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Characterization of a Psychrophilic Metagenome Esterase EM2L8 and Production of a Chiral Intermediate for Hyperlipemia Drug (메타게놈유래의 저온성 에스터라제 EM2L8의 효소적 특성과 이를 활용한 고지혈증 치료제 키랄소재의 생산)

  • Jung, Ji-Hye;Choi, Yun-Hee;Lee, Jung-Hyun;Kim, Hyung-Kwoun
    • Microbiology and Biotechnology Letters
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    • v.37 no.2
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    • pp.118-124
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    • 2009
  • Esterase EM2L8 gene isolated from deep sea sediment was expressed in Escherichia coli BL21 (DE3) and the esterase activity of the cell-free extract was assayed using p-nitrophenyl butyrate-spectrophotometric method. Its optimum temperature was $40-45^{\circ}C$ and 45% activity of the maximum activity was retained at $15^{\circ}C$. The activation energy at $15-45^{\circ}C$ was calculated to be 4.9 kcal/mol showing that esterase EM2L8 was a typical cold-adapted enzyme. Enzyme activity was maintained for 6 h and 4 weeks at $30^{\circ}C$ and $4^{\circ}C$, respectively. When each ethanol, methanol, and acetone was added to the reaction mixture to 15% concentration, enzyme activity was maintained. In the case of DMSO, enzyme activity was kept up to 40% concentration. (S)-4-Chloro-3-hydroxy butyric acid is a chiral intermediate for the synthesis of Atorvastatin, a hyperlipemia drug. When esterase EM2L8 (40 U) was added to buffer solution (1.2 mL, pH 9.0) containing ethyl-(R,S)-4-chloro-3-hydroxybutyrate (38 mM), it was hydrolyzed into 4-chloro-3-hydroxy butyric acid with a rate of $6.8\;{\mu}mole/h$. The enzyme hydrolyzed (S)-substrate more rapidly than (R)-substrate. When conversion yield was 80%, e.e.s value was 40%. When DMSO was added, hydrolysis rate increased to $10.4\;{\mu}mole/h$. The plots of conversion yield vs e.e.s in the presence or absence of DMSO were almost same, implying that the reaction enantioselectivity was not changed by the addition of DMSO. Taken together, esterase EM2L8 had high activity and stability at low temperatures as well as in various organic solvents/aqueous solutions. These properties suggested that it could be used as a biocatalyst in the synthesis of useful pharmaceuticals.

A study of neuropeptides related to headaches in children with meningeal irritation signs (수막 자극 증상으로 내원한 환자에서 두통과 관련된 신경 펩타이드에 대한 연구)

  • Seo, Bo Gil;Yoo, Myung Hwan;Shim, Jae Won;Shim, Jung Yeon;Jung, Hye Lim;Park, Moon Soo;Kim, Deok-Soo
    • Clinical and Experimental Pediatrics
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    • v.49 no.5
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    • pp.533-538
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    • 2006
  • Purpose : The headache, one of the symptoms of meningitis, is related to abrupt elevation of intracranial pressure(ICP) or stimulation of intracranial nociceptive structure. However, in cases of mild elevation of ICP or normal findings of cerebrospinal fluid(CSF) analysis, patients sometimes complain of headaches. Therefore, other pathways may contribute to the occurrence of headaches in aseptic meningitis or meningismus. We intend to investigate the role of substance P(SP) and calcitonin gene-related peptide(CGRP) in aseptic meningitis or meningismus. Methods : We measured leukocyte count, the concentration of protein and glucose in CSF and ICP of patients with meningeal irritation sign. We also measured SP and CGRP levels by using immunoassay. We analyzed the relationship between the presence of headache and the value of SP and CGRP. Results : The concentrations of CGRP($18.8{\pm}10.5ng/mL$) in CSF and ICP($14.8{\pm}4.5cmH_2O$) in aseptic meningitis group were significantly higher than in those($14.1{\pm}7.4ng/mL$ and $12.0{\pm}5.1cmH_2O$, respectively) of the meningismus group(P<0.05). There was no significant difference in the SP levels between the two groups. In the aseptic meningitis group, the concentrations of SP and CGRP were significantly higher in the normal ICP group than in the elevated ICP group(P<0.05). Conclusion : Headaches in children with aseptic meningitis or meningismus is considered to be related to the elevation of the CSF levels of SP and CGRP.

