Multimodal Imaging Probe Development for Pancreatic ß Cells: From Fluorescence to PET.

Pancreatic ß cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic ß cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

A validated UPLC-MS/MS method for pharmacokinetic study of inflachromene, a novel microglia inhibitor.

Inflachromene (ICM), a novel microglia inhibitor, is under development to treat neuroinflammatory disorders. For the pharmacokinetic evaluation of ICM, we developed quantitative ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method in rat plasma. Protein precipitation method with acetonitrile (ACN) was used for plasma sample preparation. The analyte and carbamazepine (the internal standard) were separated by the chromatography on an ACQUITY UPLC™ BEH C18 column running gradient mobile phase system made up of 0.1% [v/v] formic acid in water and 0.1% [v/v] formic acid in acetonitrile. For the quantification of ICM, the ion transitions, m/z 378.2→187.1 (ICM) and m/z 237.1→194.1 (IS) were monitored in multiple reaction monitoring (MRM) mode. Standard calibration curve showed excellent linearity with the correlation coefficient (R2) of 0.99 within the concentration range of 0.05-10 µg/mL. The precision and accuracy were within acceptable limits (all < 20%). ICM was degraded time and temperature dependently in rat plasma. The analytical method was successfully utilized in a pharmacokinetic study following intravenous and oral administration of ICM in rats.

Quantitative Evaluation of Cytochrome P450 3A4 Inhibition and Hepatotoxicity in HepaRG 3-D Spheroids.

Predicting drug-drug interactions (DDIs) is an important step during drug development to avoid unexpected side effects. Cytochrome P450 (CYP) 3A4 is the most abundant human hepatic phase I enzyme, which metabolizes >50% of therapeutic drugs. Therefore, it is essential to test the potential of a drug candidate to induce CYP3A4 expression or inhibit its activity. Recently, 3-dimensional (3-D) mammalian cell culture models have been adopted in drug discovery research to assess toxicity, DDIs, and pharmacokinetics. In this study, we applied a human 3-D spheroid culture protocol using HepaRG cells combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess its ability to predict CYP3A4 inhibition. Levels of midazolam, a specific substrate of CYP3A4, were used to determine the long-term metabolic capacity of CYP3A4. Midazolam was decreased in the 3-D HepaRG culture system by ∼80% over 7 days, whereas its primary metabolite, 1-hydroxymidazolam, increased by ∼40%. Next, we assessed hepatotoxicity by determining the cytotoxicity of known hepatotoxicants in HepaRG spheroids, HepG2 cells, and primary human hepatocytes. Significant differences in cytotoxicity were detected in the system using 3-D HepaRG spheroids. These results suggest that 3-D HepaRG spheroids are a good model for prediction of CYP inhibition and hepatotoxicity in screening of early drug candidates.

Zebrafish as a Screening Model for Testing the Permeability of Blood-Brain Barrier to Small Molecules.

The objective of this study was to evaluate the permeability of small molecules into the brain via the blood-brain barrier in zebrafish and to investigate the possibility of using this animal model as a screening tool during the early stages of drug discovery. Fifteen compounds were used to understand the permeation into the brain in zebrafish and mice. The ratio of brain-to-plasma concentration was compared between the two animal models. The partition coefficient (Kp,brain), estimated using the concentration ratio at designated times (0.167, 0.25, 0.5, or 2 h) after oral administrations (per os, p.o), ranged from 0.099 to 5.68 in zebrafish and from 0.080 to 11.8 in mice. A correlation was observed between the Kp,brain values obtained from the zebrafish and mice, suggesting that zebrafish can be used to estimate Kp,brain to predict drug penetration in humans. Furthermore, in vivo transport experiments to understand the permeability glycoprotein (P-gp) transporter-mediated behavior of loperamide (LPM) in zebrafish were performed. The zebrafish, Kp,brain,30min of LPM was determined to be 0.099 ± 0.069 after dosing with LPM alone, which increased to 0.180 ± 0.115 after dosing with LPM and tariquidar (TRQ, an inhibitor of P-gp). In mouse, the Kp,brain,30min of LPM was determined to be 0.080 ± 0.004 after dosing with LPM alone and 0.237 ± 0.013 after dosing with LPM and TRQ. These findings indicate that the zebrafish could be used as an effective screening tool during the discovery stages of new drugs to estimate their distribution in the brain.

