Improvement of absorbability, osteoconductivity, and strength of a ß-tricalcium phosphate spacer for opening wedge high tibial osteotomy: clinical evaluations with 106 patients.

BACKGROUND: An ideal synthetic spacer for medial opening wedge high tibial osteotomy (MOWHTO) has not yet been developed. The authors have developed a new ß-tricalcium phosphate (ß-TCP) spacer with 60% porosity (N-CP60) by modifying the micro- and macro-pore structures of a conventional ß-TCP spacer (CP60) that is widely used in clinical practice. The purpose of this study was to compare the absorbability, osteoconductivity, and in vivo strength of the N-CP60 spacer with those of the CP60 spacer, when used in MOWHTO. METHODS: First, the porosity, diameter distribution of macro- and micropores, and compressive strength of each ß-TCP block were examined using methodology of biomaterial science. Secondly, a clinical study was performed using a total of 106 patients (106 knees) with MOWHTO, who were followed up for 18 months after surgery. In these knees, the N-CP60 and CP-60 spacers were implanted into 49 tibias and 57 tibias, respectively. The absorbability and osteoconductivity were radiologically evaluated by measuring the area of the implanted spacer remaining unabsorbed and assessing with the Hemert's score, respectively. The incidence of cracking in the implanted spacers was determined using computed radiography. Statistical comparisons were made with non-parametric tests. The significance level was set at p = 0.05. RESULTS: The N-CP60 and CP60 blocks had almost the same porosity (mean, 61.0% and 58.7%, respectively). The diameter of macropores was significantly larger (p < 0.0001) in the N-CP60 block than in the CP60 block, while the diameter of micropores was significantly smaller (p = 0.019) in the N-CP60 block. The ultimate strength of the N-CP60 block (median, 36.8 MPa) was significantly greater (p < 0.01) than that of the CP60 block (31.6 MPa). As for the clinical evaluations, the absorption rate of the N-CP60 spacer at 18 months after implantation (mean, 48.0%) was significantly greater (p < 0.001) than that of the CP60 spacer (29.0%). The osteoconductivity of the N-CP60 spacer was slightly but significantly higher (p = 0.0408) than that of the CP60 spacer only in zone 1. The incidence of in vivo cracking of the posteriorly located N-CP60 spacer at one month (mean, 75.5%) was significantly lower (p = 0.0035) than that of the CP60 spacer (91.2%). CONCLUSIONS: The absorbability, osteoconductivity, and compressive strength of the new N-CP60 spacer were significantly improved by modifying the macro- and micro-pore structures, compared with the conventional CP60 spacer. The N-CP60 spacer is more clinically useful than the CP60 spacer. TRIAL REGISTRATION NUMBER: H29-0002.

Preclinical metabolism and the disposition of vornorexant/TS-142, a novel dual orexin 1/2 receptor antagonist for the treatment of insomnia.

We investigated the metabolism and disposition of vornorexant, a novel dual orexin receptor antagonist, in rats and dogs, and clarified in vitro metabolite profiles in humans. Furthermore, we investigated the pharmacokinetics of active metabolites in rats and dogs and their CNS distribution in rats to elucidate its contribution to drug efficacy. [14 C]vornorexant was rapidly and mostly absorbed after the oral administration in rats and dogs. The drug-derived radioactivity, including metabolites, was distributed to major organs such as the liver, kidneys in rats, and was almost eliminated within 24 h post-dose in both species. Metabolite profiling revealed that main clearance mechanism of vornorexant was metabolism via multiple pathways by oxidation. The major circulating components were the cleaved metabolites (M10, M12) in rats, and the unchanged form in dogs, followed by M1, and then M3. Incubation with human hepatocytes resulted in formation of metabolites, including M1, M3, M10, and M12. The metabolic pathways were similar in all tested species. Resulting from the PK and CNS distribution of active metabolites (M1 and M3) with weaker pharmacological activity, the concentration of the unchanged form was higher than that of active metabolites in rat CSF and dog plasma, suggesting that the unchanged form mainly contributed to the drug efficacy. These findings demonstrate that vornorexant is absorbed immediately after administration, and vornorexant and its metabolites are rapidly and completely eliminated in rats and dogs. Thus, vornorexant may have favorable pharmacokinetic profiles as a hypnotic drug to provide rapid onset of action and minimal next-day residual effects in humans.

