Prognostic impact of radiological tumor burden in patients with metastatic urothelial carcinoma treated with pembrolizumab.

PURPOSE: Radiological tumor burden has been reported to be prognostic in many malignancies in the immunotherapy era, yet whether it is prognostic in patients with metastatic urothelial carcinoma (mUC) treated with pembrolizumab remains uninvestigated. We sought to assess the predictive and prognostic value of radiological tumor burden in patients with mUC. METHODS: We performed a retrospective analysis of 308 patients with mUC treated with pembrolizumab. Radiological tumor burden was represented by baseline tumor size (BTS) and baseline tumor number (BTN). Optimal cut-off value of BTS was determined as 50 mm using the Youden index (small BTS: n = 194, large BTS: n = 114). Overall (OS), cancer-specific (CSS), progression-free survival (PFS), and objective response rate (ORR) were compared. Non-linear associations between BTS and OS and CSS were evaluated using restricted cubic splines. RESULTS: Patients with large BTS were less likely to have undergone the surgical resection of the primary tumor (P = 0.01), and more likely to have liver metastasis (P < 0.001) and more metastatic lesions (P < 0.001). On multivariable analyses controlling for the effects of confounders (resection of primary tumor, metastatic site, number of metastases and lactate dehydrogenase level), large BTS and high BTN were independently associated with worse OS (HR 1.52; P = 0.015, and HR 1.69; P = 0.018, respectively) and CSS (HR 1.59; P = 0.01, and HR 1.66; P = 0.031, respectively), but not PFS. Restricted cubic splines revealed BTS was correlated with OS and CSS in linear relationships. Additionally, large BTS was significantly predictive of lower ORR and complete response rate on univariable analyses (P = 0.041 and P = 0.032, respectively), but its association disappeared on multivariable analyses. CONCLUSION: Radiological tumor burden has independent prognostic value with a linear relationship in pembrolizumab-treated patients with mUC and might help drive the earlier introduction of second-line pembrolizumab and/or switching to subsequent therapies.

Discovery of novel N-(1-benzyl-1H-imidazol-2-yl)amide derivatives as melanocortin 1 receptor agonists.

Melanocortin-1 receptor (MC1R) is primarily activated by α-melanocyte-stimulating hormone (α-MSH) and plays a crucial role, such as keeping homeostasis in the skin against melanogenesis and external stimuli, anti-inflammatory effects, and tissue fibrosis suppression. Afamelanotide, an α-MSH analog MC1R agonist, is clinically used for treating erythroblastic protoporphyria (EPP) by subcutaneous implantation administration. Therefore, we initiated an investigation aimed at orally available small molecule nonpeptide MC1R agonists. Optimization from the internal hit compound 6a finally resulted in the discovery of N-(1-benzyl-1H-imidazol-2-yl)amide derivative 9g bearing isonipecotinic acid moiety, which demonstrated good MC1R agonistic activity and metabolic stability.

Health impact assessment of pet cats caused by organohalogen contaminants by serum metabolomics and thyroid hormone analysis.

Companion animals are in close contact with the human surroundings, and there is growing concern about the effects of harmful substances on the health of pet cats. In this study, we investigated the potential health effects of organohalogen compounds (OHCs) on thyroid hormone (TH) homeostasis and metabolomics in Japanese pet cats. There was a significant negative correlation between concentrations of several contaminants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), hydroxylated PCBs (OH-PCBs), hydroxylated PBDEs (OH-PBDEs), and THs in cat serum samples. These results suggested that exposure to OHCs causes a decrease in serum TH levels in pet cats. In this metabolomics study, each exposure level of parent compounds (PCBs and PBDEs) and their hydroxylated compounds (OH-PCBs and OH-PBDEs) were associated with their own unique primary metabolic pathways, suggesting that parent and phenolic compounds exhibit different mechanisms of action and biological effects. PCBs were associated with many metabolic pathways, including glutathione and purine metabolism, and the effects were replicated in in-vivo cat PCB administration studies. These results demonstrated that OHC exposure causes chronic oxidative stress in pet cats. PBDEs were positively associated with alanine, aspartate, and glutamate metabolism. Due to the chronic exposure of cats to mixtures of these contaminants, the combination of their respective metabolic pathways may have a synergistic effect.

