Synthesis, SAR study, and biological evaluation of novel 2,3-dihydro-1H-imidazo[1,2-a]benzimidazole derivatives as phosphodiesterase 10A inhibitors.

Phosphodiesterase 10A (PDE10A) inhibitors were designed and synthesized based on the dihydro-imidazobenzimidazole scaffold. Compound 5a showed moderate inhibitory activity and good permeability, but unfavorable high P-glycoprotein (P-gp) liability for brain penetration. We performed an optimization study to improve both the P-gp efflux ratio and PDE10A inhibitory activity. As a result, 6d was identified with improved P-gp liability and high PDE10A inhibitory activity. Compound 6d also showed satisfactory brain penetration, suppressed phencyclidine-induced hyperlocomotion and improved MK-801-induced working memory deficit.

Addressing phototoxicity observed in a novel series of biaryl derivatives: discovery of potent, selective and orally active phosphodiesterase 10A inhibitor ASP9436.

We synthesized several biaryl derivatives as PDE10A inhibitors to prevent phototoxicity of 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1) and found that the energy difference between the energy-minimized conformation and the coplanar conformation of the biaryl moiety helped facilitate prediction of the phototoxic potential of biaryl compounds. Replacement of the quinoline ring of 1 with N-methyl benzimidazole increased this energy difference and prevented phototoxicity in the 3T3 NRU test. Further optimization identified 1-methyl-5-(1-methyl-3-{[4-(1-methyl-1H-benzimidazol-4-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (38b). Compound 38b exhibited good selectivity against other PDEs, and oral administration of 38b improved visual-recognition memory deficit in mice at doses of 0.001 and 0.003mg/kg in the novel object recognition test. ASP9436 (sesquiphosphate of 38b) may therefore be used for the treatment of schizophrenia with a low risk of phototoxicity.

Synthesis, SAR study, and biological evaluation of novel quinoline derivatives as phosphodiesterase 10A inhibitors with reduced CYP3A4 inhibition.

A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyridone ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A PET study with (11)C-labeled 42b indicated that 42b exhibited good brain penetration and specifically accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated phencyclidine-induced hyperlocomotion in mice with an ED50 value of 2.0mg/kg and improved visual-recognition memory impairment at 0.1 and 0.3mg/kg in mice novel object recognition test.

Species differences in intestinal glucuronidation activities between humans, rats, dogs and monkeys.

1. Glucuronidation via UDP-glucuronosyltransferase (UGT) in the intestine has been reported to influence the pharmacokinetics (PK) of drugs; however, information concerning the differences in activity between species is limited. Here, we investigated the in vitro and in vivo activities of intestinal glucuronidation for 17 UGT substrates in humans, rats, dogs and monkeys. 2. Although in vitro intrinsic clearance (CLint,u,UGT) in intestinal microsomes showed a good correlation between humans and laboratory animals, values tended to be lower in humans than in laboratory animals. The ratio of CLint,u,UGT in the absence and presence of bovine serum albumin differed between species. In vivo, the fraction of drug absorbed (FaFg) in humans correlated with that in dogs and monkeys, but not in rats. 3. While an inverse correlation between CLint,u,UGT and FaFg was observed in each species, the CLint,u,UGT values in the intestinal microsomes corresponding to FaFg values in dogs were three to four times higher than in other animals. 4. These results indicate the need for a degree of caution when extrapolating PK data from laboratory animals to humans.

Hyper alginate gel microbead formation by molecular diffusion at the hydrogel/droplet interface.

We report a simple method for forming monodispersed, uniformly shaped gel microbeads with precisely controlled sizes. The basis of our method is the placement of monodispersed sodium alginate droplets, formed by a microfluidic device, on an agarose slab gel containing a high-osmotic-pressure gelation agent (CaCl(2) aq.): (1) the droplets are cross-linked (gelated) due to the diffusion of the gelation agent from the agarose slab gel to the sodium alginate droplets and (2) the droplets simultaneously shrink to a fraction of their original size (<100 µm in diameter) due to the diffusion of water molecules from the sodium alginate droplets to the agarose slab gel. We verified the mass transfer mechanism between the droplet and the agarose slab gel. This method circumvents the limitations of gel microbead formation, such as the need to prepare microchannels of various sizes, microchannel clogging, and the deformation of the produced gel microbeads.