Degree of loss in the tissue thickness, microvascular density, specific perimetry and standard perimetry in early glaucoma.

OBJECTIVE: To identify the degree of loss of the circumpapillary retinal nerve fibre layer (cpRNFL), the layer from the macular RNFL to the inner plexiform layer (mGCL++), circumpapillary (cpVD) and macular vascular density (mVD), Pulsar perimetry and standard perimetry in early glaucoma. METHODS: In this cross-sectional study, one eye from each of 96 healthy controls and 90 eyes with open-angle glaucoma were measured with cpRNFL, mGCL++, cpVD, mVD, Pulsar perimetry with Octopus P32 test (Pulsar) and standard perimetry with Humphrey field analyser 24-2 test (HFA). For direct comparison, all parameters were converted to relative change values adjusted in both their dynamic range and age-corrected normal value. RESULTS: The degree of loss in mGCL++ (-24.7%) and cpRNFL (-25.8%) was greater than that in mVD (-17.3%), cpVD (-14.9%), Pulsar (-10.1%) and HFA (-5.9%) (each p<0.01); the degree of loss in mVD and cpVD was greater than that in Pulsar and HFA (each p<0.01); and the degree of loss in Pulsar was greater than that in HFA (p<0.01). The discrimination ability between glaucomatous and healthy eyes (area under the curve) was higher for mGCL++ (0.90) and cpRNFL (0.93) than for mVD (0.78), cpVD (0.78), Pulsar (0.78) and HFA (0.79). CONCLUSION: The degree of loss of cpRNFL and mGCL++ thickness preceded by approximately 7%-10% and 15%-20% compared with the micro-VD and visual fields in early glaucoma, respectively. TRIAL REGISTRATION NUMBER: UMIN Clinical Trials Registry (http://www.umin.ac.jp/; R000046076 UMIN000040372).

Preclinical Metabolism and Disposition of TP0473292, a Novel Oral Prodrug of the Potent Metabotropic Glutamate 2/3 Receptor Antagonist TP0178894 for the Treatment of Depression.

TP0473292 is an adamantane carboxylic acid (ACA) ester prodrug for enhancing the oral bioavailability of the hydrophilic glutamate analog TP0178894, a novel metabotropic glutamate 2 and 3 receptor antagonist, and being developed as an antidepressant. TP0473292 showed high membrane permeability and rapid hydrolysis to TP0178894 in rat, monkey, and human liver S9 fractions, with a conversion rate of such that complete conversion by first-pass metabolism was expected. TP0473292 was also hydrolyzed in the intestinal, renal, and lung S9 fractions, coinciding with the result that TP0473292 was activated by carboxylesterase (CES) 1 and more efficiently by CES2. Despite the rapid hydrolysis of TP0473292 in the intestinal S9 fraction, TP0473292 achieved good oral bioavailability of poorly permeable TP0178894 (approximately 60%) in rats and monkeys, with no TP0473292 detected in the plasma, revealing that rapid hydrolysis in the intestine is not necessarily a disadvantage. We also confirmed the penetration of TP0178894 into the cerebrospinal fluid and its unmetabolized excretion in urine. The ester promoiety, ACA, was metabolized to chemically stable acyl glucuronide and excreted in urine in rats and monkeys, suggesting a low risk of idiosyncratic drug toxicity. TP0473292 and its metabolites did not show a drug-drug interaction potential via cytochrome P450 in humans. These results suggested that TP0473292 functions as an ideal oral prodrug in humans; this was later confirmed to be true in phase 1 clinical trials. Furthermore, ACA was firstly confirmed to be a useful promoiety for hydrophilic drugs to enhance their oral bioavailability. SIGNIFICANCE STATEMENT: Hydrolysis in the intestine reportedly has negative effects on the oral bioavailability of hydrophilic active metabolites of ester prodrugs. This study reports the preclinical pharmacokinetics of a hydrophilic metabotropic glutamate 2/3 receptor antagonist, TP0178894, and its ester prodrug TP0473292, which was found to act as an oral prodrug despite being activated predominantly in the intestine. Furthermore, this study firstly reports that adamantane carboxylic acid is useful as the ester promoiety of a prodrug for increasing lipophilicity and oral bioavailability.