Comparison of Radioactivity Measurement with Radionuclide Calibrators in Nuclear Medicine Centers (의료용 방사능측정기의 측정 정확도 평가)

  • Son, Hye-Kyung;Kim, Ji-Hye;Lim, Chun-Il;Yang, Hyun-Kyu;Park, Ki-Jung;Oh, Heon-Jin;Kim, Hyeog-Ju;Kim, Dong-Sup
    • Progress in Medical Physics
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    • v.21 no.1
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    • pp.16-21
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    • 2010
  • To acquire good image quality and to minimize unnecessary radiation dose to patients, it is important to ensure that the radiopharmaceutical administered is accurately measured. Quality control of radionuclide calibrators should be performed to achieve these goals. The purpose of this study is to support the quality control of radionuclide calibrators in nuclear medicine centers and to investigate the level of measurement accuracy of the radionuclide calibrators. 58 radionuclide calibrators from 45 nuclear medicine centers, 74 radionuclide calibrators from 58 nuclear medicine centers, and 60 radionuclide calibrators from 45 nuclear medicine centers were tested with I-131, Tc-99m and I-123, respectively. The results showed that 81% of calibrators for I-131, 61% of calibrators for Tc-99m and 67% of calibrators for I-123 were within ${\pm}5%$. 17% of calibrators for I-131, 20% of calibrators for Tc-99m and 15% of calibrators for I-123 had a deviation in the range 5%< $|{\Delta}|{\leq}10%$. 2% of calibrators for I-131, 19% of calibrators for Tc-99m and 18% of calibrators for I-123 had a deviation of $|{\Delta}|$ >10%. Follow-up measurements were performed on the calibrators whose error exceeded the ${\pm}10%$ limit. As a result, some of the calibrator showed an improvement and their deviation decreased below the ${\pm}10%$ limit. The results have shown that such comparisons are necessary to improve the accuracy of the measurement and to identify malfunctioning radionuclide calibrators.

Development trend of the mushroom industry (버섯 산업의 발달 동향)

  • Yoo, Young Bok;Oh, Min Ji;Oh, Youn Lee;Shin, Pyung Gyun;Jang, Kab Yeul;Kong, Won Sik
    • Journal of Mushroom
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    • v.14 no.4
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    • pp.142-154
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    • 2016
  • Worldwide production of mushrooms has been increasing by 10-20% every year. Recently, Pleurotus eryngii and P. nebrodensis have become popular mushroom species for cultivation. In particular, China exceeded 8.7 million tons in 2002, which accounted for 71.5% of total world output. A similar trend was also observed in Korea. Two kinds of mushrooms-Gumji (金芝; Ganoderma) and Seoji-are described in the ancient book 'Samguksagi' (History of the three kingdoms; B.C 57~A.D 668; written by Bu Sik Kim in 1145) during the Korea-dynasty. Many kinds of mushrooms are also described in more than 17 ancient books during the Chosun-dynasty (1392~1910) in Korea. Approximately 200 commercial strains of 38 species of mushrooms were developed and distributed to cultivators. The somatic hybrid variety of oyster mushroom, 'Wonhyeong-neutari,' was developed by protoplast fusion, and distributed to growers in 1989. Further, the production of mushrooms as food was 199,829 metric tons, valued at 850 billion Korean Won (one trillion won if mushroom factory products are included) in 2015. In Korea, the major cultivated species are P. ostreatus, P. eryngii, Flammulina velutipes, Lentinula edodes, Agaricus bisporus, and Ganoderma lucidum, which account for 90% of the total production. Since mushroom export was initiated in 1960, the export and import of mushrooms have increased in Korea. Technology was developed for liquid spawn production, and automatic cultivation systems led to the reduction of production cost, resulting in the increase in mushroom export. However, some species were imported owing to high production costs for effective cultivation methods. In academia, RDA scientists have conducted mushroom genome projects since 1997. One of the main outcomes is the whole genome sequencing of Flammulina velutipes for molecular breeding. With regard to medicinal mushrooms, we have been conducting genome research on Cordyceps and its related species for developing functional foods. There are various kinds of beneficial substances in mushrooms; mushroom products, including pharmaceuticals, tonics, healthy beverages, functional biotransformants, and processed foods have also became available on the market. In addition, compost and feed can likewise be made from mushroom substrates after harvest.