Usefulness of a humanized tricellular static transwell blood-brain barrier model as a microphysiological system for drug development applications. - A case study based on the benchmark evaluations of blood-brain barrier microphysiological system.

Microphysiological system (MPS), a new technology for in vitro testing platforms, have been acknowledged as a strong tool for drug development. In the central nervous system (CNS), the blood‒brain barrier (BBB) limits the permeation of circulating substances from the blood vessels to the brain, thereby protecting the CNS from circulating xenobiotic compounds. At the same time, the BBB hinders drug development by introducing challenges at various stages, such as pharmacokinetics/pharmacodynamics (PK/PD), safety assessment, and efficacy assessment. To solve these problems, efforts are being made to develop a BBB MPS, particularly of a humanized type. In this study, we suggested minimal essential benchmark items to establish the BBB-likeness of a BBB MPS; these criteria support end users in determining the appropriate range of applications for a candidate BBB MPS. Furthermore, we examined these benchmark items in a two-dimensional (2D) humanized tricellular static transwell BBB MPS, the most conventional design of BBB MPS with human cell lines. Among the benchmark items, the efflux ratios of P-gp and BCRP showed high reproducibility in two independent facilities, while the directional transports meditated through Glut1 or TfR were not confirmed. We have organized the protocols of the experiments described above as standard operating procedures (SOPs). We here provide the SOPs with the flow chart including entire procedure and how to apply each SOP. Our study is important developmental step of BBB MPS towards the social acceptance, which enable end users to check and compare the performance the BBB MPSs.

Quantitative prediction of human intestinal glucuronidation effects on intestinal availability of UDP-glucuronosyltransferase substrates using in vitro data.

We investigated whether the effects of intestinal glucuronidation on the first-pass effect can be predicted from in vitro data for UDP-glucuronosyltransferase (UGT) substrates. Human in vitro intrinsic glucuronidation clearance (CL(int, UGT)) for 11 UGT substrates was evaluated using pooled intestinal microsomes (4.00-4620 µl · min⁻¹ · mg⁻¹) and corrected by the free fraction in the microsomal mixture (CLu(int), (UGT) = 5.2-5133 µl · min⁻¹ · mg⁻¹). Eleven UGT substrates were stable against intestinal cytochrome P450, indicating intestinal glucuronidation has a main effect on human intestinal availability. Oral absorbability intestinal availability (F(a)F(g)) values were calculated from in vivo pharmacokinetic parameters in the literature (F(a)F(g) = 0.01-1.0). It was found that CLu(int, UGT) and human F(a)F(g) have an inverse relationship that can be fitted to a simplified intestinal availability model. Experiments using Supersomes from insect cells expressing UGT isoforms showed that the substrates used were conjugated by various UGT isoforms. These results suggest that combining the simplified intestinal availability model and in vitro conjugation assay make it possible to predict human F(a)F(g) regardless of UGT isoform.

A lithography-free procedure for fabricating three-dimensional microchannels using hydrogel molds.

We present a lithography-free procedure for fabricating intrinsically three-dimensional smooth-walled microchannels within poly(dimethylsiloxane) (PDMS) elastomer using hydrogel molds. In the fabrication process, small pieces of agarose gel ("wires" or "chips") are embedded in uncured PDMS composite, arranged in the shape of the desired microchannels, and used as molds to form the microchannels. The point of the process is that molds for creating junctions of microchannels such as T-junctions or cross-junctions can be robustly formed by simply grafting gel wires in uncured PDMS composite without using adhesive agents. The technical advantage of this method is that three-dimensional microstructures such as microchannels with circular cross sections, three-dimensionally arranged junctions or interchanges of microchannels can be flexibly designed and fabricated with a straightforward procedure without the need for any specialized equipment or layer-by-layer assemblage processes. This method provides a low-cost, green procedure for fabricating microfluidic devices and promises to make microfluidic processes more accessible and easy to implement in a variety of scientific fields.