Chiral LC-MS/MS method for the simultaneous determination of (R,S)-ketamine, (R,S)-norketamine, and (2R,6R;2S,6S)-hydroxynorketamine in mouse plasma and brain.

A convenient LC-MS/MS assay method to simultaneously and sensitively determine (R,S)-ketamine (Ket), (R,S)-norketamine (NK), and (2R,6R;2S,6S)-hydroxynorketamine (HNK) enantiomers in plasma and brain from mice was developed. This method enables the chiral separations of these six enantiomers in one analysis by constructing a column-switching system composed of one achiral column and two chiral columns with a relatively short analysis time (17 min). The chromatography involves the separation of (2R,6R;2S,6S)-HNK from (R,S)-Ket and (R,S)-NK on an octadecyl-silica column, followed by chiral separations on a CHIRALPAK AY-RH column for (2R,6R;2S,6S)-HNK or on a CHIRALPAK AS-RH column for the other analytes. The calibration curves for plasma and brain showed a good linearity in the range of 3-1000 ng/mL and 1.5-500 ng/g, respectively. The accuracy ranged from 90.0% to 104.0% in within-run and between-run. This validated method was applicable to determine the stereoselective pharmacokinetic profiles of (R,S)-Ket, (R,S)-NK, and (2R,6R;2S,6S)-HNK in plasma and brain collected from individual mice after a single intraperitoneal dosing of racemic Ket at an antidepressant dose. It is hoped that this assay will greatly help for understanding the relationship between the antidepressant actions of (R,S)-Ket enantiomers or their metabolites and their pharmacokinetics.

Elucidation of clearance mechanism of TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor: does a species difference in excretion routes exist between humans and animals?

1. TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor, is reportedly excreted predominantly through urinary excretion in an unchanged form in humans, with partial biliary excretion also possible. However, the clearance mechanisms remain unclear. The aim of this study was to investigate the clearance mechanisms in humans and to assess species differences in the excretion routes.2. TP0463518 was not metabolised in rat, dog, or human hepatocytes. TP0463518 is a substrate for human BCRP, OATP1B1, OATP1B3, and OAT3, suggesting that renal uptake by OAT3 is probably the predominant clearance route, with hepatic uptake by OATP1B1 and OATP1B3 contributing partially to clearance in humans.3. A species difference in excretion routes was observed. The unchanged urinary excretion rates in humans, male rats, female rats, dogs, and monkeys were 80.7%, 0.1%, 40.9%, 15.2%, and 72.6%, respectively. Urinary excretion was predominant in humans and monkeys, while only biliary excretion was observed in male rats. Uptake studies using hepatocytes showed that the hepatic uptake clearance in rats was 13.6-fold higher than that in humans. Therefore, not only reabsorption via renal tubules, but also hepatic uptake seems to be involved in the species differences in excretion routes between rats and humans.

Structure-Function Relationships and Glaucoma Detection with Magnification Correction of OCT Angiography.

Purpose: To investigate the effects of adjusting the ocular magnification during OCT-based angiography imaging on structure-function relationships and glaucoma detection. Design: Cross-sectional study. Participants: A total of 96 healthy control participants and 90 patients with open-angle glaucoma were included. Methods: One eye of each patient in the control group and the patient group was evaluated. The layers comprising the macula vascular density (VD) and circumpapillary VD were derived from swept-source OCT angiography imaging. The mean sensitivity (MS) of the standard automated perimetry was measured using the Humphrey 24-2 test. Structure-function relationships were evaluated with simple and partial correlation coefficients. A receiver operating characteristic analysis was performed to evaluate the diagnostic accuracy for glaucoma using the area under the receiver operating characteristic curve (AUC). Ocular magnification was adjusted using Littmann's formula modified by Bennett. Main Outcome Measures: The association between the axial length and VD, structure-function relationships, and glaucoma detection with and without magnification correction. Results: The superficial layer of the macular region was not significantly correlated to the axial length without magnification correction (r = 0.0011; P = 0.99); however, it was negatively correlated to the axial length with magnification correction (r = -0.22; P = 0.028). Regarding the nerve head layer in the circumpapillary region, a negative correlation to the axial length without magnification correction was observed (r = -0.22; P = 0.031); however, this significant correlation disappeared with magnification correction. The superficial layer of the macula and the nerve head layer of the circumpapillary region were significantly correlated to Humphrey 24-2 MS values without magnification correction (r = 0.22 and r = 0.32, respectively); however, these correlations did not improve after magnification correction (r = 0.20 and r = 0.33, respectively). Glaucoma diagnostic accuracy in the superficial layer (AUC, 0.63) and nerve head layer (AUC, 0.70) without magnification correction did not improve after magnification correction (AUC, 0.62 and 0.69, respectively). Conclusions: Adjustment of the ocular magnification is important for accurate VD measurements; however, it may not significantly impact structure-function relationships and glaucoma detection.