Tow-Year Follow-up Study for Clinical Feature and Immunity of The Children, Vaccinated by 47 Passaged Oka Strain Live Attenuated Varicella Vaccine (47계대 Oka주 약독화 생백신 접종 후 2년간 임상 및 면역성의 추적 연구)

  • Kang, Jin Han;Kim, Jong Hyun;Hur, Jae Kyun;Woo, Koo
    • Pediatric Infection and Vaccine
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    • v.7 no.1
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    • pp.129-135
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    • 2000
  • Purpose : We previously reported the short-term immunogenecity and safety of 47 passaged Oka strain live attenuated varicella vaccine in healthy children in 1997. Now, we conducted this two-year follow-up study to confirm the maintenance of immunity, the occurrence of natural varicella infection and the activation of vaccine induced latent infection on the same vaccine. Methods : 99 children who had been immunized by 47 passaged Oka strain live attenuated varicella vaccine in 1997 were followed up by questionnaire, and 46 children out of study group were followed up serologically. They were asked to report any instance of varicella or herpes zoster since they had been immunized. If there was any evidence of varicella or herpes zoster, they should be clinically or serologically confirmed by doctor. Also, those patients' parents were asked to report any instance of varicella or herpes zoster in their family, playmate, kindergarten, school, or other settings. The immunity to VZV was confirmed by EIA and FAMA test. Results : 6 recipients developed breakthrough varicella after exposure to VZV in family, kindergarten and school during follow-up period. However, clinical features of those patients were very mild and self limited without therapy. And none of the recipients developed herpes zoster during this observation period. The results of EIA test showed that study subjects were all seropositive except one, and the antibody titers and GMT of FAMA test were seropositively maintained in all subjects. Statistically, the antibody titers of EIA and FAMA test confirmed two years after vaccination were higher than those results confirmed one month after vaccination. Conclusion : Our study results suggest that the immunity of 47 passaged Oka strain live attenuated varicella is well maintained until 2 years later after vaccination, and mild natural infection after exposure to VZV can be occurred with low rate. There were not developing zoster in study vaccine after vaccination for two-years.

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Safety and Reactogenicity of the Inactivated Poliomyelitis Vaccine (PoliorixTM) in Korea (2006-2012) (우리나라 영아에서 주사용 소아마비 백신(PoliorixTM)의 안전성 및 이상반응에 대한 연구)

  • Sin, Jong Beom;Park, Moon Sung;Ma, Sang Hyuk;Choi, Young Youn;Shin, Son Moon;Kim, Won Duck;Kuriyakose, Sherine;Ulianov, Liliana;Hardt, Karin
    • Pediatric Infection and Vaccine
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    • v.20 no.3
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    • pp.139-146
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    • 2013
  • Objective: As per the requirement of Korean Food and Drug Administration, this post-marketing surveillance was conducted in Korea to evaluate the safety and reactogenicity of Poliorix$^{TM}$ following its introduction in 2006. Methods: In this open, multicenter study, the vaccine was administered as per the current practice of Korean doctors and in reference to the guidebook by the Korean Pediatric Society and as indicated in the Korean label which was as follows - for primary vaccination three doses were given to infants at ages 2, 4 and 6 months whereas, for the booster dose a single dose was given to children aged 4-6 years. Safety data during this six year surveillance was collected using diary cards which were distributed to the parents to record adverse events. Results: A total of 639 subjects were enrolled into the study. Of these, 617 subjects and 22 subjects received the vaccine as a primary and booster dose, respectively. At least one unsolicited symptom was reported in 11.4% (73/639) of the subjects during the 7-day follow-up period; upper respiratory tract infection (2.5%;16/639) was the most frequently reported unsolicited symptom. One subject reported at least one unsolicited symptom (gastroenteritis) of grade 3 intensity within the 31-day post-vaccination period. Approximately 1.7% (11/639) of subjects reported 13 serious adverse events (SAEs). All SAEs were resolved by the end of the study. Conclusion: In Korea, primary and booster vaccination with Poliorix$^{TM}$ was well-tolerated in healthy subjects when administered according to the prescribing information as part of routine clinical practice.

