The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans.

V-ATPase, which comprises 13-14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.

Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A.

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Design, synthesis, and study of novel phenethyl-based antitumor phospholipids downregulating p38 mitogen-activated protein kinase.

Phenethyl-based edelfosine-analogs with saturated, monounsaturated, or polyunsaturated alkoxy substituents on phenyl ring were designed as novel antitumor lipids modulating p38 MAPK. Evaluation of the synthesised compounds against nine panels of diverse cancer cells presented saturated and monounsaturated alkoxy-substituted derivatives as the most active than other derivatives. In addition, ortho-substituted compounds were more active than meta- or ortho-substituted compounds. They were potential anticancer agents against blood, lung, colon, CNS, ovary, renal, and prostate cancers but not against skin nor breast cancers. Compounds, 1b and 1a emerged as the most potential anticancer agents. Assessment of compound 1b impact on p38 MAPK and AKT confirmed it as an inhibitor of p38 MAPK but not AKT. In silico study suggested compounds 1b and 1a as possible binders to the lipid binding pocket of p38 MAPK. Overall, compounds 1b and 1a as novel broad spectrum antitumor lipids modulating activity of p38 MAPK for further development.

Development of a Rapid LC-MS/MS Method for Simultaneous Quantification of Donepezil and Tadalafil in Rat Plasma: Its Application in a Pharmacokinetic Interaction Study after Oral Administration in Rats.

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats.

In vitro and in vivo neuroprotective effect of novel mPGES-1 inhibitor in animal model of Parkinson's disease.

mPGES-1 is found to be up-regulated in the dopaminergic neurons of the substantia nigra pars compacta (SNpc) of postmortem brain tissue from Parkinson's disease (PD) patients and neurotoxin 6-hydroxydopamine (6-OHDA)-induced PD mice. Since the genetic deletion of mPGES-1 abolished 6-OHDA-induced PGE2 production and 6-OHDA-induced dopaminergic neurodegeneration in vitro and in vivo models, mPGES-1 enzyme has the potential to be an important target for PD therapy. In the present work, we investigated whether a small organic molecule as mPGES-1 inhibitor could exhibit the neuroprotective effects against 6-OHDA-induced neurotoxicity in in vitro and in vivo models. For this research goal, a new series of arylsulfonyl hydrazide derivatives was prepared and investigated whether these compounds may protect neurons against 6-OHDA-induced neurotoxicity in both in vitro and in vivo studies. Among them, compound 7s (MPO-0144) as a mPGES-1 inhibitor (PGE2 IC50 = 41.77 nM; mPGES-1 IC50 = 1.16 nM) exhibited a potent neuroprotection (ED50 = 3.0 nM) against 6-OHDA-induced in PC12 cells without its own neurotoxicity (IC50 = >10 µM). In a 6-OHDA-induced mouse model of PD, administration of compound 7s (1 mg/kg/day, for 7 days, i.p.) ameliorated motor impairments and dopaminergic neuronal damage. These significant biological effects of compound 7s provided the first pharmacological evidence that mPGES-1 inhibitor could be a promising therapeutic agent for PD patients.

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails.

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC50 of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.

Dieckol Isolated from Eisenia bicyclis Ameliorates Wrinkling and Improves Skin Hydration via MAPK/AP-1 and TGF-ß/Smad Signaling Pathways in UVB-Irradiated Hairless Mice.

Repetitive exposure to ultraviolet B (UVB) is one of the main causes of skin photoaging. We previously reported that dieckol isolated from Eisenia bicyclis extract has potential anti-photoaging effects in UVB-irradiated Hs68 cells. Here, we aimed to evaluate the anti-photoaging activity of dieckol in a UVB-irradiated hairless mouse model. In this study, hairless mice were exposed to UVB for eight weeks. At the same time, dieckol at two doses (5 or 10 mg/kg) was administered orally three times a week. We found that dieckol suppressed UVB-induced collagen degradation and matrix metalloproteinases (MMPs)-1, -3, and -9 expression by regulating transforming growth factor beta (TGF-ß)/Smad2/3 and mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) signaling. In addition, dieckol rescued the production of hyaluronic acid (HA) and effectively restored the mRNA expression of hyaluronan synthase (HAS)-1/-2 and hyaluronidase (HYAL)-1/-2 in UVB-irradiated hairless mice. We observed a significant reduction in transepidermal water loss (TEWL), epidermal/dermal thickness, and wrinkle formation in hairless mice administered dieckol. Based on these results, we suggest that dieckol, due to its anti-photoaging role, may be used as a nutricosmetic ingredient for improving skin health.

