}$이나 $Mg^{++}$ 이온이 필요하였다. Uropygial gland malic enzyme의 경우에 있어서 malate와 $NADP^+$에 대한 Km은 각각 $370{\mu}M$, $10.6{\mu}M$ 이었으며, liver malic enzyme의 경우는 $606{\mu}M$, $20.8{\mu}M$이었다. Uropygial gland와 liver 효소에 대한 product inhibition pattern은 서로 같은 양상을 나타나는데 즉, bicarbonate는 malate와 $NADP^+$에 대하여 모두 uncompetitive inhibitor이었고, pyruvate에 대하여서는 두 기질 모두 uncompetitive inhibitor이었으며, NADPH는 malate에 대하여 noncompetitive inhibitor이었으나 $NADP^+$에 대해서는 competitive inhibitor이었다. 그러나 uropygial gland 효소의 product (bicarbonate, pyruvate, NADPH)에 대한 Ki 값은 liver 효소의 Ki 값보다 2-3배정도의 높은 값을 나타냈다. Initial velocity plot과 produt inhibition pattern을 조사해본 결과 효소에 $NADP^+$가 먼저 결합한 후 다음에 malate가 들어와서 결합하게 되면 생성물이 $CO_2$, pyruvate, NADPH 순서로 유리되는 ordered kinetic mechanism을 갖는다는 사실을 알아 내었다. Uropygial gland에서 분리한 malic enzyme은 간에서 분리한 같은 효소에 대하여 이상과 같은 차이점이 있음은 물론 전기이동에 의하여서도 이동거리가 서로 다른 것으로 보아 하나의 isozyme인 것으로 예측되며 uropygial gland의 특별한 역할에 잘 맞도록 구성된 것으로 믿어진다. Malic enzyme from the uropygial gland of duck was purified by the combination of 2',5'-ADP agarose and DEAE-Sephacel in the electrophoretically homogeneous form. Purification fold and specific activity were 183 and 174 unit/mg protein, respectively. Molecular size of subunit determined by SDS-polyacrylamide gel electrophoresis was 60,000 dalton. Optimal pH was 7.5 and $Mn^{++}$ or $Mg^{++}$ was required for activity. For the enzyme in uropygial gland the apparent Km for malate and $NADP^+$ were $370{\mu}M$ and $10.6{\mu}M$ respectively. In case of liver enzyme apparent Km for malate and $NADP^+$ were $606{\mu}M$ and $20.8{\mu}M$, respectively. In uropygial gland and liver, the following product inhibition patterns were observed with malate and $NADP^+$ as variable substrates: NADPH, noncompetitive ($Ki_G$, $166{\mu}M$; $Ki_L$, $97{\mu}M$) and competitive($Ki_G$, $43{\mu}M$; $Ki_L$, $22{\mu}M$): pyruvate, uncompetitive($Ki_G$, $26{\mu}M$; $Ki_L$, $11{\mu}M$) and uncompetitive ($Ki_G$, $32{\mu}M$; $Ki_L$, $20{\mu}M$): bicarbonate, noncompetitive ($Ki_G$, $112{\mu}M$; $Ki_L$, $36{\mu}M$) and noncompetitive ($Ki_G$, $183{\mu}M$; $Ki_L$, $63{\mu}M$). In addition to this kinetic difference between the enzymes from the gland and liver, electrophoretic mobility of the gland enzyme was also different from that of the liver enzyme, suggesting that these enzymes are isozymes each other and the gland enzymes are specially designed to meet the spcialized function of the unopygial gland.

오리의 Uropygial Gland 로부터 Malic Enzyme 의 정제 및 그 특성

박완제,김유삼 ( Wan Je Park,Yu Sam Kim ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.1