Establishment of a New Qualitative Evaluation Method for Articular Cartilage by Dynamic T2w MRI Using a Novel Contrast Medium as a Water Tracer.

OBJECTIVE: In the early stages of cartilage damage, diagnostic methods focusing on the mechanism of maintaining the hydrostatic pressure of cartilage are thought to be useful. 17O-labeled water, which is a stable isotope of oxygen, has the advantage of no radiation exposure or allergic reactions and can be detected by magnetic resonance imaging (MRI). This study aimed to evaluate MRI images using 17O-labeled water in a rabbit model. DESIGN: Contrast MRI with 17O-labeled water and macroscopic and histological evaluations were performed 4 and 8 weeks after anterior cruciate ligament transection surgery in rabbits. A total of 18 T2-weighted images were acquired, and 17O-labeled water was manually administered on the third scan. The 17O concentration in each phase was calculated from the signal intensity at the articular cartilage. Macroscopic and histological grades were evaluated and compared with the 17O concentration. RESULTS: An increase in 17O concentration in the macroscopic and histologically injured areas was observed by MRI. Macroscopic evaluation showed that the 17O concentration significantly increased in the damaged site group. Histological evaluations also showed that 17O concentrations significantly increased at 36 minutes 30 seconds after initiating MRI scanning in the Osteoarthritis Research Society International (OARSI) grade 3 (0.493 in grade 0, 0.659 in grade 1, 0.4651 in grade 2, and 0.9964 in grade 3, P < 0.05). CONCLUSION: 17O-labeled water could visualize earlier articular cartilage damage, which is difficult to detect by conventional methods.

Psychological Factors That Suppress Help-Seeking among Middle-Aged and Older Adults Living Alone.

Help-seeking among destitute adults has not been adequately investigated. Therefore, this study clarifies the mechanisms that suppress help-seeking in middle-aged and older adults living alone. Data were collected from 1274 individuals (aged 50-79 years) who were living alone, using a survey that measured future time perspective, barriers to help-seeking, help-seeking intentions, and current and childhood economic statuses. Men living alone experienced lower help-seeking intention than women, were more likely to try to solve problems by themselves, and experienced greater distrust in others. No sex differences were observed in "future anxiety" and "resignation to the future." Poor economic status was associated with high "resignation to the future," "future anxiety," and "distrust of others" for both sexes. "Resignation to the future" was particularly higher among men with a poorer current economic status, which suppressed help-seeking. Abandoning hope for the future, which is characteristic of middle-aged and older men living alone, may inhibit help-seeking behavior.

High Rate of Radiographic Union at the Fibular Osteotomy Site With No Complications After an Acute Oblique Osteotomy and Ligation Procedure to Shorten the Fibula in High Tibial Osteotomy.

