Cardiovascular Safety Pharmacology of Sibutramine

Sibutramine is an anorectic that has been banned since 2010 due to cardiovascular safety issues. However, counterfeit drugs or slimming products that include sibutramine are still available in the market. It has been reported that illegal sibutramine-contained pharmaceutical products induce cardiovascular crisis. However, the mechanism underlying sibutramine-induced cardiovascular adverse effect has not been fully evaluated yet. In this study, we performed cardiovascular safety pharmacology studies of sibutramine systemically using by hERG channel inhibition, action potential duration, and telemetry assays. Sibutramine inhibited hERG channel current of HEK293 cells with an IC50 of 3.92 muM in patch clamp assay and increased the heart rate and blood pressure (76 Deltabpm in heart rate and 51 DeltammHg in blood pressure) in beagle dogs at a dose of 30 mg/kg (per oral), while it shortened action potential duration (at 10 muM and 30 muM, resulted in 15% and 29% decreases in APD50, and 9% and 17% decreases in APD90, respectively) in the Purkinje fibers of rabbits and had no effects on the QTc interval in beagle dogs. These results suggest that sibutramine has a considerable adverse effect on the cardiovascular system and may contribute to accurate drug safety regulation.

Measurement of Human Cytochrome P450 Enzyme Induction Based on Mesalazine and Mosapride Citrate Treatments Using a Luminescent Assay

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

Dependence Potential of Tramadol: Behavioral Pharmacology in Rodents

Tramadol is an opioid analgesic agent that has been the subject of a series of case reports suggesting potential for misuse or abuse. However, it is not a controlled substance and is not generally considered addictive in Korea. In this study, we examined the dependence potential and abuse liability of tramadol as well as its effect on the dopaminergic and serotonergic systems in rodents. In animal behavioral tests, tramadol did not show any positive effects on the experimental animals in climbing, jumping, and head twitch tests. However, in the conditioned place preference and self-administration tests, the experimental animals showed significant positive responses. Taken together, tramadol affected the neurological systems related to abuse liability and has the potential to lead psychological dependence.

Dependence Potential of the Synthetic Cannabinoids JWH-073, JWH-081, and JWH-210: In Vivo and In Vitro Approaches

Synthetic cannabinoids (CBs) such as the JWH series have caused social problems concerning their abuse liability. Because the JWH series produces euphoric and hallucinogenic effects, they have been distributed illegally under street names such as "Spice" and "Smoke". Many countries including Korea have started to schedule some of the JWH series compounds as controlled substances, but there are a number of JWH series chemicals that remain uncontrolled by law. In this study, three synthetic CBs with different binding affinities to the CB1 receptor (JWH-073, 081, and 210) and Delta9-tetrahydrocannabinol (Delta9-THC) were evaluated for their potential for psychological dependence. The conditioned place preference test (unbiased method) and self-administration test (fixed ratio of 1) using rodents were conducted. Ki values of the three synthetic cannabinoids were calculated as supplementary data using a receptor binding assay and overexpressed CB1 protein membranes to compare dependence potential with CB1 receptor binding affinity. All mice administered JWH-073, 081, or 210 showed significantly increased time spent at unpreferred space in a dose-dependence manner in the conditioned place preference test. In contrast, all tested substances except Delta9-THC showed aversion phenomenon at high doses in the conditioned place preference test. The order of affinity to the CB1 receptor in the receptor binding assay was JWH-210 > JWH-081 >> JWH-073, which was in agreement with the results from the conditioned place preference test. However, no change in self-administration was observed. These findings suggest the possibility to predict dependence potential of synthetic CBs through a receptor binding assay at the screening level.

Prevalence of Laboratory Animal Allergy in Laboratory Workers / 한국실험동물학회지

This study was designed to analyze the prevalence rates of laboratory animal allergy (LAA) in laboratory workers who perform researches with animals, and detect the mouse urinary allergen (Mus m 1) level in animal facilities for the purpose of establishing program for prevention of exposure to allergen. Study subjects were 240 employees who were working for two animal research institutions in Korea. Then the questionnaire and skin prick tests (SPTs) using twenty allergens were conducted with them. Presence of Mus m 1 in each air borne sample collected from animal facility was determined by using enzyme-linked immunosorbent assay. Through 240 questionnaire sheets, we found that; (1) 17.0% of workers in the direct exposure group answered that they had allergic symptoms due to laboratory animals; and (2) 6.2% of them had asthmatic symptoms. Twenty one subjects (27.6%) among the subjects with common allergens positive result and five subjects (6.6%) among the subjects with negative result showed a positive response to LAA under the SPTs. The Mus m 1 concentration (1.339 ng/m3) in the sample collected during cage exchange in mouse breeding room was up to 2.8 times higher than its concentration (0.483 ng/m3) in the sample collected at the stationary state. We suggest that LAA management programs including control of exposure to laboratory animal allergens should be considered as a measure to reduce the incidence of LAA and relieve the laboratory worker's allergic sensitivity to laboratory animals.

