Ultra-high-field pharmacological functional MRI of dopamine D1 receptor-related interventions in anesthetized rats.

The dopamine D1 receptor (D1R) is associated with schizophrenia, Parkinson's disease, and attention deficit hyperactivity disorder. Although the receptor is considered a therapeutic target for these diseases, its neurophysiological function has not been fully elucidated. Pharmacological functional MRI (phfMRI) has been used to evaluate regional brain hemodynamic changes induced by neurovascular coupling resulting from pharmacological interventions, thus phfMRI studies can be used to help understand the neurophysiological function of specific receptors. Herein, the blood oxygenation level-dependent (BOLD) signal changes associated with D1R action in anesthetized rats was investigated by using a preclinical ultra-high-field 11.7-T MRI scanner. PhfMRI was performed before and after administration of the D1-like receptor agonist (SKF82958), antagonist (SCH39166), or physiological saline subcutaneously. Compared to saline, the D1-agonist induced a BOLD signal increase in the striatum, thalamus, prefrontal cortex, and cerebellum. At the same time, the D1-antagonist reduced the BOLD signal in the striatum, thalamus, and cerebellum by evaluating temporal profiles. PhfMRI detected D1R-related BOLD signal changes in the brain regions associated with high expression of D1R. We also measured the early expression of c-fos at the mRNA level to evaluate the effects of SKF82958 and isoflurane anesthesia on neuronal activity. Regardless of the presence of isoflurane anesthesia, c-fos expression level was increased in the region where positive BOLD responses were observed with administration of SKF82958. These findings demonstrated that phfMRI could be used to identify the effects of direct D1 blockade on physiological brain functions and also for neurophysiological assessment of dopamine receptor functions in living animals.

Application of Zwitterionic Polymer Hydrogels to Optical Tissue Clearing for 3D Fluorescence Imaging.

Zwitterionic polymers have both anion and cation groups in the side chain and have been used in various biomedical applications because of the unique properties. In this study, zwitterionic polymer hydrogels are applied to optical tissue clearing for 3D fluorescence imaging. Polyacrylamide hydrogels have been employed in Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tissue-hYdrogel method. Zwitterionic polymer hydrogels are produced using zwitterionic monomers, such as 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate (DAPS) and 2-methacryloyloxyethyl phosphorylcholine (MPC), and crosslinkers. The hydrogels made from poly(DAPS-co-acrylamide) and MPC homopolymers afford the most transparent tumor tissues. However, the tissues cleared using DAPS copolymers-containing hydrogels became turbid in a refractive index-matching solution, which are unable to obtain clear 3D fluorescence images. In contrast, the 3D fluorescence imaging is achieved in the MPC polymer-treated 2-mm-thick brain slices after immunostaining. The 3D fluorescence imaging of lung metastasis that is cleared by the MPC hydrogel to demonstrate the possible application to cancer diagnosis is performed. The results indicate the increased potentials of zwitterionic polymer hydrogels, especially MPC polymer hydrogels, in biomedical applications.

A simple scoring system using type IV collagen 7S and aspartate aminotransferase for diagnosing nonalcoholic steatohepatitis and related fibrosis.

BACKGROUND: Recently we reported novel noninvasive scoring systems for diagnosing nonalcoholic steatohepatitis (NASH) and related fibrosis, namely FM-NASH index and FM-fibro index. They are highly accurate, however, they contain some items not widely used in clinical practice and require six or more items to diagnose both NASH and related fibrosis. By focusing on widely used items, we tried to identify convenient markers in common with the both diagnoses. METHODS: To explore the markers for NASH and related fibrosis in nonalcoholic fatty liver disease (NAFLD) patients, we used data of 24 clinical items in our previous report. By logistic regression analysis, we identified items suitable for the both diagnoses. We then evaluated their accuracies by area under the receiver operator characteristic curves (AUROCs) on independent validation data. RESULTS: We identified the combination of type IV collagen 7S and aspartate aminotransferase (AST) as the predictor both for NASH and related fibrosis. We developed a scoring system based on the combination and evaluated the prediction accuracy: the AUROCs for training/validation data sets are 0.857/0.769 for NASH and 0.918/0.842 for NASH-related fibrosis. The former was higher than that of NAFIC score, and the latter was higher than those of existing fibrosis markers: BARD score, FIB-4 index and NAFLD fibrosis score but lower than FM-fibro index. CONCLUSIONS: The scoring system using type IV collagen 7S and AST named CA index can predict both NASH and related fibrosis in NAFLD patients with sufficient accuracy and could be a convenient diagnostic and screening tool for NASH and related fibrosis. The scoring system needs to be validated in independent larger populations from multiple clinical centers.

Identification of novel noninvasive markers for diagnosing nonalcoholic steatohepatitis and related fibrosis by data mining.

UNLABELLED: It is important that patients with nonalcoholic steatohepatitis (NASH) are diagnosed and treated early to prevent serious complications, such as liver cirrhosis or hepatocellular carcinoma. However, current methods for NASH diagnosis are invasive given that they rely on liver biopsy, making early diagnosis difficult. In this study, we developed novel noninvasive markers for the diagnosis of NASH and NASH-related fibrosis. A total of 132 Japanese patients with nonalcoholic fatty liver disease were included in this study. Blood samples were collected, and 261 biomolecules were quantified in serum. Using cluster and pathway analyses, we identified biomolecule modules connected to biological events that occur with disease progression to NASH. The modules were used as variables for diagnosis, leading to a NASH diagnostic marker associated with two biological events, that is, protective response to hepatic steatosis and hepatitis-causing innate immune response. Regarding the NASH-related fibrosis marker, immunological responses to hepatocyte injury were identified as a biological event. To develop diagnostic markers for NASH and NASH-related fibrosis, specific biomolecules were selected from each biomolecule module. The former marker was obtained by averaging the levels of four biomolecules, whereas the latter was obtained by averaging the levels of two biomolecules. Both markers achieved a diagnostic accuracy of almost 0.9 of the area under the receiver operating characteristic curve, and the latter exhibited equivalent performance in an independent group of 62 prospectively recruited patients. CONCLUSION: We developed highly accurate markers for the diagnosis of both NASH and NASH-related fibrosis (i.e., FM-NASH index and FM-fibro index, respectively). These markers may be used as an alternative diagnostic tool to liver biopsy.