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Detection of Multidrug Resistance Using Molecular Nuclear Technique (분자핵의학 기법을 이용한 다약제내성 진단)

  • Lee, Jae-Tae;Ahn, Byeong-Cheol
    • The Korean Journal of Nuclear Medicine
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    • v.38 no.2
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    • pp.180-189
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    • 2004
  • Although the outcome of cancer patients after cytotoxic chemotherapy is related diverse mechanisms, multidrug resistance (MDR) for chemotherapeutic drugs due to cellular P-glycoprotein (Pgp) or multidrug-resistance associated protein (MRP) is most important factor in the chemotherapy failure to cancer. A large number of pharmacologic compounds, including verapamil, quinidine, tamoxifen, cyclosporin A and quinolone derivatives have been reported to overcome MDR. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are available for the detection of Pgp and MRP-mediated transporter. $^{99m}Tc$-MIBI and other $^{99m}Tc$-radiopharmaceuticals are substrates for Pgp and MRP, and have been used in clinical studies for tumor imaging, and to visualize blockade of PgP-mediated transport after modulation of Pgp pump. Colchicine, verapamil and daunorubicin labeled with $^{11}C$ have been evaluated for the quantification of Pgp-mediated transport with PET in vivo and reported to be feasible substrates with which to image Pgp function in tumors. Leukotrienes are specific substrates for MRP and $N-[^{11}C]acetyl-leukotriene$ E4 provides an opportunity to study MRP function non-invasively in vivo. SPECT and PET pharmaceuticals have successfully used to evaluate pharmacologic effects of MDR modulators. Imaging of MDR and reversal of MDR with bioluminescence in a living animal is also evaluated for future clinical trial. We have described recent advances in molecular imaging of MDR and reviewed recent publications regarding feasibility of SPECT and PET imaging to study the functionality of MDR transporters in vivo.

Study on Overcoming Interference Factor by Automatic Synthesizer in Endotoxin Test (내독소 검사에서 자동합성장치에 따른 간섭요인 극복에 대한 연구)

  • Kim, Dong Il;Kim, Si Hwal;Chi, Yong Gi;Seok, Jae Dong
    • The Korean Journal of Nuclear Medicine Technology
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    • v.16 no.2
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    • pp.3-6
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    • 2012
  • Purpose : Samsung medical ceter shall find a cause of the interference factor and suggest a solution for it. Materials and Methods : A sample of $^{18}F$-FDG, radioactive pharmaceuticals produced by TRACERlab MX and FASTlab synthesizer. Gel-clot method uses Positive control tube and single test tube. Kinetic chromogenic method uses ENDOSAFE-PTS produced by Charles River. Results : According to Gel clot method of Endotoxin Tests at FASTlab, both turbidity and viscosity increased at 40-fold dilution and Gel clot was detected. In case of TRACERlab MX, Gel clot was detected in most of samples but intermittently not in a few of them. When using ENDOSAFE-PTS, sample CV (Coefficient of Variation) of FASTlab is 0% at all dilution rates whereas spike CV is 0% at 1-fold dilution, 0~35% at 10-fold, 3.6~12.9% at 20-fold, 5.2~7.1% at 30-fold, 1.1~17.4% at 40-fold, spike recovery; 0% at one-fold, 25 ~ 58% at 10-fold, 50 ~ 86% at 20-fold, 70~92% at 30-fold, and 75~120% at 40-fold. Sample CV of TRACERlab MX, is 0% at all dilution rates whereas spike CV is 1.4~4.8% at one-fold dilution, 0.6~19.9% at 10-fold, spike recovery; 35~72% at one-fold dilution and 77~107% at 10-fold. Conclusion : Gel clot does not seem to occur probably to H3PO4 which engages in bonding with Mg2+ion contributing gelation inside PCT. Dilution which is identical to reducing the amount of H3PO4, could remove interfering effects accordingly. Spike recovery was obtained within 70~150% - recommended values of supplier - at 40-fold dilution even in kinetic chromogenic method.