Identification of 4-quinolone derivatives as inhibitors of reactive oxygen species production from human umbilical vein endothelial cells.

Oxidative stress is widely recognized as being associated with a number of disorders, including metabolic dysfunction and atherosclerosis. A series of substituted 4-quinolone derivatives were prepared and evaluated as inhibitors of reactive oxygen species (ROS) production from human umbilical vein endothelial cells (HUVECs). One compound in particular, 2-({[4-(3-hydroxy-3-methylbutoxy)pyridin-2-yl]oxy}methyl)-3-methylquinolin-4(1H)-one (25b), inhibited ROS production from HUVECs with an IC(50) of 140 nM. This compound also exhibited low CYP2D6 inhibitory activity, high aqueous solubility, and good in vitro metabolic stability. An in vivo pharmacokinetic study of this compound in SD rats revealed high oral bioavailability and a long plasma half-life.

Parallel multipoint recording of aligned and cultured neurons on micro channel array toward cellular network analysis.

This paper describes an advanced Micro Channel Array (MCA) for recording electrophysiological signals of neuronal networks at multiple points simultaneously. The developed MCA is designed for neuronal network analysis which has been studied by the co-authors using the Micro Electrode Arrays (MEA) system, and employs the principles of extracellular recordings. A prerequisite for extracellular recordings with good signal-to-noise ratio is a tight contact between cells and electrodes. The MCA described herein has the following advantages. The electrodes integrated around individual micro channels are electrically isolated to enable parallel multipoint recording. Reliable clamping of a targeted cell through micro channels is expected to improve the cellular selectivity and the attachment between the cell and the electrode toward steady electrophysiological recordings. We cultured hippocampal neurons on the developed MCA. As a result, the spontaneous and evoked spike potentials could be recorded by sucking and clamping the cells at multiple points. In this paper, we describe the design and fabrication of the MCA and the successful electrophysiological recordings leading to the development of an effective cellular network analysis device.

A Microfluidic Platform Based on Robust Gas and Liquid Exchange for Long-term Culturing of Explanted Tissues.

Microfluidic devices are important platforms to culture and observe biological tissues. Compared with conventional setups, microfluidic devices have advantages in perfusion, including an enhanced delivery of nutrients and gases to tissues. However, explanted tissues can maintain their functions for only hours to days in microfluidic devices, although their observations are desired for weeks. The suprachiasmatic nucleus (SCN) is a brain region composed of heterogeneous cells to control the biological clock system through synchronizing individual cells in this region. The synchronized and complicated cell-cell interactions of SCN cells are difficult to reproduce from seeded cells. Thus, the viability of explanted SCN contributes to the study of SCN functions. In this paper, we propose a new perfusion platform combining a PDMS microfluidic device with a porous membrane to culture an explanted SCN for 25 days. We expect that this platform will provide a universal interface for microfluidic manipulation of tissue explants.

Two-dimensional network formation of cardiac myocytes in agar microculture chip with 1480 nm infrared laser photo-thermal etching.

We have developed a new method that enables agar microstructures to be used to cultivate cells and that allows cell network patterns to be controlled. The method makes use of non-contact three-dimensional photo-thermal etching with a 1480 nm infrared focused laser beam, which is strongly absorbed by water and agar gel, to form the shapes of agar microstructures. It allows microstructures to be easily formed in an agar layer within a few minutes, with cell-culture holes formed by the spot heating of a 100 mW laser and tunnels by the tracing of a 100 microm s(-1), 40 mW laser. We cultivated rat cardiac myocytes in adjacent microstructures and observed synchronized beating in them 90 min after they had made physical contact. Our results indicate that the system can make and use microstructures for cell-network cultivation in a minimal amount of time without any expensive microfabrication facilities or complicated procedures.

An agar-microchamber cell-cultivation system: flexible change of microchamber shapes during cultivation by photo-thermal etching.