Background: A fibular shortening osteotomy is needed to perform lateral closing-wedge high tibial osteotomy (LCW-HTO). To achieve this shortening, we have recently developed an acute oblique osteotomy and ligation (AO/L) procedure for the center of the fibular shaft, based on the AO procedure. Purpose: To compare the 2-year follow-up outcomes between the AO/L procedure and the AO procedure. Study Design: Cohort study; Level of evidence, 2. Methods: A prospective comparative cohort study was conducted involving 83 patients (83 knees) who underwent shortening osteotomy of the fibula in LCW-HTO between April 2017 and March 2019. The first consecutive 41 knees (AO group) underwent fibular osteotomy with the AO procedure. The remaining 42 knees (AO/L group) underwent fibular osteotomy with the AO/L procedure. All of the patients were evaluated for at least 2 years postoperatively via clinical and radiological assessments. To determine the time needed for complete union at the osteotomy site, we evaluated the radiographs using a radiographic union score for tibial fractures, which was modified for fibular osteotomy. Comparison of outcomes between the 2 groups was performed using the Student t test for continuous variables and the Mann-Whitney U test or Fisher exact test for discrete variables. Results: Around the fibular osteotomy site, no perioperative complications were found in either group. The radiographic union score was significantly higher in the AO/L group than in the AO group (P < .0001 at 2, 3, and 6 months; P = .0290 at 12 and 24 months). The union rate at the fibular osteotomy site was significantly higher in the AO/L group (97.6%) than in the AO group (82.9%) at 12 months (P = .0290). Conclusion: The AO/L procedure significantly accelerated the formation of bridging callus at the fibular osteotomy site and provided a significantly higher union rate compared with the AO procedure. Both AO/L and AO procedures were free from perioperative complications. These results suggest that the AO/L procedure is clinically useful as an osteotomy procedure to shorten the fibula in LCW-HTO.

Optical coherence tomography evaluation of the spatiotemporal effects of 3D bone marrow stromal cell culture using a bioreactor.

Performing cell culture in a three-dimensional (3D) environment has various advantages. In cartilage tissue engineering, 3D in vitro cultures utilizing biomaterials and bioreactors can mimic the biological environment. However, the biggest drawback of these 3D culture systems is a limited ability to evaluate 3D cell distribution. Optical coherence tomography (OCT) has recently been used to evaluate 3D cellular morphology and structure in a timely manner. Here, we showed that OCT could be used to visually assess the distribution and the morphology of bone marrow stromal cells under chondrogenic 3D cultivation using alginate gels and rotary culture. In particular, OCT was able to visualize living cells embedded in alginate gels in a non-destructive and 3D manner, as well as quantitatively evaluate cell distribution and spheroid volume. We also found that cells were centralized in rotary culture but peripherally distributed in static culture, while rotary culture enhanced the hypertrophy of marrow stromal cells (MSCs) embedded in alginate gels. Together, our findings demonstrate that OCT can be used to evaluate the spatiotemporal effects of 3D cultivation using alginate gels and rotary culture. Therefore, this method may allow the observation of pre-cultured tissue over time and the optimization of culture conditions for regenerative tissue engineering.

Bottom-up physiologically based pharmacokinetic modelling for predicting the human pharmacokinetic profiles of the ester prodrug MGS0274 and its active metabolite MGS0008, a metabotropic glutamate 2/3 receptor agonist.

For ester prodrugs that are used to improve the gastrointestinal absorption of highly hydrophilic, pharmacologically active substances, it is challenging to predict the human pharmacokinetics (PK) of the prodrugs and their parent compounds using only preclinical data.This research was aimed at constructing a PBPK model for predicting the human PK of the ester prodrug MGS0274 and its parent compound MGS0008 after a single oral administration of MGS0274 besylate.First, we identified carboxylesterase 1 (CES1) as the major enzyme involved in the hydrolysis of MGS0274. Second, we constructed a new compartment model to estimate the passive diffusion clearance (CLpd) of MGS0008, a critical parameter for predicting the PK of highly hydrophilic compounds, based on in vivo monkey PK data. Finally, we constructed a permeability-limited liver PBPK model incorporating the CLpd assumed to be the same in humans.We confirmed that our method reliably predicted the human PK and that the estimated CLpd was comparable to that calculated retrospectively using the PBPK model, suggesting that the methodology for estimating the CLpd was valid.Our proposed methodology is expected to be helpful for human PK prediction of ester prodrugs hydrolysed by CES1 and their hydrophilic parent compounds even during the preclinical phase.

Extrusion of the medial meniscus is a potential predisposing factor for post-arthroscopy osteonecrosis of the knee.