Discovery of Apararenone (MT-3995) as a Highly Selective, Potent, and Novel Nonsteroidal Mineralocorticoid Receptor Antagonist.

Overactivation of the mineralocorticoid receptor (MR) is involved in many diseases, such as hypertension, kidney disease, and heart failure. Thus, MR antagonists (MRAs) are expected to be beneficial to patients with these diseases. In order to identify novel nonsteroidal MRAs that overcome the issues of already marketed steroidal MRAs, we searched for new compounds guided by our hypothesis that T-shaped compounds with a hydrophobic core structure, two polar functional groups at both extremities able to interact with MR, and a bulky substituent that can interfere with the folding of the C-terminal helix 12 may exhibit antagonist activity toward MR. We discovered that the novel 1,4-benzoxazin-3-one derivative 19 (apararenone: MT-3995) acted as a highly selective and potent nonsteroidal MRA. Apararenone exhibited a more potent antihypertensive and organ-protective activity than steroidal MRA eplerenone in a primary aldosteronism rat model obtained by infusing aldosterone in uninephrectomized rats.

Pituitary adenylate cyclase-activating polypeptide promotes cutaneous dendritic cell functions in contact hypersensitivity.

BACKGROUND: Sensory nerves regulate cutaneous local inflammation indirectly through induction of pruritus and directly by acting on local immune cells. The underlying mechanisms for how sensory nerves influence cutaneous acquired immune responses remain to be clarified. OBJECTIVE: This study aimed to explore the effect of peripheral nerves on cutaneous immune cells in cutaneous acquired immune responses. METHODS: We analyzed contact hypersensitivity (CHS) responses as a murine model of delayed-type hypersensitivity in absence or presence of resiniferatoxin-induced sensory nerve denervation. We conducted ear thickness measurements, flow cytometric analyses, and mRNA expression analyses in CHS. RESULTS: CHS responses were attenuated in mice that were denervated during the sensitization phase of CHS. By screening neuropeptides, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA expression was decreased in the dorsal root ganglia after denervation. Administration of PACAP restored attenuated CHS response in resiniferatoxin-treated mice, and pharmacological inhibition of PACAP suppressed CHS. Flow cytometric analysis of skin-draining lymph nodes showed that cutaneous dendritic cell migration and maturation were reduced in both denervated mice and PACAP antagonist-treated mice. The expression of chemokine receptors CCR7 and CXCR4 of dendritic cell s was enhanced by addition of PACAP in vitro. CONCLUSION: These findings indicate that a neuropeptide PACAP promotes the development of CHS responses by inducing cutaneous dendritic cell functions during the sensitization phase.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits significant anti-inflammatory effect while maintaining bone mineral density in mice.

Classic glucocorticoids have been prescribed for various inflammatory diseases, such as rheumatoid arthritis, due to their outstanding anti-inflammatory effects. However, glucocorticoids cause numerous unwanted side effects, including osteoporosis and diabetes. Hence, selective glucocorticoid receptor modulators (SGRMs), which retain anti-inflammatory effects with minimized side effects, are among the most anticipated drugs in the clinical field. The assumption is that there are two major mechanisms of action via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, anti-inflammatory effects of glucocorticoids are largely due to TR, while the side effects associated with glucocorticoids are mostly mediated through TA. We previously reported that JTP-117968, a novel SGRM, maintained partial TR activity while remarkably reducing the TA activity. In this study, we investigated the anti-inflammatory effect of JTP-117968 on a lipopolysaccharide (LPS) challenge model and collagen-induced arthritis (CIA) model in mice. Meanwhile, we tested the effect of JTP-117968 on the bone mineral density (BMD) in mouse femur to evaluate the side effect. Based on the evaluation, JTP-117968 reduced the plasma levels of tumor necrosis factor α induced by LPS challenge in mice significantly. Remarkably, CIA development was suppressed by JTP-117968 comparably with prednisolone and PF-802, an active form of fosdagrocorat that has been developed clinically as an orally available SGRM. Strikingly, the side effect of JTP-117968 on mouse femoral BMD was much lower than those of PF-802 and prednisolone. Therefore, JTP-117968 has attractive potential as a new therapeutic option against inflammatory diseases with minimized side effects compared to classic glucocorticoids.