A new type of cell-cultivation system based on photo-thermal etching has been developed for the on-chip cultivation of living cells using an agarose microchamber array. The method can be used to flexibly change the chamber structure by photo-thermal etching, even during the cultivation of cells, depending upon the progress in cell growth. We used an infrared (1064 nm) focused laser beam as a heat source to melt and remove agar gel at the heated spot on a thin chromium layer. The melting of the agar occurred just near the chromium thin layer, and the size of the photo-thermally etched area depended almost linearly on the power of the irradiated laser beam from 2 microm to 50 microm. Thus by using photo-thermal etching with adequate laser power we could easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar-layer even during cell cultivation. After 48 h of cultivation of nerve cells, the nerve cells in two adjacent chambers made fiber connections through the fabricated narrow tunnel-shaped channels. These results suggest that photo-thermal etching occurred only in the area where an absorbing material was used, which means that it is possible to photo-thermally etch lines without damaging the cells in the microchambers. The results also suggest that the agar-microchamber cell cultivation system in combination with photo-thermal etching can potentially be used for the next stage of single cell cultivation including the real-time control of the interaction of cells during cell cultivation.

Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture.

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks.

Induced current pharmacological split stimulation system for neuronal networks.

Magnetic stimulation noninvasively modulates neuronal activity through a magnetically induced current. However, despite the usefulness and popularity of this method, the effects of neuronal activity in the nonstimulated regions on the stimulus responses are unknown. Here, we report that the induced current-evoked responses were affected by neuronal activities in the nonstimulated regions. Our experiment used a Mu-metal-based localized induced current stimulation (LICS) system combined with the microfabricated cell culture chamber system and a microelectrode array (MEA). The cell culture chamber system has radiating microtunnels connecting one central and eight outer chambers, which were fabricated using soft lithography and a replica modeling technique with SU-8 photoresist and polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in the chambers and formed functional synaptic connections through the microtunnels. By applying a biphasic alternating pulsed magnetic field to the Mu-metal located in the central chamber, induced currents were mainly generated near the cultured neurons and modified the neuronal activities, which were recorded through MEA. Furthermore, we confirmed that the evoked responses were modified by localized pharmacological stimulation (LPS) in the outer chambers. These results suggest that our system would be promising tool for analyzing the effect of magnetic stimulation on interacting neuronal activity.

Protocadherin-17 mediates collective axon extension by recruiting actin regulator complexes to interaxonal contacts.

In the process of neuronal wiring, axons derived from the same functional group typically extend together, resulting in fascicle formation. How these axons communicate with one another remains largely unknown. Here, we show that protocadherin-17 (Pcdh17) supports this group extension by recruiting actin polymerization regulators to interaxonal contact sites. Pcdh17 is expressed by a subset of amygdala neurons, and it accumulates at axon-axon boundaries because of homophilic binding. Pcdh17 knockout in mice suppressed the extension of these axons. Ectopically expressed Pcdh17 altered the pattern of axon extension. In in-vitro cultures, wild-type growth cones normally migrate along other axons, whereas Pcdh17 null growth cones do not. Pcdh17 recruits the WAVE complex, Lamellipodin, and Ena/VASP to cell-cell contacts, converting these sites into motile structures. We propose that, through these mechanisms, Pcdh17 maintains the migration of growth cones that are in contact with other axons, thereby supporting their collective extension.

In vitro reconstruction and functional development of the superior colliculus in the retinotectal pathway.

In order to examine the formation of a neural network and the functional development of a visual pathway, we performed in vitro reconstruction of the retinotectal pathway using organotypic explants and co-culture methods. Retinas and superior colliculus (SC) slices obtained from embryonic rats were co-cultured on microelectrode array (MEA) substrates for four weeks. We observed retinal ganglion cell neurites innervating SC slices that evoked responses in retinas or SC slices after applying electrical stimulation. Functional connections between retinas and SC slices were formed in the cultures. At the same time, spontaneous electrical activities were recorded from both the retinas and SC slices over the four weeks. In the co-cultured SC slices, sporadic firings were initially observed at 3-4 days in vitro (DIV), and thereafter the frequency of spontaneous firing increased and synchronized activities occurred after two weeks in vitro (WIV). In most of the single-cultured SC slices, however, only sporadic firings were observed over four weeks. In addition, the retinas and SC slices were co-cultured to enable the exchange of soluble factors with each other via culture medium but not via direct neural connections. The activity patterns resembled ones of single-cultured SC slices. These results suggest that signal inputs from retinas through direct neural connections affect the development of SCs in the retinotectal pathway.