A Novel Small-Molecule Inhibitor Targeting the IL-6 Receptor ß Subunit, Glycoprotein 130.

IL-6 is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small-molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists, we screened our in-house chemical library and identified LMT-28, a novel synthetic compound, as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular target of LMT-28 were investigated. A reporter gene assay showed that LMT-28 suppressed activation of STAT3 induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, LMT-28 downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein and substantially inhibited IL-6-dependent TF-1 cell proliferation. LMT-28 antagonized IL-6-induced TNF-α production in vivo. In pathologic models, oral administration of LMT-28 alleviated collagen-induced arthritis and acute pancreatitis in mice. Based on the observation of upstream IL-6 signal inhibition by LMT-28, we hypothesized IL-6, IL-6Rα, or gp130 to be putative molecular targets. We subsequently demonstrated direct interaction of LMT-28 with gp130 and specific reduction of IL-6/IL-6Rα complex binding to gp130 in the presence of LMT-28, which was measured by surface plasmon resonance analysis. Taken together, our data suggest that LMT-28 is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.

In vitro permeability, pharmacokinetics and brain uptake of WAY-100635 and FCWAY in rats using liquid chromatography electrospray ionization tandem mass spectrometry.

Positron emission tomography (PET) is a sensitive non-invasive imaging technique. To reduce imaging measurements of defects, there is a demand for proper LC-ESI-MS/MS method to carry out with its specificity and sensitivity. This study describes a rapid and simple liquid chromatography electrospray ionization tandem-MS/MS (LC-ESI-MS/MS) method for determination of both PET tracers: N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY-100635) and 4-fluoro-N-[2-[4-(2-methoxylphenyl)-1-piperazino]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (FCWAY). Both target compounds were prepared by one-step protein precipitation with acetonitrile and methanol (1:1, v/v), and analyzed using a C18 column. This simple method has an excellent linearity, selectivity and sensitivity. Precision and accuracy values for the intra-day and inter-day validation were below 12%. The limit of quantification (LOQ) for both target compounds was defined as 1 ng/mL in plasma and 5 ng/mL in brain homogenate. The stability of both compounds is considered stable under a various experimental conditions. The in vitro MDR-MDCK cell permeability showed the both compounds have high permeability (Papp, A→B ≥ 20 × 10(-6 )cm/s) and low efflux ratio (≤2.0). Brain to blood (AUCbrain/AUCblood) distribution ratios in rats were 3.15 ± 0.42 for WAY-100635 and 2.20 ± 0.34 for FCWAY, respectively, and these results suggest that LC-ESI-MS/MS method might be a supplementary way for the identifying and understanding of radiopharmaceuticals.

Determination of mesoridazine by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study in rats.

The object of the present study was to develop and validate an assay method of mesoridazine in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples from rats were prepared by simple protein precipitation and injected onto the LC-MS/MS system for quantification. Mesoridazine and chlorpromazine as an internal standard (IS) were separated by a reversed phase C18 column. A mobile phase was composed of 10mM ammonium formate in water and acetonitrile (ACN) (v/v) by a linear gradient system, increasing the percentage of ACN from 2% at 0.4min to 98% at 2.5min with 4min total run time. The ion transitions monitored in positive-ion mode [M+H](+) of multiple-reaction monitoring (MRM) were m/z 387>126 for mesoridazine and m/z 319>86 for IS. The detector response was specific and linear for mesoridazine at concentrations within the range 0.001-4µg/ml and the correlation coefficient (R(2)) was greater than 0.999 and the signal-to-noise ratios for the samples were ≥10. The intra- and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The matrix effects were approximately 101 and 99.5% from rat plasma for mesoridazine and chlorpromazine, respectively. Mesoridazine was stable under various processing and/or handling conditions. Mesoridazine concentrations were readily measured in rat plasma samples after intravenous and oral administration. This assay method can be practically useful to the pharmacokinetic and/or toxicokinetic studies of mesoridazine.

Preclinical pharmacokinetic characterization of 2-(4-(4-(5-(2-phenyl-5-(trifluoromethyl)oxazole-4-carboxamido)-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexyl) acetic acid, a novel DGAT-1 inhibitor.