Discovery of N-amido-phenylsulfonamide derivatives as novel microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors.

Our previous research showed that N-carboxy-phenylsulfonyl hydrazide (scaffold A) could reduce LPS-stimulated PGE2 levels in RAW 264.7 macrophage cells by an inhibition of mPGES-1 enzyme. However, a number of scaffold A derivatives showed the drawbacks such as the formation of regioisomers and poor liver metabolic stability. In order to overcome these synthetic and metabolic problems, therefore, we decided to replace N-carboxy-phenylsulfonyl hydrazide (scaffold A) with N-carboxy-phenylsulfonamide (scaffold B) or N-amido-phenylsulfonamide frameworks (scaffold C) as a bioisosteric replacement. Among them, MPO-0186 (scaffold C) inhibited the production of PGE2 (IC50: 0.24 µM) in A549 cells via inhibition of mPGES-1 (IC50: 0.49 µM in a cell-free assay) and was found to be approximately 9- and 8-fold more potent than MK-886 as a reference inhibitor, respectively. A molecular docking study theoretically suggests that MPO-0186 could inhibit PGE2 production by blocking the PGH2 binding site of mPGES-1 enzyme. Furthermore, MPO-0186 demonstrated good liver metabolic stability and no significant inhibition observed in clinically relevant CYP isoforms except CYP2C19. This result provides a potential starting point for the development of selective and potent mPGES-1 inhibitor with a novel scaffold.

Design, synthesis and anti-inflammatory activity of imidazol-5-yl pyridine derivatives as p38α/MAPK14 inhibitor.

P38α/MAPK14 is intracellular signalling regulator involved in biosynthesis of inflammatory mediator cytokines (TNF-α, IL-1, IL-6, and IL-1b), which induce the production of inflammatory proteins (iNOS, NF-kB, and COX-2). In this study, drug repurposing strategies were followed to repositioning of a series of B-RAF V600E imidazol-5-yl pyridine inhibitors to inhibit P38α kinase. A group 25 reported P38α kinase inhibitors were used to build a pharmacophore model for mapping the target compounds and proving their affinity for binding in P38α active site. Target compounds were evaluated for their potency against P38α kinase, compounds 11a and 11d were the most potent inhibitors (IC50 = 47 nM and 45 nM, respectively). In addition, compound 11d effectively inhibited the production of proinflammatory cytokinesTNF-α, 1L-6, and 1L-1ß in LPS-induced RAW 264.7 macrophages with IC50 values of 78.03 nM, 17.6 µM and 82.15 nM, respectively. The target compounds were tested for their anti-inflammatory activity by detecting the reduction of Nitric oxide (NO) and prostaglandin (PGE2) production in LPS-stimulated RAW 264.7 macrophages. Compound 11d exhibited satisfied inhibitory activity of the production of PGE2 and NO with IC50 values of 0.29 µM and 0.61 µM, respectively. Molecular dynamics simulations of the most potent inhibitor 11d were carried out to illustrate its conformational stability in the binding site of P38α kinase.

Eisenia bicyclis Extract Repairs UVB-Induced Skin Photoaging In Vitro and In Vivo: Photoprotective Effects.