High-throughput evaluation method for drug association with pregnane X receptor (PXR) using differential scanning fluorometry.

The pregnane xenobiotic receptor (PXR) is a key transcriptional regulator of cytochrome P450 (CYP) 3A, a crucial enzyme in the metabolism and detoxification of xenobiotics and endobiotics. PXR is activated by a wide variety of chemicals and serves as a master regulator of detoxification in mammals. Here, we report a fast evaluation method for PXR-drug interactions using differential scanning fluorometry (DSF). DSF analysis revealed that PXR associates with a fluorescence dye in the native state as well as in the unfolded state, which prevented precise evaluation of any shift in the transition midpoint (ΔT (m)) due to association with a drug. Hence, we defined a new parameter, (dF/dT)(50), where F is fluorescence intensity and T is temperature, to describe the ligand concentration. (dF/dT)(50) exhibited better correlation with EC(50) (r(2) = 0.84) than with ΔT m (r(2) = 0.71). The correlation of ΔT m measured using differential scanning calorimetry (DSC) with EC(50) (r(2) = 0.86) was similar to the above (dF/dT)(50) correlation. Therefore, the use of (dF/dT)(50) enables DSF to be used for the rapid evaluation of PXR-drug interactions and could provide prescreening to narrow down the collection of candidate ligands that most likely result in transcriptional activation of CYP3A4.

Network-wide integration of stem cell-derived neurons and mouse cortical neurons using microfabricated co-culture devices.

Regeneration of damaged central nervous systems (CNS) is an important topic in neuroscience and neuroengineering. Grafting new neurons derived from pluripotent stem cells into damaged regions can be done to restore functions after injury. Little is known, however, about network-wide interactions between stem-cell-derived neurons and CNS neurons. In this study, we developed a co-culture method of stem cell-derived neuronal networks and CNS networks and observed spontaneous activity in the co-culture samples. By using a microfabricated poly(dimethylsiloxane) device having two culture compartments and 20 connecting microconduits, we are able to compartmentalize P19-derived neurons and mouse cortical neurons and connect them via the microconduits. Furthermore, we combined the co-culture device and a microelectrode array (MEA)-based recording system and recorded spontaneous activity in the co-cultured networks. We found that periodic synchronized bursting spreading over both neuronal networks occurred during the second week in vitro and that P19-derived neurons in the co-cultured networks had different developmental processes compared with those grown in monoculture. These findings suggest that functional interactions form between P19-dervived neurons and mouse cortical neurons and that the co-culture method is useful for exploring the network-wide integrations between stem cell-derived neurons and CNS neurons.

Quantitative prediction of intestinal glucuronidation of drugs in rats using in vitro metabolic clearance data.

UDP-glucuronosyltransferase (UGT) is highly expressed in the small intestine and catalyzes the glucuronidation of small molecules, which may affect the oral bioavailability of drugs. However, no method of predicting the in vivo observed fraction of absorbed drug (F(a)F(g)) affected by UGT has yet been established. Here, we investigated the relationship between F(a)F(g) and in vitro clearance of nine UGT substrates (ketoprofen, tolcapone, telmisartan, raloxifene, entacapone, resveratrol, buprenorphine, quercetin, and ezetimibe) via UGT in intestinal microsomes (CL(int, UGT)) in rats. F(a)F(g) was calculated from pharmacokinetic parameters after intravenous and oral administration or using the portal-systemic concentration difference method, with values ranging from 0.027 (ezetimibe) to 1 (tolcapone). Glucuronides of model compounds were observed in the portal plasma after oral administration, with CL(int, UGT) values ranging from 57.8 (tolcapone) to 19,200 µL/min/mg (resveratrol). An inverse correlation between F(a)F(g) and CL(int, UGT) was observed for most compounds and was described using a simplified intestinal availability model reported previously. This model gave accurate predictions of F(a)F(g) values for three in-house compounds. Our results show that F(a)F(g) in rats is affected by UGT and can be predicted using CL(int, UGT). This work should hasten the development of a method to predict F(a)F(g) in humans.