Malic enzyme from the uropygial gland of duck was purified by the combination of 2`,5`-ADP agarose and DEAF-Sephacel in the electrophoretically homogeneous form. Purification fold and specific activity were 183 and 174 unit/㎎ protein, respectively. Molecular size of subunit determined by SDS-polyacrylamide gel electrophoresis was 60, 000 dalton. Optimal pH was 7, 5 and Mn^(++) or Mg^(++) was required for activity. For the enzyme in uropygial gland the apparent Km for malate and NADP+ were 370μM and 10. 6μM respectively. In case of liver enzyme apparent Km for malate and NADP^+ were 606μM and 20.8μM, respectively. In uropygial gland and liver, the following product inhibition patterns were observed with malate and NADP^+ as variable substrates: NADPH, noncompetitive (Ki_G, 166μM; Ki_L, 97μM) and competitive (Ki_G, 43μM; Ki_L, 22μM) : pyruvate, uncompetitive (Ki_G, 26mM; Ki_L, 11mM) and uncompetitive (Ki_G, 32mM; Ki_L, 20mM) : bicarbonate, noncompetitive (Ki_G, 112mM; Ki_L, 36mM) and noncompetitive (Ki_G, 183mM; Ki_L, 63mM) . In addition to this kinetic difference between the enzymes from the gland and liver, electrophoretic mobility of the gland enzyme was also different from that of the liver enzyme, suggesting that these enzymes are isozymes each other and the gland enzymes are specially designed to meet the spcialized function of the unopygial gland.

CFC-101 ( 녹농균 백신 ) 의 능동 및 수동면역 효과

김영지(Young Gi Kim),김제학(Je Hak Kim),박완제(Wan Je Park),김유삼(Yu Sam Kim),함경수(Kyung Soo Hahm),조양제(Yang Je cho),박관하(Kwan Ha Park) 한국응용약물학회 1994 Biomolecules & Therapeutics(구 응용약물학회지) Vol.2 No.4

The treatment of pseudomonal infection is a perplexed problem because of its modest susceptibility to most of the major antibiotics. A novel Pseudomonas vaccine(CFC-101) was prepared from the outer membrane protein fractions of several Pseudomonas strains. In this study, we examined CFC-101`s effectiveness in both active and passive immunization models. CFC-101 in mice at 0.2 mg/kg, i.p., given three times at two-day intervals, completely prevented the death caused by Pseudomonas aeruginosa. Antibody titer, in accordance with the protective effect in this active immunization, was elevated to its peak level following three consecutive administrations of CFC-101. Thereafter, antibody titer stayed at a constantly high level. Each outer membrane protein fraction from the four CFC-101 producers, exhibited good cross-protective effects in mouse infection models against different Fisher types of P. aeruginosa. In the passive immunization model, 21∼336 ㎍/㎏ of anti-rabbit IgG to CFC-101, when mice being infected with a challenge strain, prevented the Pseudomonas-induced death up to 60%. Therefore, the preventive effect of CFC-101 was verified in both the active and passive immunization models.

마우스에서 CFC-101 ( 녹농균 백신 ) 의 감염 방어효과

김영지(Young Gi Kim),김제학(Je Hak Kim),박완제(Wan Je Park),안동호(Dong Ho Ahn),홍광희(Kwang Hee Hong),김현수(Hyun Su Kim),김유삼(Yu Sam Kim),함경수(Kyung Soo Hahm) 한국응용약물학회 1994 Biomolecules & Therapeutics(구 응용약물학회지) Vol.2 No.4

To optimize the immunological efficacy of CFC-101, an outer-membrane protein vaccine purified from relatively less pathogenic 4 different Pseudomonas aeruginosa strains, we investigated to establish its dose, administration route, interval and frequency of vaccination in mice. As expected, the 4 CFC-101 producing strains were less pathogenic than the challenging organism, P. aeruginosa GN11189. CFC-101 completely protected the death caused by P. aeruginosa at above 0.05 mg/kg vaccinized by 3 times with 7-day intervals. At the optimally effective dose of 0.2 mg/kg of CFC-101, at least 3 immunizations were necessary for complete protection against P. aeruginosa-induced death. If immunized 3 times, the immunization interval could be shortened up to 2 days to acquire the best protection against P. aeruginosa. CFC-101 was effective either by intraperitoneal, subcutaneous or intramuscular but not by oral administration. The present results show that the newly developed Pseudomonas vaccine, CFC-101, is highly effective for the protection from death caused by pseudomonal infections.

CJ-50002(비브리오백신)의 랫드 및 비글개에서의 단회투여 독성시험

이성학(Sung Hak Lee),임동문(Dong Moon Lim),김달현(Dal Hyun Kim),정상보(Sang Bo Jung),박완제(Wan Je Park),이영수(Young Soo Lee) 한국독성학회 1999 Toxicological Research Vol.15 No.2

The single dose toxicity test of CJ-50002, an oral Vibrio vaccine against Vibrio vulnificus injection, was performed in Sprague Dawley (SD) rats and beagle dogs. CJ-50002 was orally administered up to 500 mg/kg in SD rats and 167 mg/kg in beagle dogs. In these experiments, there were no death and clinical changes which were related to CJ-50002. In addition, there were no significant changes between control group and treated groups in body weights and autopsy findings. In conclusion, oral LD50 of CJ-50002 is over 500 mg/kg in SD rats and over 167 mg/kg in beagle dogs.