Evaluation of gyrB as Chromosomal Marker in Bacillus anthracis

Bacillus anthracis is generally accepted as the most potent biological warfare agent because of its highly pathogenic nature and transmission efficiency. Identification of chromosomal markers for the rapid detection of B. anthracis is difficult since significant chromosomal homology exists among B. anthracis, B. cereus and B. thuringiensis. In this study, we tested whether the gyrB sequence could be used as the target for the PCR detection of B. anthracis. The gyrB sequence, composed of 1,923 bp, was identical in 17 Korean B. anthracis isolates. The comparison of gyrB sequence between B. anthracis and B. cereus type strain showed 8.8% difference (105 bp among 1,194 bp), and the gyrB sequence similarities of B. cereus, B. thuringiensis and B. mycoides with B. anthracis were 92.3%, 86.9% and 86.1%, respectively. When polymerase chain reaction was designed and performed based on the gyrB sequence, a specific amplicon (351 bp) could be amplified. These results indicate that gyrB could be useful as a chromosomal marker for the rapid screening of B. anthracis by PCR or differentiation of B. anthracis from other related species by multiplex PCR with other plasmid markers.

The First Outbreak of Botulism in Korea / 대한임상미생물학회지

Botulism is a rare neuroparalytic disease caused by neurotoxins of Clostridium species. A ten-year-old girl and her mother were admitted to a hospital with symptoms of progressive dizziness, blurred vision, slurred speech, constipation and difficulty in swallowing. These characteristic manifestations and clinical course prompted an examination of the possibility of botulism. Mouse bioassay performed with mother's stool demonstrated type A botulinum toxin and culture of the mother's stool was positive for Clostridium botulinum type A. This is the first case of botulism in Korea.

Genetic Diversity among Virulent Mega Plasmids pXO1 and pXO2 of Bacillus anthracis Isolated in Korea

We compared genetic variations in virulence mega plasmids pXO1 and pXO2 of twenty-seven Bacillus anthracis strains from Korean patients and environmental samples together with those of Bacillus anthracis Sterne, Pasteur and A2012 standard strains. Genetic variations were analyzed in twenty-three variable regions (ten and thirteen variablenumber tandem repeats and insertion/deletions in pXO1 and pXO2, respectively). The pXO1 plasmids were classified into 7 groups and pXO2 plasmids to 12 groups. Discrete phylogenic lineages could be differentiated between environmental and clinical strains by UPGMA (unweighted pair group method with average) method. In addition, clinical strains showed more variations than environmental isolates. The pXO2 plasmid appeared genetically more unstable than pXO1. A general plasmid genotype could be suggested for Korean soil isolates since they mostly clustered into a representative group.

Molecular Analysis of Clostridium difficile Isolates by Arbitrarily Primed-Polymerase Chain Reaction and Polymerase Chain Reaction-Ribotyping / 감염

BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.

Antimicrobial Resistance of Neisseria gonorrhoeae Isolated in Korea / 감염

BACKGROUND: The aim of this study was to monitor trends in antimicrobial susceptibilities of Neisseria gonorrhoeae isolates, in particular, to examine the possibility of increasing prevalence of fluoroquinolone resistance in Korea and the relationship between patterns of mutations involving gyrA and parC genes and the ciprofloxacin resistance level. METHODS: The antimicrobial susceptibilities of 489 gonoccocal isolates which were nationwide collected from patients visiting Health Centers were determined by NCCLS disk diffusion and agar plate dilution methods. PCR and direct DNA sequencing of the amplicons were performed to identify mutations within the quinolone resistance-determining regions of gyrA and parC genes. RESULTS: The proportion of resistance to penicillin, tetracycline, or both remained as high as 94%. The isolates with ciprofloxacin resistance remarkably increased from 1% in 1999 to 5% in 2000 and also resistance to cefoxitine and ceftriaxone were shown to be increased. The strains resistant to spectinomycin was little reported. Four isolates with 16 microgram/mL of MIC for ciprofloxacin all showed the same alternations of Ser-91 to Phe, Asp-95 to Gly in GyrA and Ser-87 to Arg in ParC, but ciprofloxacin-susceptible strains have little amino acid substitution. CONCLUSION: Considering the increasing prevalence of isolates with resistance to ciprofloxacin and ofloxacin, it is likely that the antibiotics such as spectinomycin, or ceftriaxone are recommended as the first-line treatment for gonoccocal infections in Korea. The results from this study suggest that mutation analysis for quinolone resistance-determining regions of gyrA and parC genes are important in epidemiological studies for the spread of ciprofloxacin resistant strains.

The Detection of Enterotoxin Gene from Bacteroides fragilis Isolates in Korea by Polymerase Chain Reaction

Bacteroides fragilis is a Gram negative nonsporulating anaerobic rod bacterium that makes up about 1 to 2% of the norrnal human colonic microflora. In 1984, Myer et al. reported that some strains of B. fragilis produce enterotoxin and cause diarrheal disease in cattle and human. Since then it has been termed enterotoxigenic B. fragilis (ETBF). In this study, we tried to detect enterotoxin gene from 37 B. fragilis strains, isolated in Korean patients, to confirm the existence of ETBF and usefulness of PCR as a rapid diagnosis method. By this method, we identified 9 ETBF strains and confirmed their pathogenesis by cytotoxicity test. No significant cross- reactivity with other anaerobes or aerobes was observed. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobic infections. And the entire amplified PCR mixture was ligated into a pT7Blue T-vector and transformed into E. coli. When the nucleotide sequences of cloned PCR products were compared with reported enterotoxin gene, pBF529 inserted DNA sequence was nearly in good agreement with it but pBF570 inserted DNA sequence showed some difference at nucleotide 270-300. A search for nucleotide sequence homologies revealed that pBF529 exhibited 99%, but pBF570 indicated only 90% identity with reported enterotoxin gene. According to these results, it was suggested that ETBF toxin can be differentiated into at least 2 subtypes.

Identification of new serovar yeonchon and hongchon belonging to leptospira interrogans icterohaemorrhagiae serogroup

No abstract available.