Simple pharmacokinetic models accounting for drug monitoring results of atomoxetine and its 4-hydroxylated metabolites in Japanese pediatric patients genotyped for cytochrome P450 2D6.

Atomoxetine is an approved medicine for attention-deficit/hyperactivity disorder and a cytochrome P450 2D6 (CYP2D6) probe substrate. Simple physiologically based pharmacokinetic (PBPK) models and compartment models were set up to account for drug monitoring results of 33 Japanese patients (6-15 years of age) to help establish the correct dosage for the evaluation of clinical outcomes. The steady-state one-point drug monitoring data for the most participants indicated the extensive biotransformation of atomoxetine to 4-hydroxyatomoxetine under individually prescribed doses of atomoxetine. However, 5 participants (with impaired CYP2D6 activity scores based on the CYP2D6 genotypes) showed high plasma concentrations of atomoxetine (0.53-1.5 µM) compared with those of total 4-hydroxyatomoxetine (0.49-1.4 µM). Results from full PBPK models using the in-built Japanese pediatric system of software Simcyp, one-compartment models, and new simple PBPK models (using parameters that reflected the subjects' small body size and normal/reduced CYP2D6-dependent clearance) could overlay one-point measured drug/metabolite plasma concentrations from almost common 28 participants within threefold ranges. Validated one-compartment or simple PBPK models can be used to predict steady-state plasma concentrations of atomoxetine and/or its primary metabolites in Japanese pediatric patients (>6 years) who took a variety of individualized doses in a clinical setting.

Adult and infant pharmacokinetic profiling of dihydrocodeine using physiologically based pharmacokinetic modeling.

We previously analysed the serum concentrations of dihydrocodeine in a 1-month-old infant with respiratory depression after being prescribed dihydrocodeine phosphate 2.0 mg/day divided t.i.d. for 2 days. The purpose was to develop a full physiologically based pharmacokinetic (PBPK) model that could account for these and other drug monitoring results. Based on experiments in Caco-2 cell monolayers, the effective permeability of dihydrocodeine in human jejunum was established as 1.28 × 10-4 cm/s. The in vitro Vmax /Km values for dihydrocodeine demethylation mediated by recombinant cytochrome P450 2D6 and 3A4 were 0.19 and 0.066 µl/min/pmol, respectively, and for dihydrocodeine 6-O-glucuronidation mediated by recombinant UGT2B4 and 2B7, the Vmax /Km values were 0.14 and 0.22 µl/min/mg protein, respectively. Renal clearance was calculated as 5.37 L/h on the total clearance value multiplied by the fraction recovered in urine. The reported plasma concentration-time profiles of dihydrocodeine after intravenous administration in healthy volunteers were used to adjust the tissue partitioning ratios. The developed model simulated the pharmacokinetic profiles of dihydrocodeine after single and multiple oral administrations reasonably well in the same population. Subsequently, the validated model was used to simulate pharmacokinetic profiles for five pediatric cases, including the 1-month-old Japanese boy and a 14-year-old Japanese girl who took an overdose of dihydrocodeine phosphate (37 mg). The simulated pharmacokinetic profiles for five virtual pediatric subjects matching the age, gender, and P450 2D6 phenotype of each case approximately reflected the observed values. These results suggested that our dihydrocodeine PBPK model reproduced the results of clinical cases reasonably well for subjects.

Magnetic Phase Transition in a Mixture of Two Interacting Superfluid Bose Gases at Finite Temperature.

The miscibility condition for a binary mixture of two interacting Bose-Einstein condensates is shown to be deeply affected by interaction driven thermal fluctuations. These give rise to a first order phase transition to a demixed phase with full spatial separation of the two condensates, even if the mixture is miscible at zero temperature. Explicit predictions for the isothermal compressibility, the spin susceptibility, and the phase transition temperature T_{M} are obtained in the framework of Popov theory, which properly includes beyond mean-field quantum and thermal fluctuations in both the spin and density channels. For a mixture of two sodium condensates occupying the hyperfine states |F=1⟩,|m_{F}=1⟩ and |F=1,m_{F}=-1⟩, respectively, T_{M} is predicted to occur at about 0.7 times the usual BEC critical temperature.

Pharmacokinetics of anticoagulants apixaban, dabigatran, edoxaban and rivaroxaban in elderly Japanese patients with atrial fibrillation treated in one general hospital.

Steady-state plasma concentrations of anticoagulants and the time since the previous administration in mainly outpatients with atrial fibrillation administered standard or reduced doses were analyzed for 110 elderly Japanese subjects (mean age, 76 years) treated with apixaban (2.5 or 5.0 mg twice daily), dabigatran etexilate (110 or 150 mg twice daily), edoxaban (30 or 60 mg once daily) or rivaroxaban (10 or 15 mg once daily) at one general hospital. The pharmacokinetics in patients treated with standard and reduced doses of the four anticoagulants using liquid chromatography-tandem mass spectrometry was compared with the concentration ranges estimated using physiologically based pharmacokinetic modeling. Reduced doses of anticoagulants resulted in relatively small pharmacokinetic variations compared with the standard dose. Statistical analyses revealed that renal impairment is likely not the sole determinant factor for high plasma concentrations of apixaban, dabigatran, edoxaban and rivaroxaban. Patients with atrial fibrillation should be treated with the correct doses of oral anticoagulants as specified in the package inserts (e.g. reduced doses for elderly patients, patients with low body weights and in combination with P-glycoprotein inhibitor drugs) to avoid excessive or insufficient doses of direct oral anticoagulants.

