• Journal of Pharmaceutical Investigation
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• v.30 no.3
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• pp.191-199
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• 2000

Genomics is providing targets faster than we can validate them and combinatorial chemistry is providing new chemical entities faster than we can screen them. Historically, the drug discovery cascade has been established as a sequential process initiated with a potency screening against a selected biological target. In this sequential process, pharmacokinetics was often regarded as a low-throughput activity. Typically, limited pharmacokinetics studies would be conducted prior to acceptance of a compound for safety evaluation and, as a result, compounds often failed to reach a clinical testing due to unfavorable pharmacokinetic characteristics. A new paradigm in drug discovery has emerged in which the entire sample collection is rapidly screened using robotized high-throughput assays at the outset of the program. Higher-throughput pharmacokinetics (HTPK) is being achieved through introduction of new techniques, including automation for sample preparation and new experimental approaches. A number of in vitro and in vivo methods are being developed for the HTPK. In vitro studies, in which many cell lines are used to screen absorption and metabolism, are generally faster than in vivo screening, and, in this sense, in vitro screening is often considered as a real HTPK. Despite the elegance of the in vitro models, however, in vivo screenings are always essential for the final confirmation. Among these in vivo methods, cassette dosing technique, is believed the methods that is applicable in the screening of pharmacokinetics of many compounds at a time. The widespread use of liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) allowed the feasibility of the cassette dosing technique. Another approach to increase the throughput of in vivo screening of pharmacokinetics is to reduce the number of sample analysis. Two common approaches are used for this purpose. First, samples from identical study designs but that contain different drug candidate can be pooled to produce single set of samples, thus, reducing sample to be analyzed. Second, for a single test compound, serial plasma samples can be pooled to produce a single composite sample for analysis. In this review, we validated the issue whether the second method can be applied to practical screening of in vivo pharmacokinetics using data from seven of our previous bioequivalence studies. For a given drug, equally spaced serial plasma samples were pooled to achieve a 'Pooled Concentration' for the drug. An area under the plasma drug concentration-time curve (AUC) was then calculated theoretically using the pooled concentration and the predicted AUC value was statistically compared with the traditionally calculated AUC value. The comparison revealed that the sample pooling method generated reasonably accurate AUC values when compared with those obtained by the traditional approach. It is especially noteworthy that the accuracy was obtained by the analysis of only one sample instead of analyses of a number of samples that necessitates a significant man-power and time. Thus, we propose the sample pooling method as an alternative to in vivo pharmacokinetic approach in the selection potential lead(s) from combinatorial libraries.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.5
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• pp.175-182
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• 2007

Members of prostaglandin(PG) E-series elicit cellular effects mainly through adenylyl cyclase-cAMP signaling. The role of $PGE_2$-induced increase in cAMP has been shown to be compartmentalized in the cardiac myocytes: $PGE_2$-induced increase of cAMP is not involved in the control of cardiomyocytic contraction. The purpose of the present study was to define the effect of $PGE_1$ on the cGMP levels and the role of $PGE_1$ in the atrial secretory function. Experiments were performed in perfused beating rabbit atria and atrial contractile responses, cGMP and cAMP efflux, and atrial natriuretic peptide(ANP) secretion were measured. $PGE_1$ increased cGMP as well as cAMP efflux concentration in a concentration-dependent manner, however, no significant changes in atrial secretory responses were observed(with $1.0{\mu}M\;PGE_1$; for cGMP, $144.76{\pm}37.5%$, n=11 versus $-16.81{\pm}4.76%$, n=6, control, p<0.01; for cAMP, $187.60{\pm}41.52%$, n=11 versus $7.38{\pm}19.44%$, n=6, control, p<0.01). $PGE_1$ decreased atrial dynamics slightly but transiently, whereas $PGE_2$ showed similar effects but with lower potency. Isoproterenol increased atrial cAMP efflux(with 2.0 nM; $145.71{\pm}41.89$, n=5 versus $7.38{\pm}19.44%$, n=6, control, p<0.05) and mechanical dynamics and decreased ANP secretion. The $PGE_1$-induced increase in cGMP efflux showed a bell-shaped concentration-response curve. $PGE_1$-induced increase of cGMP efflux was not observed in the presence of L-NAME, an inhibitor of nitric oxide(NO) synthase, or ODQ, an inhibitor of NO-sensitive guanylyl cyclase. L-NAME and ODQ showed no significant effect on the $PGE_1$-induced transient decrease of atrial dynamics. These data indicate that $PGE_1$ increases cGMP levels via NO-soluble GC signaling in the cardiac atrium and also show that $PGE_1$-induced increases in cGMP and cAMP levels are not involved in the regulation of atrial secretory and contractile functions.