1. A novel diacylglyceride acyltransferase-1 (DGAT-1) inhibitor, 2-(4-(4-(5-(2-phenyl-5-(trifluoromethyl) oxazole-4-carboxamido)-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexyl) acetic acid (KR-69232), was synthesized for a potential therapeutic use against several metabolic disorders, such as obesity, insulin resistance, and type II diabetes, characterized by excessive triglycerides (TGs) in the blood. 2. The half-lives against phase I metabolism were measured as 75.3 ± 20.9 min and over 120 min in rat and human liver microsomes, respectively. In Caco-2 cell monolayers, extremely low permeability (<0.13 × 10⁻6cm/s) was seen in the absorptive direction, predicting limited intestinal absorption of KR-69232. This compound was highly bound to rat and human plasma proteins (>99.8%). 3. With the intravenous administration of KR-69232 in rats (1, 2, and 5 mg/kg), non-linear kinetics were observed at the highest dose, with significantly higher systemic clearance, higher volume of distribution, and lower dose-normalized AUC. Following oral administration, it exhibited low bioavailability (<10%) and was absorbed slowly (T(max), 3.8-5.2 h) over the dose range. We also confirmed that considerable KR-69232 remained in the intestine at T(max), demonstrating its limited absorption into the systemic circulation.

Pharmacokinetic characterization of the novel TAZ modulator TM-25659 using a multicompartment kinetic model in rats and a possibility of its drug-drug interactions in humans.

This study evaluated the pharmacokinetics of the novel TAZ modulator TM-25659 in rats following intravenous and oral administration at dose ranges of 0.5-5 mg/kg and 2-10 mg/kg, respectively. Plasma protein binding, plasma stability, liver microsomal stability, CYP inhibition, and transport in Caco-2 cells were also evaluated. After intravenous injection, systemic clearance, steady-state volumes of distribution, and half-life were dose-independent, with values ranging from 0.434-0.890 mL · h(-1) · kg(-1), 2.02-4.22 mL/kg, and 4.60-7.40 h, respectively. Mean absolute oral bioavailability was 50.9% and was not dose dependent. Recovery of TM-25659 was 43.6% in bile and <1% in urine. In pharmacokinetic modeling studies, the three-compartment (3C) model was appropriate for understanding these parameters in rats. TM-25659 was stable in plasma. Plasma protein binding was approximately 99.2%, and was concentration-independent. TM-25659 showed high permeation of Caco-2 cells and did not appear to inhibit CYP450. TM-25659 was metabolized in phase I and II steps in rat liver microsomes. In conclusion, the pharmacokinetics of TM-25659 was characterized for intravenous and oral administration at doses of 0.5-5 and 2-10 mg/kg, respectively. TM-25659 was eliminated primarily by hepatic metabolism and urinary excretion.

Association study between polymorphisms of CD28, CTLA4 and ICOS and non-segmental vitiligo in a Korean population.

CD28 molecule (CD28), cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and inducible T-cell co-stimulator (ICOS) are important regulators of the immune system. Vitiligo, a common autoimmune skin disorder, is characterized by a loss of melanocytes that results in cutaneous white patches. The aim of the present study was to determine whether or not polymorphisms of the CD28, CTLA4 and ICOS genes are associated with non-segmental vitiligo in a Korean population. To determine the relationships between CD28, CTLA4 and ICOS genes and vitiligo, four single nucleotide polymorphisms (SNPs) associated with the CD28 gene [rs1879877 (promoter, -1198), rs3181097 (promoter, -1059), rs2140148 (intron 1) and rs3116494 (intron 2)], two SNPs associated with the CTLA4 gene [rs231777 (intron 1) and rs231779 (intron 1)] and five SNPs associated with the ICOS gene [rs4270326 (intron 3), rs11571314 (intron 3), rs10183087 (3' untranslated region; UTR), rs4404254 (3'UTR) and rs1559931 (3'UTR)] were selected. Two hundred and thirty-one patients with non-segmental vitiligo (NSV) and 405 healthy controls were enrolled. Genotyping was performed using the restriction fragment length polymorphism technique and direct sequencing. SNPStats, Haploview 4.2 and SPSS 18.0 were used to conduct the analyses. Significant differences were noted between CTLA4 (p<0.05) and NSV, but not CD28 and ICOS (p>0.05). However, these associations disappeared after Bonferroni correction. The CD28, CTLA4 and ICOS genes may not be associated with NSV.