Chronic exposure to ultraviolet B (UVB) is a major cause of skin aging. The aim of the present study was to determine the photoprotective effect of a 30% ethanol extract of Eisenia bicyclis (Kjellman) Setchell (EEB) against UVB-induced skin aging. By treating human dermal fibroblasts (Hs68) with EEB after UVB irradiation, we found that EEB had a cytoprotective effect. EEB treatment significantly decreased UVB-induced matrix metalloproteinase-1 (MMP-1) production by suppressing the activation of mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) signaling and enhancing the protein expression of tissue inhibitors of metalloproteinases (TIMPs). EEB was also found to recover the UVB-induced degradation of pro-collagen by upregulating Smad signaling. Moreover, EEB increased the mRNA expression of filaggrin, involucrin, and loricrin in UVB-irradiated human epidermal keratinocytes (HaCaT). EEB decreased UVB-induced reactive oxygen species (ROS) generation by upregulating glutathione peroxidase 1 (GPx1) and heme oxygenase-1 (HO-1) expression via nuclear factor erythroid-2-related factor 2 (Nrf2) activation in Hs68 cells. In a UVB-induced HR-1 hairless mouse model, the oral administration of EEB mitigated photoaging lesions including wrinkle formation, skin thickness, and skin dryness by downregulating MMP-1 production and upregulating the expression of pro-collagen type I alpha 1 chain (pro-COL1A1). Collectively, our findings revealed that EEB prevents UVB-induced skin damage by regulating MMP-1 and pro-collagen type I production through MAPK/AP-1 and Smad pathways.

Synthesis of Halogenated 1,5-Diarylimidazoles and Their Inhibitory Effects on LPS-Induced PGE2 Production in RAW 264.7 Cells.

A series of halogenated 1,5-diarylimidazole compounds were synthesized and their inhibitory effects on LPS-induced PGE2 production in RAW 264.7 cells were evaluated. A wide variety of 2,4-, 4-, and 2-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles were synthesized for SAR study via two different pathways. Overall, 4-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles, regardless of the species of halogen, exhibited very strong inhibitory activities of PGE2 production. Among them, 4-chloro-5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazole (3, IC50 3.3 nM ± 2.93), and 4-chloro-5-(4-chlorophenyl)-1-(4-methylsulfonylphenyl)imidazole (13, IC50 5.3 nM ± 0.23) showed the best results.

Evaluation of the Inhibitory Effects of Pyridylpyrazole Derivatives on LPS-Induced PGE2 Productions and Nitric Oxide in Murine RAW 264.7 Macrophages.

A series of thirteen triarylpyrazole analogs were investigated as inhibitors of lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 macrophages. The target compounds 1a-m have first been assessed for cytotoxicity against RAW 264.7 macrophages to determine their non-cytotoxic concentration(s) for anti-inflammatory testing to make sure that the inhibition of PGE2 and NO production would not be caused by cytotoxicity. It was found that compounds 1f and 1m were the most potent PGE2 inhibitors with IC50 values of 7.1 and 1.1 µM, respectively. In addition, these compounds also showed inhibitory effects of 11.6% and 37.19% on LPS-induced NO production, respectively. The western blots analysis of COX-2 and iNOS showed that the PGE2 and NO inhibitory effect of compound 1m are attributed to inhibition of COX-2 and iNOS protein expression through inactivation of p38.

A Pharmacokinetic Study of Ephedrine and Pseudoephedrine after Oral Administration of Ojeok-San by Validated LC-MS/MS Method in Human Plasma.

A sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was developed and fully validated for the simultaneous determination of ephedrine and pseudoephedrine in human plasma after oral administration of the herbal prescription Ojeok-san (OJS); 2-phenylethylamine was used as the internal standard (IS). Both compounds presented a linear calibration curve (r2 ≥ 0.99) over a concentration range of 0.2-50 ng/mL. The developed method was fully validated in terms of selectivity, lower limit of quantitation, precision, accuracy, recovery, matrix effect, and stability, according to the regulatory guidelines from the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety. This validated method was successfully applied for the pharmacokinetic assessment of ephedrine and pseudoephedrine in 20 healthy Korean volunteers administered OJS.

Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide and PGE2 productions in murine RAW 264.7 macrophages.

In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as inhibitors of nitric oxide (NO) and prostaglandin E2 (PGE2) release induced by lipopolysaccharide (LPS) in murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE2 production were not caused by non-specific cytotoxicity. Compounds 1h and 2f were the most active PGE2 inhibitors with IC50 values of 2.94 µM and 4.21 µM, respectively. Western blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein expression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-induced murine RAW 264.7 macrophages.

Synthesis and cytotoxic effects of 2-thio-3,4-dihydroquinazoline derivatives as novel T-type calcium channel blockers.