CJ-50002(비브리오 백신)에 대한 변이원성시험

강재구(Jae-Ku Kang),이성학(Sung Hak Lee),김달현(Dal Hyun Kim),이나경(Na-Gyong Lee),박완제(Wan Je Park),이영수(Young Soo Lee) 한국독성학회 1999 Toxicological Research Vol.15 No.2

We have been developing a vaccine (CJ-50002) against Vibrio vulnificus, composed of whole cell lysate of a V. vulnificus O-antigen serotype 4-strain. In order to evaluate the mutagenic potential of CJ-50002, 3 sets of mutagenicity tests were performed. In the reverse mutation test using Salmonella typhimurium TA1535, TA1537, TA98 and TA100, CJ-50002 did not increase the number of revertant at any concentration tested in this study (309.6, 154.8, 77.4, 38.7, 19.4 and 9.7 ㎍/plate). CJ-50002, at concentrations of 309.6, 154.8 and 77.4 ㎍/ml, did not increase the number of cells having structural or numerical chromosome aberration in cytogenic test using Chinese Hamster Lung cells. In mouse micronucleus test, no significant increase in the occurrence of micro nucleated polychromatic erythrocytes was observed in ICR male mice orally administered with CJ-50002 at the doses of 500, 250 and 125 mg/kg. These results indicate that CJ-50002 has no mutagenic potential in these in vitro and in vivo systems.

경구투여한 V. vulnificus 백신의 면역원성 및 감염방어효능

이나경(Na Gyong Lee),정상보(Sang Bo Jung),안보영(Bo Young Ahn),김영지(Young Gi Kim),이윤하(Youn Ha Lee),전영중(Young Joong Jeon),박완제(Wan Je Park) 한국응용약물학회 1998 Biomolecules & Therapeutics(구 응용약물학회지) Vol.6 No.2

Vibrion vulnificus is an estuarine gram0negative human pathogen that affects people with chronic hepatitis, alcoholic cirrhosis, diabetes mellitus or other underlying diseases, V. vulnificus infection is mediated primarily by consumption of raw fish or by exposure of pre-existing wounds to seawater, causing permanent tissue damages or fatal septic shock. We have been developing a vaccine against V. vulnificus composed of whole cell lysate of a V. vulnificus O-antigen serotype 4 strain. Oral administration of the V. vulnificus vaccine elicited a high serum antibody response in rabbits. The induced antibodies were reactive not only to the homologous strain but also to heterologous O-antigen serotype strains, indicating cross-reactivities among serotypes. Western blot analysis revealed that the antibodies are mainly specific for outer membrane proteins (OMPs) and reacted equally well with OPMs purified from 9 O-antigen serotypes The rabbit antisera showed opsonophagocytic killing activity against heterologous strains as well as the homologous strain. Passively transferred rabbit antisera into mice were protective against a lethal V. vulnificus infection. These data demonstrate that oral administration of the V. vulnificus infections, suggesting that this vaccine preparation could be used to develop an oral vaccine against V. vulnificus. Keywords □ V. vulnificus. oral vaccine, immunogenicity, protective efficacy

유전자 재조합 B 형 간염 바이러스 표면 항원 , CJC-50100 의 일반약리작용

정성학(Seong Hak Jeong),최재묵(Jae Mook Choi),이남중(Nam Jung Lee),전형수(Hyung Soo Jeon),김연희(Yon Hee Kim),김재승(Jae Seung Kim),하석훈(Suk Hoon Ha),김영훈(Young Hoon Kim),이나경(Na Gyung Lee),김제학(Je Hak Kim),박완제(Wan Je Park 한국응용약물학회 2001 Biomolecules & Therapeutics(구 응용약물학회지) Vol.9 No.1

N/A CJC-50100 is a recombinant hepatitis B virus surface antigen (HBsAg) expressed in yeast. The general pharmacological properties of CJC-50100 were evaluated in mice, rats, dogs and isolated guinea pig ileum. The doses were 0.33∼33.3 ㎍/㎏ i.m. for mice and rats and 3.3∼9.9 ㎍/㎏ i.v. for dogs. The concentrations of 0.002∼0.02 ㎍/mlwere used for the assay with guinea pig ileum. Intramuscular administration of CJC-50100 at the doses did not alter general behavior and the responses for central nervous system, smooth muscle, gastrointestinal system, cardiovascular and respiratory system, and water and electrolytes excretion. In summary, CJC-50100 had no pharmacological effect in these studies even up to the 100-fold of the expected clinical dose, 20 ㎍/man/60 kg.