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Characteristics of Percutaneous Absorption of Glycol ethers (Glycol ethers에 대한 피부 투과 특성)

  • Lee, Han-Seob;Choi, Sung-Boo;Kim, Nac-Joo;Keun, Jang-Hyoun;Hwang, Hyun-Suk;Baek, Jung-Hun;Choi, Jin-Ho;Lee, Ho-Joon
    • Journal of the Korean Applied Science and Technology
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    • v.30 no.1
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    • pp.116-126
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    • 2013
  • Glycol ethers are a group of solvents based on alkyl ethers of ethylene glycol commonly used in paints. These solvents typically have a higher boiling point, together with the favorable solvent properties of lower-molecular weight ethers and alcohols. The word "Glycol ethers" was registered as a United States trademark by Union Carbide Corp. Typically, glycol ethers are found in pharmaceuticals, sunscreens, cosmetics, inks, dyes and water based paints. On the other hand, glycol ethers are used in degreasers, cleaners, aerosol paints and adhesives. Most glycol ethers are relatively water soluble, biodegradable and only a few are considered toxic. Therefore, they are unlikely to pose an adverse risk to the environment. Recent study suggests that occupational exposure to glycol ethers is related to low motile sperm count in men, but the finding has been disputed by others. In this study, skin permeation of 3 types glycol ethers were studied in vitro using matrix such as solvent and detergent. The absorption of glycol ethers[methyl glycol ethers(MC), ethyl glycol ethers(EC) and butyl glycol ethers(BC)] has been measured in vitro through rat skin. Epidermal membranes were set up in Franz diffusion cells and their permeability to PBS measured to establish the integrity of the skin before the glycol ethers were applied to the epidermal surface. Absorption rates for each glycol ethers were determined and permeability assessment made to quantify any irreversible alterations in barrier function due to contact with the esters. Types of glycol ethers in vitro experimental results on MC> EC> BC quickly appeared in the following order: skin permeation was beneficial to the skin permeation small molecular weight, the difference in chemical structure, such as hydrophilic, because with the partition coefficient and solubility mechanisms and passive diffusion to increase the speed at which transmission is considered.

Skin Improvement Effects and Development of Liposome Capsule Technology Using Centella Asiatica Extract Powder (센텔라아시아티카정량추출물의 리포좀 캡슐기술 개발과 피부개선효과)

  • Kim, Seong Jang;Ju, Yeon Jeong;Kim, In-Young
    • Journal of the Korean Applied Science and Technology
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    • v.37 no.5
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    • pp.1285-1297
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    • 2020
  • In this study, we report the results of a study on the clinical evaluation of wrinkle improvement by developing a method for liposome of high-purity Centella asiatica extract used in pharmaceuticals and cosmetics, and a cream using the same. In order to make Centellasome-10EX stabilizing centella asiatica extract in liposome lamella vesicle, it could be completed using 5% hydrogenated lecithin and 2% sucrose distearate. The appearance of Centellasome-10EX was a creamy form of low viscosity, the color was pale yellow, and the odor had the inherent odor of the raw material. The pH was about 6.12, the specific gravity was 1.09, and the acid value was about 0.35. The content of the main constituents of centella asiatica extract contained in the liposome vesicle contains 10,800 ppm of asiatic acid, 10,900 ppm of asiaticoside, 6,000 ppm of madecasic acid, and 1,600 ppm of madecassoside, and long-term storage. There was no discoloration even at the time, and it was found that the main component remained stable thermodynamically. To mechanistically analyze the structure of the liposome vesicle of Centellasome-10EX, as a result of observation with a transmission electron microscope (Cryo-TEM), the multilayer vesicles are formed and filled with moisture, and there are 10 to 60 multilayers around it. It was confirmed that the liposome lamella vesicle was formed. As a clinical trial (in-vivo) test, the moisturizing effect of centellasome cream after application for 5 weeks was 28.3%, which was significantly increased compared to placebo. The skin elasticity effect was 13.6%, which significantly increased the moisturizing power than the placebo. The effect of improving fine wrinkles around the eyes was improved by 23.52% compared to placebo cream. Through the results of this study, it was possible to study the formulation and manufacturing method for encapsulation and stabilization of the developed Centellasome-10EX in the liposome vesicle. It is expected that the results obtained through clinical research on the wrinkle improvement effect of the cream using this can be widely used to study skin science in the cosmetic industry and to develop high-quality cosmetics with high efficacy.