BACKGROUND: Post-arthroscopic osteonecrosis of the knee (PAONK) is a rare condition. No studies have analyzed the relationship between the meniscus extrusion and PAONK. The purpose of this retrospective study is to test a hypothesis that the degree of the medial meniscus (MM) extrusion might be significantly greater in the knees with PAONK than in the matched control knees both before and after the meniscectomy. METHODS: Ten knees with PAONK were detected out of a total of 876 knees which had undergone arthroscopic partial meniscectomy of the MM. Ten matched control knees were randomly selected out of the remaining 866 knees without PAONK. The clinical data of these 20 patients were retrospectively collected from the medical records. To evaluate the location of the menisci on the joint line, Extrusion width and Inner width were defined on a coronal section of magnetic resonance imaging (MRI). The intra- and inter-rater reliability was evaluated by calculating the intra- and inter-class coefficients. Statistical comparisons between the 2 groups were made using the 3 non-parametric tests. RESULTS: Before the meniscectomy, the Extrusion width of the MM (mean 4.7 ± 1.4 mm) was significantly greater than that (3.0 ± 1.3 mm) in the Control group (P = 0.0195). In the MRI taken in a range from 3 to 50 weeks after the meniscectomy, the Extrusion width of the MM (5.9 ± 1.1 mm) in the PAONK group was significantly greater than that (3.4 ± 1.4 mm) in the Control group (P = 0.0009), and the Inner width of the MM (0.6 ± 1.7 mm) in the PAONK group was significantly less than that (3.9 ± 1.0 mm) in the Control group (P = 0.0001). CONCLUSION: A significant relationship was found between the degree of the MM extrusion and the onset of PAONK. This study suggested that the extrusion of the MM is a potential predisposing factor for PAONK.

Guaiazulene derivative 1,2,3,4-tetrahydroazuleno[1,2-b] tropone reduces the production of ATP by inhibiting electron transfer complex II.

Molecularly targeted therapy has been used for treatment of various types of cancer. However, cancer cells often acquire resistance to molecularly targeted drugs that inhibit specific molecular abnormalities, such as constitutive activation of kinases. Even in cancer cells that have acquired resistance, enhanced anabolism, including the synthesis of nucleotides, amino acids and lipids, is common to normal cancer cells. Therefore, there is a renewed interest in effectively eliminating cancer cells by specifically targeting their abnormal energy metabolism. Multiple strategies are currently being developed for mitochondrial-targeted cancer therapy, with agents targeting oxidative phosphorylation, glycolysis, the tricarboxylic acid cycle and apoptosis. In this study, we found that one of the guaiazulene derivatives, namely, 1,2,3,4-tetrahydroazuleno[1,2-b] tropone (TAT), inhibited the proliferation of cancer cell lines stronger than that of normal cells. In addition, we showed that TAT inhibited energy production in cancer cell lines, resulting in apoptosis. Analyses done in cancer cell lines and in the animal model Caenorhabditis elegans suggested that TAT acts on the mitochondrial electron transfer complex II and suppresses cellular energy production by inhibiting oxidative phosphorylation across species. These results suggest that TAT could represent a novel anticancer agent that selectively targets mitochondria.

Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

Discovery of MGS0274, an ester prodrug of a metabotropic glutamate receptor 2/3 agonist with improved oral bioavailability.

We previously reported that MGS0008 is a selective group II metabotropic glutamate receptor (mGlu2/3 receptor) agonist that is effective in animal models of schizophrenia. MGS0008 is a highly hydrophilic glutamate analog and is therefore expected to show low oral bioavailability in humans. To improve the oral bioavailability of MGS0008, ester prodrugs of MGS0008 were synthesized and their usefulness was evaluated. Among the prodrugs, the l-menthol-ester prodrug 4h demonstrated preferable lipophilicity, good chemical stability, and a high conversion rate to MGS0008 in human and monkey liver microsomes. A pharmacokinetic study in monkeys revealed that the oral bioavailability of MGS0008 after oral dosing of compound 4h was approximately 15-fold higher than that after oral dosing of MGS0008. Based on these findings, a diastereomer of compound 4h (compound 4j, or MGS0274), was selected as a candidate for clinical drug development, and its besylate is currently under development for the treatment of schizophrenia (Development code: TS-134).

Chronic stressors, stress coping and depressive tendencies among older adults.

