HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13 . The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs

Effects of Piperazine Derivative on Paclitaxel Pharmacokinetics

Paclitaxel (PTX) is an anticancer agent that is used to treat many cancers but it has a very low oral bioavailability due, at least in part, to the drug efflux transporter, P-glycoprotein (P-gp). Therefore, this study was performed to enhance oral bioavailability of PTX. In this study, we investigated the effects of several piperazine derivatives on P-gp function in vitro. Compound 4 was selected as the most potent P-gp inhibitor from the in vitro results for examining the pharmacokinetic (PK) changes of PTX in rats. Compound 4 increased the AUCinf of PTX without alterations in the Cmax value. The elimination half-life was extended and the oral clearance decreased. Additionally, the Tmax was delayed or widened in the treatment groups. Therefore, the bioavailability (BA) of PTX was improved 2.1-fold following the co-administration of 5 mg/kg of the derivative. A piperazine derivative, compound 4, which was confirmed as a substantial P-gp inhibitor in vitro increased the BA of PTX up to 2-fold by a lingering absorption, in part due to inhibition of intestinal P-gp and a low oral clearance of PTX. These results suggest that co-administering compound 4 may change the PK profile of PTX by inhibiting P-gp activity in the body

Pharmacokinetic Alteration of Paclitaxel by Ferulic Acid Derivative

P-glycoprotein (P-gp) is known to be involved in multidrug resistance (MDR) and modulation of pharmacokinetic (PK) profiles of substrate drugs. Here, we studied the effects of synthesized ferulic acid (FA) derivatives on P-gp function in vitro and examined PK alteration of paclitaxel (PTX), a well-known P-gp substrate drug by the derivative. Compound 5c, the FA derivative chosen as a significant P-gp inhibitor among eight FA candidates by in vitro results, increased PTX AUCinf as much as twofold versus the control by reducing PTX elimination in rats. These results suggest that FA derivative can increase PTX bioavailability by inhibiting P-gp existing in eliminating organs

In Vitro and in Vivo Evaluation of Phenylbutenoid Dimers as Inhibitors of P‑Glycoprotein

The expression of P-glycoprotein (P-gp), an ATP-dependent efflux transporter, is closely associated with the failure of chemotherapy and drug absorption. Two synthesized optically active phenylbutenoid dimers, 3<i>S</i>-(3,4-dimethoxyphenyl)-4<i>R</i>-{(<i>E</i>)-3,4-dimethoxystyryl}­cyclohex-1-ene (<b>1</b>) and 3<i>R</i>-(3,4-dimethoxyphenyl)-4<i>S</i>-{(<i>E</i>)-3,4-dimethoxystyryl}­cyclohex-1-ene (<b>2</b>), were tested for their P-gp inhibitory effects by measuring cellular accumulation and efflux of daunomycin in P-gp-overexpressed human breast cancer cells (MCF-7/ADR). Compound <b>2</b> significantly increased the accumulation of daunomycin (539%) and decreased the efflux of this compound (55.4%), and similar results were observed for <b>1</b>. ATPase assays and Western blot analysis were performed to identify the mechanisms by which compounds <b>1</b> and <b>2</b> inhibit P-gp. In addition, changes in the pharmacokinetic profile of paclitaxel coadministered with <b>2</b> in rats were evaluated. Paclitaxel (25 mg/kg) when orally administered with <b>2</b> (5 mg/kg) improved its relative bioavailability by 185%. Compound <b>2</b> effectively improved cellular accumulation by reducing the efflux of daunomycin and significantly enhanced oral exposure to paclitaxel. Therefore, compound <b>2</b> may be useful for improving oral exposure and cellular availability of drugs that are also substrates of P-gp

Immunosuppressive Drug Measurement by Liquid Chromatography Coupled to Tandem Mass Spectrometry: Interlaboratory Comparison in the Korean Clinical Laboratories

Background: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is increasingly used for immunosuppressive drug tests. However, most LC-MS/MS tests are laboratory-developed and their agreement is unknown in different Korean laboratories. This interlaboratory comparison study evaluated test reproducibility and identified potential error sources. Methods: Test samples containing three concentrations of tacrolimus, sirolimus, everolimus, cyclosporine, and mycophenolic acid were prepared by pooling surplus samples from patients undergoing routine therapeutic drug monitoring and tested in duplicate in the participating 10 clinical laboratories. Reconstitution and storage experiments were conducted for the commonly used commercial calibrator set. The robust estimators of reproducibility parameters were calculated. Spearman's rank correlation coefficient (rho, rho) was used to evaluate the correlation between drugs. Multiple linear regression was used to determine whether the experimental conditions alter the calibration curves. Results: The reproducibility coefficient of variation exceeded 10% only for sirolimus concentrations 1 and 2 (10.8% and 12.5%, respectively) and everolimus concentrations 1 and 2 (12.3% and 11.4%, respectively). The percent difference values showed weak correlations between sirolimus and everolimus (rho=0.334, P=0.175). The everolimus calibration curve slope was significantly altered after reconstitution following prolonged 5 degrees C storage (P=0.015 for 14 days; P=0.025 for 28 days); the expected differences at 6 ng/mL were 0.598% for 14 days and 0.384% for 28 days. Conclusions: LC-MS/MS test reproducibility for immunosuppressive drugs seems to be good in the Korean clinical laboratories. Continuous efforts are required to achieve test standardization and harmonization, especially for sirolimus and everolimus.11Nsciescopu

A newly developed capture-based sequencing panel for genomic assay of lung cancer

Background The increase in genetic alterations targeted by specific chemotherapy in lung cancer has led to the need for universal use of more comprehensive genetic testing, which has highlighted the development of a lung cancer diagnostic panel using next-generation sequencing. Objective We developed a hybridization capture-based massively parallel sequencing assay named Friendly, Integrated, Research-based, Smart and Trustworthy (FIRST)-lung cancer panel (LCP), and evaluated its performance. Methods FIRST-LCP comprises 64 lung cancer-related genes to test for various kinds of genetic alterations including single nucleotide variations (SNVs), insertions and deletions (indels), copy number variations (CNVs), and structural variations. To assess the performance of FIRST-LCP, we compiled test sets using HapMap samples or tumor cell lines with disclosed genetic information, and also tested our clinical lung cancer samples whose genetic alterations were known by conventional methods. Results FIRST-LCP accomplished high sensitivity (99.4%) and specificity (100%) of the detection of SNVs. High precision was also achieved, with intra- or inter-run concordance rate of 0.99, respectively. FIRST-LCP detected indels and CNVs close to the expected allele frequency and magnitude, respectively. Tests with samples from lung cancer patients also identified all SNVs, indels and fusions. Conclusion Based on the current state of the art, continuous application of the panel design and analysis pipeline following up-to-date knowledge could ensure precision medicine for lung cancer patients.