In our previous work, a series of 2-amino-3,4-dihydroquinazoline derivativesusing an electron acceptor group was reported to be potent T-type calcium channel blockers and exhibit strong cytotoxic effects against various cancerous cell lines. To investigate the role of the guanidine moiety in the 2-amino-3,4-dihydroquinazoline scaffold as a pharmacophore for dual biological activity, a new series of 2-thio-3,4-dihydroquniazoline derivatives using an electron donor group at the C2-position was synthesized and evaluated for T-type calcium channel blocking activity and cytotoxic effects against two human cancerous cell lines (lung cancer A549 and colon cancer HCT-116). Among them, compound 6g showed potent inhibition of Cav3.2 currents (83% inhibition) at 10 µM concentrations. The compound also exhibited IC50 values of 5.0 and 6.4 µM against A549 and HCT-116 cell lines, respectively, which are comparable to the parental lead compound KYS05090. These results indicate that the isothiourea moiety similar to the guanidine moiety of 2-amino-3,4-dihydroquinazoline derivatives may be an essential pharmacophore for the desired biological activities. Therefore, our preliminary work can provide the opportunity to expand a chemical repertoire to improve affinity and selectivity for T-type calcium channels.

Diarylurea derivatives comprising 2,4-diarylpyrimidines: Discovery of novel potential anticancer agents via combined failed-ligands repurposing and molecular hybridization approaches.

A series of diarylurea derivatives comprising 2,4-diarylpyrimidines were synthesized based on a combination of postulated molecular hybridization design and failed-ligands repurposing approaches, which enabled the discovery of novel potential antiproliferative agents. Towards credible biological evaluation, an in vitro anticancer activity assay was conducted employing a library of 60 cancer cell lines constituting nine panels representing blood, lung, colon, CNS, skin, ovary, renal, prostate, and breast cancers. The results revealed high effectiveness and broad-spectrum anticancer activity of compounds 4m and 4g. Five-dose assay of compounds 4m and 4g proved their high potency that surpassed that of four standard kinase inhibitors FDA-approved anticancer drugs against many cancer cells. Towards the identification of their molecular target, screening of kinase inhibitory profile employing a panel of 51 kinases involved in cancer revealed inhibition of several kinases from the platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) kinase family, which might mediate, at least in part, the antiproliferative activity. Molecular docking of 4g into the crystal structure of the Feline McDonough Sarcoma (FMS) kinase predicted that it binds to a pocket formed by the juxtamembrane domain, the catalytic loop, and the αE helix, thus stabilizing the inhibited conformation of the kinase. Flow cytometric study of the cytotoxic effects of compound 4g in A549 cells showed it induces dose- and time-dependent apoptotic events leading to cell death. Collectively, this work presents compound 4g as a potential broad-spectrum anticancer agent against multiple cancer types.

Quantitation of plasma and biliary cefpiramide concentrations in human samples using high-performance liquid chromatography.

Cefpiramide is frequently used to treat biliary infections. However, no bioanalytical method has been validated to quantitate cefpiramide in human samples, particularly in bile. Therefore, this study was conducted to develop a simple, selective and validated high-performance liquid chromatographic method to determine cefpiramide in human plasma and bile. A protein precipitation procedure was used to extract cefpiramide and cefoperazone (internal standard, IS) from 200 µl of plasma and bile. Utilizing a Capcell Pak C18 column (4.6 × 250 mm), cefpiramide and IS were separated using the timed-gradient mobile phase consisting of 0.1 m sodium acetate (pH 5.2) and acetonitrile at a flow rate of 1 ml/min with photodiode array detector (wavelength set at 273 nm). The calibration curves showed linearity at concentrations ranging from 1 to 150 µg/ml in both plasma and bile (r2 > 0.999). The within- and between-run coefficients of variation (CVs) for plasma samples were 0.570-4.43 and 1.10-2.76%, respectively; for bile samples, the within- and between-day precision (CV) was 0.814-6.34 and 2.05-4.00%, respectively. Our newly developed bioanalytical method was successfully employed to quantify cefpiramide concentrations in both plasma and bile at multiple time points in patients with acute cholangitis.

Isoflavones Isolated from the Seeds of Millettia ferruginea Induced Apoptotic Cell Death in Human Ovarian Cancer Cells.