AIMS: This study aimed to clarify the association between chronic stressors, stress coping and depressive tendencies among older adults. METHODS: We carried out a survey with 500 participants aged in their 70s, who were randomly selected from the basic resident register. Of these, 304 residents responded to the survey (60.8%). RESULTS: Findings from a factor analysis and cluster analysis revealed five groups of chronic stressors: physical, economic and interpersonal stressor; physical and economic stressor; interpersonal stressor; physical stressor; and low stressor. The factor analysis revealed four factors of coping: cognitive coping, support-seeking, positive problem solving and escape-avoidance. Furthermore, a multiple logistic regression analysis showed that both physical, economic and interpersonal stressor, and physical and economic stressor had negative associations with depressive tendencies. With respect to stress coping, positive problem solving had a positive association with depressive tendencies, whereas escape-avoidance had a negative association with depressive tendencies. CONCLUSIONS: For older individuals, the more complex the overlapping chronic stressors that are experienced, the higher the depressive tendencies. Furthermore, although older individuals selectively use various coping strategies, they do not alleviate the stress responses induced by chronic stressors. Geriatr Gerontol Int 2020; 20: 297-303.

Haemoglobin(ßK120C)-albumin trimer as an artificial O2 carrier with sufficient haemoglobin allostery.

The allosteric O2 release of haemoglobin (Hb) allows for efficient O2 delivery from the lungs to the tissues. However, allostery is weakened in Hb-based O2 carriers because the chemical modifications of the Lys- and Cys-ß93 residues prevent the quaternary transition of Hb. In this paper, we describe the synthesis and O2 binding properties of a recombinant Hb [rHb(ßK120C)]-albumin heterotrimer that maintains sufficient Hb allostery. The rHb(ßK120C) core, with two additional cysteine residues at the symmetrical positions on its protein surface, was expressed using yeast cells. The mutations did not influence either the O2 binding characteristics or the quaternary transition of Hb. Maleimide-activated human serum albumins (HSAs) were coupled with rHb(ßK120C) at the two Cys-ß120 positions, yielding the rHb(ßK120C)-HSA2 trimer, in which the Cys-ß93 residues were unreacted. Molecular dynamics simulation demonstrated that the HSA moiety does not interact with the amino acid residues around the haem pockets and the α1ß2 surfaces of the rHb(ßK120C) core, the alteration of which retards Hb allostery. Circular dichroism spectroscopy demonstrated that the quaternary transition between the relaxed (R) state and the tense (T) state of the Hb core occurred upon both the association and dissociation of O2. In phosphate-buffered saline solution (pH 7.4) at 37 °C, the rHb(ßK120C)-HSA2 trimer exhibited a sigmoidal O2 equilibrium curve with the O2 affinity and cooperativity identical to those of native Hb (p 50 = 12 Torr, n = 2.4). Moreover, we observed an equal Bohr effect and 2,3-diphosphoglycerate response in the rHb(ßK120C)-HSA2 trimer compared with naked Hb.

Preclinical disposition of MGS0274 besylate, a prodrug of a potent group II metabotropic glutamate receptor agonist MGS0008 for the treatment of schizophrenia.

MGS0274 besylate is an ester-based lipophilic prodrug of a metabotropic glutamate (mGlu) 2 and mGlu3 receptor agonist MGS0008 and being developed for the treatment of schizophrenia. We investigated the disposition of these compounds in rats and monkeys and in vitro metabolism in humans to evaluate whether MGS0274 besylate could be useful as a prodrug in humans. After the oral administration of MGS0274 besylate to monkeys (2.89 mg/kg), MGS0008 was immediately found in plasma, reached a maximum concentration at 4 hours postdose, and decreased with a terminal half-life of 16.7 hours; MGS0274 was barely detectable. The oral bioavailability as MGS0008 was 83.7%, which was approximately 20-fold greater than that after oral dosing of MGS0008 (3.8%). In rats, MGS0008 penetrated the cerebrospinal fluid and was eliminated slower than from plasma. The in vitro metabolism study indicated that MGS0274 was rapidly hydrolyzed to MGS0008, which was not further metabolized. After the intravenous administration of MGS0008 to rats and monkeys, almost all the dose was excreted unchanged in urine. These results suggested that MGS0274 was, as expected, presystemically hydrolyzed to MGS0008 after gastrointestinal absorption and that MGS0008 was distributed throughout the body without further metabolism and ultimately excreted in urine in the animals. Furthermore, the hydrolytic activity against MGS0274 in the human liver S9 fraction was comparable to that in monkeys, suggesting the possibility of the rapid presystemic hydrolysis of MGS0274 to MGS0008 in humans, as it is in monkeys. Consequently, MGS0274 besylate is expected to function as a preferable prodrug in humans.