• Journal of Korean Society of Environmental Engineers
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• v.28 no.7
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• pp.697-703
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• 2006

Endocrine disruptors were measured with GC/MS in effluents discharged from sewage treatment processes in pilot scale for the purpose of water reuse. From that analysis, we compared the removal rate of them by treatment processes. Nonylphenol was mainly detected in effluents and high concentration from 0.36 to 0.94 ${\mu}g/L$. $17{\beta}$-estradiol(E2) and $17{\alpha}$-ethynylestradiol(EE2) were detected as below the limit of detection in effluent. Endocrine disruptors were removed effectively in the range from 50 to 100% by treatment process. EC50 value($9.0{\times}10^{-3}$ M) of $17{\beta}$-estradiol(E2) by dose response curve of E-screen assay has higher than that of bisphenol A($2.736{\times}10^{-5}M$) and p-octylphenol($9.760{\times}10^{-6}$ M). These results showed that alkylphenols have lower relative estrogen potency than other estrogens such as $17{\beta}$-estradiol(E2). Calculated estrogenic activity(ng-EEQ/L) was 2 times higher than measured total estrogenic activity which estimated by E-screen assay. Moreover estrogenic activity of effluent by treatment process showed very low as below 1 ng-EEQ/L.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.499-510
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• 2013

Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine ${\gamma}$-lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at $37-40^{\circ}C$, with a $T_m$ value of $70.1^{\circ}C$. The enzyme showed clear catalytic and thermal stability below $40^{\circ}C$, with $T_{1/2}$ values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. Additionally, the enzyme $K_r$ values were 0.002, 0.054, 0.097, 0.184, and 0.341 $S^{-1}$ at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuria-related diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine ($K_m$ 2.46 mM, $K_{cat}\;1.39{\times}10^{-3}\;s^{-1}$), methionine ($K_m$ 4.1 mM, $K_{cat}\;0.97{\times}10^{-3}\;s^{-1}$), and cysteine ($K_m$ 4.9 m M, $K_{cat}\;0.77{\times}10^{-3}\;s^{-1}$). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5'-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.

• Environmental Analysis Health and Toxicology
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• v.12 no.3_4
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• pp.31-41
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• 1997

Public concerns about hazardous health effect from the exposure to organic by-products of the chlorination have been increased. There are numerous studies reporting that chlorination of drinking water produces numerous chlorinated organic by-products including THMs, HAAs, HANs. Some of these products are known to be animal carcinogens. The purpose of this study was to estimate health risk of DBPs by chlorinated drinking water ingestion in Seoul based on methodologies that have been developed for conducting risk assessment of complex-chemical-mixture. The drinking water sample was collected seperately at six water treatment plant in Seoul at March, April, 1996. In tap water of households in Seoul, DBPs were measured wilfh the mean value of 36.6 $\mu$g/L. Risk assessment processes,. which include processes for the estimation of human cancer potency using animal bioassay data and calculation of human exposure, entail uncertainties. In the exposure assessment process, exposure scenarios with various assumptions could affect the exposure amount and excess cancer risk. The reference dose of haloacetonitriles was estimated to be 0.0023 mg/kg/day by applying dibromoacetonitrile NOAEL and uncertainty factor to the mean concentration. In the first case, human excess cancer risk was estimated by the US EPA method used to set the MCL (maximum contaminant level). In the second and third case, the risk was estimated for multi-route exposure with and without adopting Monte-Carlo simulation, respectively. In the second case, exposure input parameters and cancer potencies used probability distributions, and in the third case, those values used point estimates (mean, and maximum or 95% upper-bound value). As a result, while the excess cancer risk estimated by US EPA method considering only direct ingestion tended to be underestimated, the risk which was estimated by considering multi-route exposure without Monte-Carlo simulation and then using the maximum or 95% upper-bound value as input parameters tended to be overestimated. In risk assessment for Trihalomethanes, considering multi-route exposure with adopting Monte-Carlo analysis seems to provide the most reasonable estimations.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.2
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• pp.179-184
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• 2002

Induction of cytochrome P45O (CYP) and 7-etholqresorufin-O-deethylase (EROD) in the microsome exposed to 3-methylcholan-throne (MC), $\beta$-naphthoflavone (BNF) and phenobarbital-Na (PB) was investigated, Microsome was isolated from digestive gland of clam (Coelomactra antiquata) and then exposed to each chemical in concentration range of 0.1 to 1.0 mM for 7 hours. The CYP content and EROD activity in the microsome exposed to each chemical significantly increased compared to the control group. The overall CYP and EROD induction potency was in order of MC>BNF>PB. The induction response of EROD was two times higher than that of CYP level in the microsome exposed to MC, but the induction response of EROD was slightly higher than that of CYP level in BNF and PB exposure groups.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.2
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• pp.191-195
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• 2002

In previous reports the authors have screened the inhibition effects of marine algae extracts on halitosis, and demonstrated that a brown algae, Eisenia bicyclis (' Daehwang') possess not only strong deodorant effect bug also considerable anticariogenic activities. In this study, we screened antibacterial effects of various marine algae, and measured minimum inhibitory concentration (MIC) value of them against mutans streptococci in vitro. Among the 27 species of marine algae, $80\%$ ethanol extract of dried sea-mustard, Laminaria japonica ('Dasima') showed the strongest inhibition activity against Streptococcus mutans KCTC 3300. The extracts of Ulva lactuca ('Galparae'), Codium fragile ('Cheonggak'), Ecklonia cava ('Gamtae'), E. stolonifera ('Gompi') and Undalia Pinnatifida ('Miyeok') showed slightly weaker inhibitory potency than L. japonica. Differences of MIC values in $80\%$ ethanol extract of some species of marine algae were observed depending on test bacterial species, i.e., S. mutans KCTC 3300 or S. sobrinus KCTC 3307. Eighty percent ethanol extract of dried L japonica was fractionated with diethyl ether, chloroform, ethyl acetate, n-buthanol and water successively, The ether-soluble fraction had inhibitory effect on S. mutans KCTC 3300, however the inhibitory effects were not found in the other fractions. The MIC values of $80\%$ ethanol extract and ether fraction were 180 and 105 $\mu$g/mL respectively, while no significant inhibition activity of water-soluble fraction was found even when the fraction was added up to 5,500 $\mu$g/mL.

• Korean Journal of Food Science and Technology
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• v.37 no.1
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• pp.78-83
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• 2005

Recently, much attention has been focused on plant antioxidants, because they are expected to protect against oxidative damage, possibly preserving biological functions of cells. Antioxidant compounds were isolated from Angelica keiskei through extraction with 80% EtOH, and fractionations were carried out sequentially with n-hexane, chloroform, ethyl acetate, n-butanol, and water. Two active compounds were isolated from ethyl acetate fraction by silica gel column chromatography, and were identified as isoquercitrin ($quercetin-3-O-{\beta}-D-glucose$) and hyperoside ($quercetin-3-O-{\beta}-D-glucose$). Isoquercitrin and hyperoside showed strong antioxidative potency, as revealed by evaluation of their ABTS, DPPH, OH, and $H_{2}O_{2}$ radical-scavenging activities, and ex vivo DNA damage-protecting effects.

• Journal of Ginseng Research
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• v.39 no.2
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• pp.169-177
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• 2015