The seeds of Millettia ferruginea are used in fishing, pesticides, and folk medicine in Ethiopia. Here, the anti-cancer effects of isoflavones isolated from M. ferruginea were evaluated in human ovarian cancer cells. We found that isoflavone ferrugone and 6,7-dimethoxy-3',4'-methylenedioxy-8-(3,3-dimethylallyl)isoflavone (DMI) had potent cytotoxic effects on human ovarian cancer cell A2780 and SKOV3. Ferrugone and DMI treatment increased the sub-G1 cell population in a dose-dependent manner in A2780 cells. The cytotoxic activity of ferrugone and DMI was associated with the induction of apoptosis, as shown by an increase in annexin V-positive cells. Z-VAD-fmk, a broad-spectrum caspase inhibitor, and z-DEVD-fmk, a caspase-3 inhibitor, significantly reversed both the ferrugone and DMI-induced apoptosis, suggesting that cell death stimulated by the isoflavones is mediated by caspase-3-dependent apoptosis. Additionally, ferrugone-induced apoptosis was found to be caspase-8-dependent, while DMI-induced apoptosis was caspase-9-dependent. Notably, DMI, but not ferrugone, increased the intracellular levels of reactive oxygen species (ROS), and antioxidant N-acetyl-L-cysteine (NAC) attenuated the pro-apoptotic activity of DMI. These data suggest that DMI induced apoptotic cell death through the intrinsic pathway via ROS production, while ferrugone stimulated the extrinsic pathway in human ovarian cancer cells.

Development and Validation of an LC-MS/MS Assay to Quantitate 2',4',6'-Trihydroxyacetophenone in Rat and Dog Plasma and its Application to a Pharmacokinetic Study.

In the present study, a simple, rapid, and reliable bioanalytical method was developed using liquid chromatography with tandem-mass spectrometry (LC-MS/MS) to quantify 2',4',6'-trihydroxyacetophenone (THAP) in rat and dog plasma with 2',4',6'-trihydroxybenzaldehyde as an internal standard (IS). The LC-MS/MS instrument was operated in the multiple reaction monitoring (MRM) mode to detect THAP at m/z transition 166.89 > 82.8 and IS at 152.89 > 82.8, respectively. A simple, one-step protein precipitation (PP) method was employed with acetonitrile for sample preparation. Utilizing a Gemini C18 column, THAP and IS were separated with an isocratic mobile phase consisting of 10 mM ammonium acetate and methanol (10:90, v/v) at a flow rate of 0.2 mL/min. Total chromatographic run time was 2.5 min per sample injection. The standard calibration curve for THAP was linear (r2 ≥ 0.9987) over the concentration range of 0.1 to 100 µg/mL with the lower limit of quantitation (LLOQ) of 0.1 µg/mL (S/N ratio > 10). According to the regulatory guidelines from the U.S. Food and Drug Administration (FDA) and the Korea Ministry of Food and Drug Safety (MFDS), our newly developed biomedical analytical method was fully and adequately validated in terms of selectivity, sensitivity, linearity, intra- and inter-day precision and accuracy, recovery, matrix effect, stability, and dilution integrity. Our validated assay was successfully utilized in a nonclinical pharmacokinetic study of THAP in rats and dogs.

A novel mPGES-1 inhibitor alleviates inflammatory responses by downregulating PGE2 in experimental models.

We previously reported the strong inhibitory potency of N-phenyl-N'-(4- benzyloxyphenoxycarbonyl)-4-chlorophenylsulfonyl hydrazide (PBCH) on lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production in macrophages. Herein, we characterized PBCH as a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor and evaluated its anti-inflammatory effects using in vivo experimental models. PBCH inhibited PGE2 production in various activated cells in addition to inhibiting the mPGES-1 activity. In the ear edema and paw edema rat models, PBCH significantly reduced ear thickness and paw swelling, respectively. Besides, in adjuvant-induced arthritis (AIA) rat model, PBCH decreased paw swelling, plasma rheumatoid factor (RF), and receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. Furthermore, while PBCH reduced the plasma prostaglandin E metabolite (PGEM) levels, it did not affect the plasma levels of prostacyclin (PGI2) and thromboxane A2 (TXA2). Our data suggest that PBCH downregulates PGE2 production by interfering with the mPGES-1 activity, thus reducing edema and arthritis in rat models.