녹동균 세포외막 단백질 백신 CFC-1-101의 안정성 및 면역원성 검토 : 임상 제 Ⅰ/Ⅱa상 시험

장인진,김익상,유경상,임동석,김형기,신상구,장우현,박완제,이나경,정상보,안동호,조양제,안보영,이윤하,김영지,남성우,김현수 대한감염학회 1998 감염 Vol.30 No.3

목적 : 제일제당에서는 녹농균의 세포외막 단밸질을 유효성분으로 하는 백신인 CFC-101을 개발하였으며, 동물시험에서 이 백신의 안전성과 유효성을 입증하였다. 본 연구에서는 이 녹농균 백신의 인체에 대한 안전성과 면역원성을 평가하는 동시에 인체 접종시의 최적 투여 용량을 결정하기 위하여 제 I/Ⅱa상 임상시험을 수행하였다. 방법 : 건강한 성인 남자를 피험자로 선별하여 각 용량군에 백신투여자 6명, 위약투여자 2명을 배정하였다. 백신 투여군은 0.25mg, 0.5mg 또는 1.0mg 용량의 녹농균 백신을 7일 간격으로 3회에 걸쳐 근육주사 하였으며, 위약 투여군에게는 세포외막 단백질을 제외한 동일한 성분을 투여하였다. 백신접종 후 국소적 또는 전신적인 반응의 발생여부를 관찰하고, 혈액시료를 체취하여 백신의 역가와 유효성을 검정하였다. 결과 : 녹농균 백신 CFC-101은 모든 접종자에서 양호한 내약성을 보였다. 또한 0.5mg 과 1.0mg 백신 투여군에서는 100%의 항체양전율을 나타내었다. 생성된 항체는 녹농균 세포외막단백질에 특이성을 보였고, 녹농균 감염에 대해 방어효능이 있었다. 결론 : 이와같은 결과로부터 이 녹농균 백신은 인체에 안전하게 투여할 수 있으며, 높은 항체 생성능으로 감염방어 효능을 보이고 0.5mg과 1.0mg이 최적용량인 것으로 판단되었다. Background : We developed a Pseudomonas aeruginosa outer membrane protein(OMP) vaccine CFC-101, and the prophylactic efficacy of which has been demonstrated in animal models. In order to evaluate the safety and immunogenicity of the P. aeruginosa vaccine, we carried out a phase I/Ⅱa clinical trial in healthy male volunteers. Methods : Groups of eight volunteers, including two placebo subjects, were vaccinated intramuscularly with three doses of 0.25, 0.5 or 1.0 mg of the vaccine at one week intervals. Sings of systemic and local reactions observed after vaccination were recorded for each vaccinee for 5 days. Physical examinations were performed on days 0, 1, 7, 8, 14, 15, 21, and 42, and clinical laboratory tests were done on days 0, 3, and 21. Blood samples for assay of serum antibody levels were obtained up to 42 days after the first vaccination. Results : The vaccine was generally well tolerated by all vaccinees, showing no significant side effects. In the three dosage groups, all vaccinees, except one receiving the 0.25 mg dose, showed significant elevation in serum IgG antibody titers against the vaccine proteins, indicating 100% seroconversion in 0.5 and 1.0 mg groups. The human antibodies induced by the vaccine were specific for P. aeruginosa OMPs, as confirmed by western blot analysis and immunoprecipitation assays. The capacity of the human antisera to enhance opsonophagocytic killing activity by polymorphonuclear leukocytes and to confer protection against P. aeruginosa infections indicates that the antibodies elicited by the vaccine have protective efficacy. Conclusion : We conclude that the P. aeruginosa OMP vaccine is safe and effective for human use and its optimal dose to be 0.5 or 1.0 mg.