Background: Ginsenoside Rd (GSRd), one of the most abundant ingredients of Panax ginseng, protects the heart via multiple mechanisms including the inhibition of $Ca^{2+}$ influx.We intended to explore the effects of GSRd on L-type $Ca^{2+}$ current ($I_{Ca,L}$) and define the mechanism of the suppression of $I_{Ca,L}$ by GSRd. Methods: Perforated-patch recording and whole-cell voltage clamp techniques were applied in isolated rat ventricular myocytes. Results: (1) GSRd reduced $I_{Ca,L}$ peak amplitude in a concentration-dependent manner [half-maximal inhibitory concentration $(IC_{50})=32.4{\pm}7.1{\mu}mol/L$] and up-shifted the current-voltage (I-V) curve. (2) GSRd ($30{\mu}mol/L$) significantly changed the steady-state activation curve of $I_{Ca,L}$ ($V_{0.5}:-19.12{\pm}0.68$ vs. $-6.26{\pm}0.38mV$; n = 5, p < 0.05) and slowed down the recovery of $I_{Ca,L}$ from inactivation [the time content (${\zeta}$) from 91 ms to 136 ms, n = 5, p < 0.01]. (3) A more significant inhibitive effect of GSRd ($100{\mu}mol/L$) was identified in perforated-patch recording when compared with whole-cell recording [$65.7{\pm}3.2%$ (n = 10) vs. $31.4{\pm}5.2%$ (n = 5), p < 0.01]. (4) Pertussis toxin ($G_i$ protein inhibitor) completely abolished the $I_{Ca,L}$ inhibition induced by GSRd. There was a significant difference in inhibition potency between the two cyclic adenosine monophosphate elevating agents (isoprenaline and forskolin) prestimulation [$55{\pm}7.8%$ (n = 5) vs. $17.2{\pm}3.5%$ (n = 5), p < 0.01]. (5) 1H-[1,2,4]Oxadiazolo[4,3-a]-quinoxalin-1-one (a guanylate cyclase inhibitor) and N-acetyl-$\small{L}$-cysteine (a nitric oxide scavenger) partly recovered the $I_{Ca,L}$ inhibition induced by GSRd. (6) Phorbol-12-myristate-13-acetate (a protein kinase C activator) and GF109203X (a protein kinase C inhibitor) did not contribute to the inhibition of GSRd. Conclusion: These findings suggest that GSRd could inhibit $I_{Ca,L}$ through pertussis toxin-sensitive G protein ($G_i$) and a nitric oxide-cyclic guanosine monophosphate-dependent mechanism.

• Archives of Pharmacal Research
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• v.23 no.4
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• pp.353-359
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• 2000

${\alpha}_2$-Adrenoceptor antagonists, which can enhance synaptic norepinephrine levels by blocking feedback inhibition processes, are potentially useful in the treatment of disease states such. as depression, memory impairment, impotence and sexual dysfunction. (10bS)-1,2,3,5,6,10b-Hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) was evaluated in several in vitro biological tests to establish its pharmacological profile of activities as an ${\alpha}_2$-adrenoceptor antagonist. Saturation binding assay revealed that$^{3}[H]$rauwolscine bound to the $\alpha$$_2$-adrenoceptors with a Kd value of 6.3$\pm$0.5 nM and a Bmax value of 25l$\pm$39 fmol/mg protein in rat cortical synaptic membranes. Competitive binding assay showed that YSL-3S inhibited the binding of$^3[H]$rauwolscine (1 nM) in a concentration-dependent manner with a Ki value of 98.2$\pm$12.1 nM while it did not inhibit the binding of [$^3$H]cytisine (1.25 nM) to neuronal nicotinic cholinergic receptors. The Ki values of yohimbine, clonidine and norepinephrine for $^3[H]$rauwolscine binding were 15.8$\pm$1.0, 40.1$\pm$5.9 and 40.0$\pm$11.5 nM, respectively. In addition, the binding affinity of YSL-3S for ${\alpha}_2$-adrenoceptors was higher than that of its antipode and the racemic mixture. The functional activity of YSL-3S at the presynaptic ${\alpha}_2$-adrenoceptors was assessed using the prostatic portion of the rat vas deferens. Clonidine inhibited field-stimulated contractions of the vas deference in a dose-dependent manner. The presence of YSL-3S or yohimbine caused a parallel, rightward the dose-response curve of clonidine in a dose-dependent manner, indicating an antagonistic action at the presynaptic ${\alpha}_2$-adrenoceptors. The $pA_2$values of yohimbine and YSL-3S were 7.66$\pm$0.13 and 6.64$\pm$0.18, respectively. The results indicate that YSL-3S acts as a competitive antagonist at presynaptic ${\alpha}_2$ -adrenoceptors with a potency approximately ten times lower than yohimbine, but is devoid of binding affinity for neuronal nicotinic cholinergic receptors.