Transient elevation of cytoplasmic calcium ion concentration at a single cell level precedes morphological changes of epidermal keratinocytes during cornification.

Epidermal keratinocytes achieve sequential differentiation from basal to granular layers, and undergo a specific programmed cell death, cornification, to form an indispensable barrier of the body. Although elevation of the cytoplasmic calcium ion concentration ([Ca2+]i) is one of the factors predicted to regulate cornification, the dynamics of [Ca2+]i in epidermal keratinocytes is largely unknown. Here using intravital imaging, we captured the dynamics of [Ca2+]i in mouse skin. [Ca2+]i was elevated in basal cells on the second time scale in three spatiotemporally distinct patterns. The transient elevation of [Ca2+]i also occurred at the most apical granular layer at a single cell level, and lasted for approximately 40 min. The transient elevation of [Ca2+]i at the granular layer was followed by cornification, which was completed within 10 min. This study demonstrates the tightly regulated elevation of [Ca2+]i preceding the cornification of epidermal keratinocytes, providing possible clues to the mechanisms of cornification.

Determination of free thyroid hormones in animal serum/plasma using ultrafiltration in combination with ultra-fast liquid chromatography-tandem mass spectrometry.

Thyroid hormones (THs), which mainly consist of 3, 3', 5-triiodo-l-thyronine (T3) and L-thyroxine (T4), play a critical role in regulating biological processes such as growth and metabolism in various animal species. Thus, accurate measurement of T3 and T4, especially physiologically active free (protein-unbound) forms, in serum/plasma is needed for the evaluation of TH homeostasis. However, such high-precision determination of free THs is lacking for non-human species. The present study aimed to develop a highly sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of six free THs in serum/plasma, which is applicable to not only humans but also non-human species. Two different physical separation steps, ultrafiltration (UF) and equilibrium dialysis (ED), were examined to obtain the free TH fraction. Several experimental conditions were carefully optimized and validated for UF or ED using the commercially available bovine serum. As a result, UF at 1100 × g and 37 °C for 30 min with a 30 kDa ultrafiltration device (Centrifree YM-30, Millipore) yielded excellent precision (CV: <10%). The optimized ED step also yielded high precision (CV: <10%) and the measurement values were approximately equal to those of UF, but at least 16 h were required to reach equilibrium. Thus, UF combined with LC-MS/MS was finally chosen, in terms of the time needed for the measurement. Acceptable accuracy (recovery: 70%-110%) and intra- and inter-day precision (CV: <10% and <12%, respectively) were obtained, when triplicate analyses in three different days were conducted using the bovine serum. The developed analytical method was successfully applied to the determination of free THs in serum/plasma samples of humans, cats, and dogs. Furthermore, comparison with free T4 concentrations measured by a common immunoassay method evidently indicated that the ultrafiltration-LC-MS/MS method developed in this study can increase the specificity and accuracy of TH measurement.

A novel JAK inhibitor JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents.

Janus kinases (JAKs) are required for several inflammatory cytokine signalling pathways and are implicated in the pathogenesis of chronic dermatitis, including atopic dermatitis and psoriasis. JAK inhibitors are therefore promising therapeutic candidates for chronic dermatitis. In this study, we evaluated the effects of the novel JAK inhibitor JTE-052 on inflammatory responses associated with chronic dermatitis, and compared its profile with those of conventional therapeutic agents in rodent models of chronic dermatitis. JTE-052 inhibited the Th1-, Th2- and Th17-type inflammatory responses of human T cells and mast cells in vitro. Oral administration of JTE-052 inhibited skin inflammation in hapten-induced chronic dermatitis in mice, associated with reduced levels of inflammatory cytokines in the skin and immunoglobulin (Ig) E in serum. In contrast, although ciclosporin partly inhibited skin inflammation, it did not reduce interleukin (IL)-4 production in skin, and enhanced IgE production in serum. Oral administration of JTE-052 also inhibited skin inflammation in mouse models of atopic dermatitis and psoriasis induced by a mite extract, thymic stromal lymphopoietin or IL-23. The maximal efficacy of JTE-052 in these dermatitis models was superior to the conventional therapeutic agents, ciclosporin and methotrexate. Topical application of JTE-052 ointment ameliorated hapten-induced chronic dermatitis in rats more effectively than tacrolimus ointment. Furthermore, JTE-052 ointment did not cause the thinning of normal skin associated with topical corticosteroids. These results indicate that JTE-052 is a promising candidate as an anti-inflammatory drug for various types of chronic dermatitis, with a distinctly different profile from conventional therapy following either oral or topical application.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits improved transrepression/transactivation dissociation.

Classic glucocorticoids that have outstanding anti-inflammatory effects are still widely prescribed for the treatment of various inflammatory and autoimmune diseases. Conversely, glucocorticoids cause numerous unwanted side effects, particularly systemically dosed glucocorticoids. Therefore, selective glucocorticoid receptor modulator (SGRM), which maintains beneficial anti-inflammatory effects while reducing the occurrence of side effects, is one of the most anticipated drugs. However, there have been no SGRMs marketed to date. The assumption is that there are two major mechanisms of action of glucocorticoids via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, the anti-inflammatory effects of glucocorticoids are mostly mediated through TR, while the side effects associated with glucocorticoids are largely caused by TA. We started to evaluate novel orally available SGRMs that maintain anti-inflammatory effects while minimizing adverse effects by favoring TR over TA. Based on this evaluation, we discovered JTP-117968, (4b'S,7'R,8a'S)-4b'-benzyl-7'-hydroxy-N-(2-methylpyridin-3-yl)-7'-(trifluoromethyl)-4b',6',7',8',8a',10'-hexahydro-5'H-spiro[cyclopropane-1,9'-phenanthrene]-2'-carboxamide, a non-steroidal SGRM. JTP-117968 has partial TR activity, but exhibits extremely low TA activity. The maximum TR efficacy of JTP-117968 was comparable to its structural analogue, PF-802, (4bS,7R,8aR)-4b-Benzyl-7-hydroxy-N-(2-methylpyridin-3-yl)-7-(trifluoromethyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide, which is the active form of Fosdagrocorat that has been developed clinically as a first-in-class orally available SGRM. Remarkably, the TA activity of JTP-117968 was much weaker than PF-802 not only in in vitro assays, but also in in vivo mice experiments. These findings indicate that JTP-117968 exhibits improved TR/TA dissociation because the compound has significantly lower TA activity compared with an already reported SGRM. Therefore, JTP-117968 is expected to be a useful compound for evaluating ideal SGRMs in the future.

JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation.

BACKGROUND: Using JAK inhibitors to inhibit cytokine signaling is presumed to be a possible means of treating skin inflammatory disorders such as contact dermatitis. OBJECTIVE: To clarify the action site of JAK inhibitors in skin inflammatory disorders. METHODS: We analyzed the mechanism of action of the JAK inhibitor JTE-052 using murine skin inflammation models, including contact hypersensitivity (CHS) and irritant contact dermatitis. Cells isolated from ear tissue or lymph node (LN) were analyzed by flow cytometry. The amounts of cytokines in the culture medium were measured by ELISA or bead array system. Proliferation of LN cells was evaluated by measurement of tritiated thymidine incorporation. RESULTS: Oral administration of JTE-052 during both sensitization and elicitation phase attenuated CHS, but did not affect croton oil-induced irritant contact dermatitis. JTE-052 potently inhibited T cell proliferation and activation by antigen presentation in vitro, and attenuated skin inflammation in a sensitized-lymphocyte transfer model without suppressing T cell migration. JTE-052 did not affect hapten-induced cutaneous dendritic cell migration into draining lymph nodes or their costimulatory molecule expressions. CONCLUSION: The JAK inhibitor JTE-052 exerts an inhibitory effect on antigen-specific T cell activation and subsequent inflammation in acquired skin immunity, such as CHS.

Quantitative characterization of highly efficient correlated photon-pair source using biexciton resonance.

A high efficiency method for the generation of correlated photon pairs accompanied by reliable means to characterize the efficiency of that process is needed in the study of entangled states, which have important potential applications in quantum information and quantum communication. In this study, we report the first characterization of the efficiency of generation of correlated photon pairs emitted from a CuCl single crystal using the biexciton-resonance hyper-parametric scattering (RHPS) method which is the highly efficient method of generation of correlated photon pairs. In order to characterize the generation efficiency and signal-to-noise ratio of correlated photon pairs using this method, we investigated the pump power dependence on the photon counting rate and coincidence counting rate under resonant excitation. The pump power dependence shows that the power characteristic of the photon counting rates changes from linear to quadratic dependence of the pump power. This behavior represents a superposition of contributions from correlated photon pairs and non-correlated photons. The analysis of the pump power dependence shows that one photon-pair is produced by a pump pulse with 2 x 106 photons. Moreover, the generation efficiency of this method obtained by calculating the number of generated photon pairs per pump power is comparable to that of several methods based on the χ(3) parametric process.

Wave Propagation Direction and c-Axis Tilt Angle Influence on the Performance of ScAlN/Sapphire-Based SAW Devices.

Some previously reported surface acoustic wave (SAW) devices using bulk piezoelectric substrates showed higher acoustic power radiated in either forward or backward wave propagation direction depending on their crystal orientations and are called natural single-phase unidirectional transducers (NSPUDT). While these reports were based on bulk piezoelectric substrates, we report directionality in the c-axis tilted 44% scandium doped aluminum nitride thin piezoelectric film-based SAW devices on sapphire. It is worth noting that our observance of directionality is specifically in Sezawa mode. We produced a c-axis tilt up to 5.5° over the single wafer and examined the directionality by comparing the forward and backward insertion loss utilizing split finger electrodes as a receiver. The wave propagation direction and c-axis tilt angle influence on the performance of SAW devices is evaluated. Furthermore, return loss and insertion loss data are presented for various SAW propagation directions and c-axis tilt angles. Finally, the comparison for both acoustic modes, i.e., Rayleigh and Sezawa, is reported.

Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase.

Choline is an essential nutrient for all living cells and is produced extracellularly by sequential degradation of phosphatidylcholine (PC). However, little is known about how choline is produced extracellularly. Here, we report that ENPP6, a choline-specific phosphodiesterase, hydrolyzes glycerophosphocholine (GPC), a degradation product of PC, as a physiological substrate and participates in choline metabolism. ENPP6 is highly expressed in liver sinusoidal endothelial cells and developing oligodendrocytes, which actively incorporate choline and synthesize PC. ENPP6-deficient mice exhibited fatty liver and hypomyelination, well known choline-deficient phenotypes. The choline moiety of GPC was incorporated into PC in an ENPP6-dependent manner both in vivo and in vitro. The crystal structure of ENPP6 in complex with phosphocholine revealed that the choline moiety of the phosphocholine is recognized by a choline-binding pocket formed by conserved aromatic and acidic residues. The present study provides the molecular basis for ENPP6-mediated choline metabolism at atomic, cellular and tissue levels.

The Janus kinase inhibitor JTE-052 improves skin barrier function through suppressing signal transducer and activator of transcription 3 signaling.

BACKGROUND: Barrier disruption and the resulting continuous exposure to allergens are presumed to be responsible for the development of atopic dermatitis (AD). However, the mechanism through which skin barrier function is disrupted in patients with AD remains unclear. OBJECTIVES: Taking into account the fact that the TH2 milieu impairs keratinocyte terminal differentiation, we sought to clarify our hypothesis that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a critical role in skin barrier function and can be a therapeutic target for AD. METHODS: We analyzed the mechanism of keratinocyte differentiation using a microarray and small interfering RNA targeting STATs. We studied the effect of the JAK inhibitor JTE-052 on keratinocyte differentiation using the human skin equivalent model and normal human epidermal keratinocytes. We applied topical JAK inhibitor onto NC/Nga mice, dry skin model mice, and human skin grafted to immunocompromised mice. RESULTS: IL-4 and IL-13 downregulated genes involved in keratinocyte differentiation. STAT3 and STAT6 are involved in keratinocyte differentiation and chemokine production by keratinocytes, respectively. Topical application of the JAK inhibitor suppressed STAT3 activation and improved skin barrier function, permitting increases in levels of terminal differentiation proteins, such as filaggrin, and natural moisturizing factors in models of AD and dry skin and in human skin. CONCLUSION: STAT3 signaling is a key element that regulates keratinocyte differentiation. The JAK inhibitor can be a new therapeutic tool for the treatment of disrupted barrier function in patients with AD.