New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide.

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter ß-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide.

Effect of dopamine D4 receptor agonists on sleep architecture in rats.

Dopamine plays a key role in the regulation of sleep-wake states, as revealed by the observation that dopamine-releasing agents such as methylphenidate have wake-promoting effects. However, the precise mechanisms for the wake-promoting effect produced by the enhancement of dopamine transmission are not fully understood. Although dopamine D1, D2, and D3 receptors are known to have differential effects on sleep architecture, the role of D4 receptors (D4Rs), and particularly the influence of D4R activation on the sleep-wake state, has not been studied so far. In this study, we investigated for the first time the effects of two structurally different D4R agonists, Ro 10-5824 and A-412997, on the sleep-wake states in rats. We found that both D4R agonists generally increased waking duration, and conversely, reduced non-rapid eye movement (NREM) sleep duration in rats. The onset of NREM sleep was also generally delayed. However, only the A-412997 agonist (but not the Ro 10-5824) influenced rapid eye movement sleep onset and duration. Furthermore, these effects were accompanied with an enhancement of EEG spectral power in the theta and the gamma bands. Our results suggest the involvement of dopamine D4R in the regulation of sleep-wake states. The activation of the D4R could enhance the arousal states as revealed by the behavioral and electrophysiological patterns in this study. Dopamine D4R may contribute to the arousal effects of dopamine-releasing agents such as methylphenidate.

Nano-LC-MS/MS for Quantification of Lyso-Gb3 and Its Analogues Reveals a Useful Biomarker for Fabry Disease.

Biomarkers useful for diagnosis and evaluation of treatment for patients with Fabry disease are urgently needed. Recently, plasma globotriaosylsphingosine (lyso-Gb3) and lyso-Gb3-related analogues have attracted attention as promising biomarkers of Fabry disease. However, the plasma concentrations of lyso-Gb3 and its analogues are extremely low or below the detection limits in some Fabry patients as well as in healthy subjects. In this paper, we introduce the novel application of a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system to the measurement of lyso-Gb3 and its analogues in plasma. Nano-LC-MS/MS requires smaller amounts of samples and is more sensitive than conventional techniques. Using this method, we measured the plasma concentrations of lyso-Gb3 and its analogues in 40 healthy subjects, 5 functional variants (males with E66Q), and various Fabry patients (9 classic Fabry males/9 mutations; 7 later-onset Fabry males/5 mutations; and 10 Fabry females/9 mutations). The results revealed that the mean lyso-Gb3 and lyso-Gb3(-2) concentrations in all the Fabry patient subgroups were statistically higher, especially in the classic Fabry males, than those in the functional variants and healthy subjects. The plasma concentrations of lyso-Gb3 and its analogues in healthy subjects, functional variants, and some Fabry patients with specific mutations (R112H and M296I) that cannot be established by conventional techniques were successfully determined by means of nano-LC-MS/MS. The lyso-Gb3 and lyso-Gb3(-2) concentrations in male patients with these mutations were lower than those in most Fabry patients having other mutations, but higher than those in the functional variants and healthy subjects. This new method is expected to be useful for sensitive determination of the plasma concentrations of lyso-Gb3 and its analogues. This study also revealed that not only lyso-Gb3 but also lyso-Gb3(-2) in plasma is a useful biomarker for the diagnosis of Fabry disease.

A novel selective androgen receptor modulator, NEP28, is efficacious in muscle and brain without serious side effects on prostate.

Age-related androgen depletion is known to be a risk factor for various diseases, such as osteoporosis and sarcopenia. Furthermore, recent studies have demonstrated that age-related androgen depletion results in accumulation of ß-amyloid protein and thereby acts as a risk factor for the development of Alzheimer's disease. Supplemental androgen therapy has been shown to be efficacious in treating osteoporosis and sarcopenia. In addition, studies in animals have demonstrated that androgens can play a protective role against Alzheimer's disease. However, androgen therapy is not used routinely for these indications, because of side effects. Selective androgen receptor modulators (SARMs) are a new class of compounds. SARMs maintain the beneficial effects of androgens on bone and muscle while reducing unwanted side effects. NEP28 is a new SARM exhibiting high selectivity for androgen receptor. To investigate the pharmacological effects of NEP28, we compared the effects on muscle, prostate, and brain with mice that were androgen depleted by orchidectomy and then treated with either placebo, NEP28, dihydrotestosterone, or methyltestosterone. We demonstrated that NEP28 showed tissue-selective effect equivalent to or higher than existing SARMs. In addition, the administration of NEP28 increased the activity of neprilysin, a known Aß-degrading enzyme. These results indicate that SARM is efficacious for the treatment of not only osteoporosis and sarcopenia, but also Alzheimer's disease.

Comparison of gene expression changes induced by biguanides in db/db mice liver.

Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.