Collisionless Sound in a Uniform Two-Dimensional Bose Gas.

Using linear response theory within the random phase approximation, we investigate the propagation of sound in a uniform two dimensional (2D) Bose gas in the collisionless regime. We show that the sudden removal of a static density perturbation produces a damped oscillatory behavior revealing that sound can propagate also in the absence of collisions, due to mean-field interaction effects. We provide explicit results for the sound velocity and damping as a function of temperature, pointing out the crucial role played by Landau damping. We support our predictions by performing numerical simulations with the stochastic (projected) Gross-Pitaevskii equation. The results are consistent with the recent experimental observation of sound in a weakly interacting 2D Bose gas both below and above the superfluid Berezinskii-Kosterlitz-Thouless transition.

Regulation of type I collagen expression by microRNA-29 following ionizing radiation.

Radiation-induced fibrosis (RIF) is thought to involve the excessive accumulation of collagen and other extracellular matrix components; previously, we reported that ionizing radiation increased the type I collagen expression and that transforming growth factor (TGF)-ß was involved in this increase through activating its downstream mediator, Smad3. A recent study found that microRNAs (miRNAs)-small, noncoding sequences approximately 20 nucleotides long-negatively regulate the gene expression posttranscriptionally, and it has been suggested that miRNAs play essential roles in cellular processes, including fibrosis. However, their role in the development of RIF remains unexplored. In the present study, we examined the effects of miRNA on the expression of type I collagen induced by ionizing radiation and the mechanisms underlying the miRNA expression observed following ionizing radiation. We analyzed the regulation of miRNA following ionizing radiation by an miRNA real-time PCR, and found that miR-29 family members were downregulated in irradiated mouse fibroblasts and directly targeted type I collagen genes by specifically binding to the 3' untranslated region. We also found that the overexpression of miR-29 inhibited the ionizing radiation-induced expression of type I collagen, whereas the knockdown of miR-29 enhanced it. In addition, TGF-ß/Smad-signaling significantly decreased the transcription of miR-29, whereas the inhibition of this signaling pathway cancelled this decrease. In conclusion, miR-29 was involved in the regulation of type I collagen expression through the TGF-ß/Smad-signaling pathway in irradiated cells, suggesting that miR-29 may be an important regulator of RIF.

Endurance performance and energy metabolism during exercise in mice with a muscle-specific defect in the control of branched-chain amino acid catabolism.

It is known that the catabolism of branched-chain amino acids (BCAAs) in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA) dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK). In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice) to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.

Muscle-specific deletion of BDK amplifies loss of myofibrillar protein during protein undernutrition.

Branched-chain amino acids (BCAAs) are essential amino acids for mammals and play key roles in the regulation of protein metabolism. However, the effect of BCAA deficiency on protein metabolism in skeletal muscle in vivo remains unclear. Here we generated mice with lower BCAA concentrations by specifically accelerating BCAA catabolism in skeletal muscle and heart (BDK-mKO mice). The mice appeared to be healthy without any obvious defects when fed a protein-rich diet; however, bolus ingestion of BCAAs showed that mTORC1 sensitivity in skeletal muscle was enhanced in BDK-mKO mice compared to the corresponding control mice. When these mice were fed a low protein diet, the concentration of myofibrillar protein was significantly decreased (but not soluble protein) and mTORC1 activity was reduced without significant change in autophagy. BCAA supplementation in drinking water attenuated the decreases in myofibrillar protein levels and mTORC1 activity. These results suggest that BCAAs are essential for maintaining myofibrillar proteins during protein undernutrition by keeping mTORC1 activity rather than by inhibiting autophagy and translation. This is the first report to reveal the importance of BCAAs for protein metabolism of skeletal muscle in vivo.

mTORC1 is involved in the regulation of branched-chain amino acid catabolism in mouse heart.

The branched-chain α-ketoacid dehydrogenase (BCKDH) complex regulates branched-chain amino acid (BCAA) catabolism by controlling the second step of this catabolic pathway. In the present study, we examined the in vivo effects of treatment with an mTORC1 inhibitor, rapamycin, on cardiac BCKDH complex activity in mice. Oral administration of leucine in control mice significantly activated the cardiac BCKDH complex with an increase in cardiac concentrations of leucine and α-ketoisocaproate. However, rapamycin treatment significantly suppressed the leucine-induced activation of the complex despite similar increases in cardiac leucine and α-ketoisocaproate levels. Rapamycin treatment fully inhibited mTORC1 activity, measured by the phosphorylation state of ribosomal protein S6 kinase 1. These results suggest that mTORC1 is involved in the regulation of cardiac BCAA catabolism.

Novel Physiological Functions of Branched-Chain Amino Acids.

Branched-chain amino acids (BCAAs) are essential amino acids for humans and are major building blocks of proteins. Recent studies indicate that BCAAs act not only as components of proteins, but also as nutrasignals. In this review, we summarize the findings of recent studies investigating the physiological functions of BCAAs in the regulation of protein and glucose metabolism and brain function.

Sp7/Osterix induces the mouse pro-α2(I) collagen gene (Col1a2) expression via the proximal promoter in osteoblastic cells.

Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.

A new murine osteoblastic cell line immortalized with the SV40 large T antigen.

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.

Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation.

Xylose isomerase (XI) is a key enzyme in the conversion of D-xylose, which is a major component of lignocellulosic biomass, to D-xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI-related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell-surface display system, and the xylose assimilation and fermentation properties of this XI-displaying yeast were examined. XI-displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions.