In Vitro Metabolism of 2-Methoxy-N-[3-[4-[3-methyl-4-[(6-methyl- 3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) by Aldehyde Oxidase and Predicting Its Percent Contribution Relative to CYP-Mediated Metabolism.

2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) is an anticancer drug that was discontinued due to hepatotoxicity found in clinical studies. Metabolite analysis of CP-724,714 was conducted using human hepatocytes, in which twelve oxidative metabolites and one hydrolyzed metabolite were formed. Among the three mono-oxidative metabolites, the formation of two was inhibited by adding 1-aminobenzotriazole, a pan-CYP inhibitor. In contrast, the remaining one was not affected by this inhibitor but partially inhibited by hydralazine, indicating that aldehyde oxidase (AO) was involved in metabolizing CP-724,714, which contains a quinazoline substructure, a heterocyclic aromatic quinazoline ring, known to be preferably metabolized by AO. One of the oxidative metabolites of CP-724,714 observed in human hepatocytes was also generated in recombinant human AO. Although CP-724,714 is metabolized by both CYPs and AO in human hepatocytes, the contribution level of AO could not be evaluated using its specific inhibitors because of low AO activity in in vitro human materials. Here, we present a metabolic pathway for CP-724,714 in human hepatocytes and the involvement of AO in CP-724,714 metabolism. We showed here a plausible workflow for predicting AO contribution to the metabolism of CP-724,714 based on DMPK screening data. SIGNIFICANCE STATEMENT: 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) was identified as a substrate of aldehyde oxidase (AO) rather than xanthine oxidase. Since CP-724,714 is also metabolized by cytochrome P450s (CYPs), the contribution levels of AO and CYPs in the metabolism of CP-724,714 were estimated simultaneously based on in vitro drug metabolism screening data.

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and ß-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.

A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.

Desbuquois dysplasia (DBQD) is an autosomal recessive heterogeneous disorder characterized by joint laxity and skeletal changes, including a distinctive monkey-wrench appearance of the femora, advanced carpal ossification, and abnormal patterning of the preaxial digits. Two genes for DBQD (CANT1 encoding calcium-activated nucleotidase-1 and XYLT1 encoding xylosyltransferase-1) have been reported. We propose a novel gene for neonatal short limb dysplasia resembling DBQD, based on the phenotype and genotype of two affected siblings. The affected boy and girl died in early infancy and shortly after birth, respectively. The clinical hallmarks included mid-face hypoplasia, thoracic hypoplasia with respiratory failure, very short stature (approximately -7 SD of birth length) with mesomelic shortening of the limbs, and multiple dislocations of the large joints. Radiological examinations showed prominent lesser trochanter, flared metaphyses of the long bones, and joint dislocations. The affected boy had preaxial digital hypoplasia, and the affected girl showed overlapping and syndactyly of the preaxial digits. Molecular analyses of the girl showed compound heterozygous variants in FAM20B (NM_014864: c.174_178delTACCT p.T59Afs*19/c.1038delG p.N347Mfs*4). FAM20B encodes glycosaminoglycan xylosylkinase, which acts downstream of xylosyltransferase-1. Given the fact that FAM20B deficiency causes skeletal phenotypes in mice and zebrafish, these variants are highly probable to be pathogenic.

Japanese General Clinical Oncologists' Knowledge and Real-world Experiences of Cancer Genomic Medicine: A Nationwide Web-based Survey Study.

Introduction: In Japan, insurance began covering two cancer gene panel tests in 2019. However, the availability of these tests remains limited to 247 facilities (as of October 2023). This survey-based study assessed the knowledge and recognition of cancer genomic medicine by physicians involved in cancer treatment. Methods: Written requests for participation in a web-based questionnaire survey were sent to 14,579 affiliated general clinical oncologists certified by the Japanese Board of Cancer Therapy. The survey was conducted from July 1 to 31st, 2021. Data between physicians affiliated with cancer genome hospitals and noncancer genome hospitals and between regions of Japan were compared. Results: In total, 2,402 valid responses were analyzed. Of the respondents, 1,296 and 1,106 were physicians working at cancer and noncancer genome hospitals, respectively. Physicians working at cancer genome hospitals showed significantly higher results for both knowledge of cancer genomic medicine and experience in cancer gene panel test performance compared with those working at noncancer genome hospitals. There were no significant regional differences in the percentage of physicians who reported having performed cancer gene panel tests. Conclusions: The survey results suggest a disparity in the knowledge of cancer genomic medicine between physicians working at cancer genome hospitals and those working at noncancer genome hospitals; this disparity should be addressed by stakeholders. Closer collaboration between these facilities may be necessary to achieve national dissemination of cancer genomic medicine.

Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase.

T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits replication of the influenza virus in vitro and in vivo. T-705 has been shown to be converted to T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) by intracellular enzymes and then functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRp) of the influenza virus. To elucidate these inhibitory mechanisms, we analyzed the enzyme kinetics of inhibition using Lineweaver-Burk plots of four natural nucleoside triphosphates and conducted polyacrylamide gel electrophoresis of the primer extension products initiated from (32)P-radiolabeled 5'Cap1 RNA. Enzyme kinetic analysis demonstrated that T-705RTP inhibited the incorporation of ATP and GTP in a competitive manner, which suggests that T-705RTP is recognized as a purine nucleotide by influenza virus RdRp and inhibited the incorporation of UTP and CTP in noncompetitive and mixed-type manners, respectively. Primer extension analysis demonstrated that a single molecule of T-705RTP was incorporated into the nascent RNA strand of the influenza virus and inhibited the subsequent incorporation of nucleotides. These results suggest that a single molecule of T-705RTP is incorporated into the nascent RNA strand as a purine nucleotide analog and inhibits strand extension, even though the natural ribose of T-705RTP has a 3'-OH group, which is essential for forming a covalent bond with the phosphate group.

T-705 (favipiravir) activity against lethal H5N1 influenza A viruses.

The neuraminidase inhibitors oseltamivir and zanamivi are used to treat H5N1 influenza. However, oseltamivir-resistant H5N1 viruses have been isolated from oseltamivir-treated patients. Moreover, reassortment between H5N1 viruses and oseltamvir-resistant human H1N1 viruses currently circulating could create oseltamivir-resistant H5N1 viruses, rendering the oseltamivir stockpile obsolete. Therefore, there is a need for unique and effective antivirals to combat H5N1 influenza viruses. The investigational drug T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has antiviral activity against seasonal influenza viruses and a mouse-adapted H5N1 influenza virus derived from a benign duck virus. However, its efficacy against highly pathogenic H5N1 viruses, which are substantially more virulent, remains unclear. Here, we demonstrate that T-705 effectively protects mice from lethal infection with oseltamivir-sensitive or -resistant highly pathogenic H5N1 viruses. Furthermore, our biochemical analysis suggests that T-705 ribofuranosyl triphosphate, an active form of T-705, acts like purines or purine nucleosides in human cells and does not inhibit human DNA synthesis. We conclude that T-705 shows promise as a therapeutic agent for the treatment of highly pathogenic H5N1 influenza patients.

Possible Significance of a Café-style Event to Introduce Advance Care Planning for General Citizens.

OBJECTIVES: We conducted a survey to determine whether the general public who participated in a café-style event to raise awareness of advance care planning (ACP) actually implemented ACP after attending the event. METHODS: On February 20, 2020, a café-style event (Tokai Blue Café: TBC) was held at the Tokai University Hospital. The TBC consisted of a lecture about ACP, "The Go-Wish Game," and a tea party. A questionnaire-based survey was conducted on ACP implementation after one month of TBC. RESULTS: Of the 14 participants (three males and 11 females), 11 agreed to answer the questionnaire and eight responded. Two respondents were male and six were female. Six of the respondents were aged ≥ 60 years. Seven of the eight respondents implemented ACP with their family members, while none did so with their family doctor, even though all of them indicated that they had a family doctor. Several respondents reported that they were uncomfortable discussing the issue with their doctors. CONCLUSION: The results indicate that a café-style event as an awareness-raising activity may have a significant effect on ACP implementation, although it suggests that there are some challenges in involving family doctors.

[Case of Marfan syndrome with microscopic hematuria].

A 36-year-old man was admitted to our hospital for investigation of microscopic hematuria. He was very tall and presented arachnodactyly of the fingers and toes. Chest computed tomography and cardiac echography revealed annuloaortic ectasia and aortic regurgitation. Based on these findings, we suspected Marfan syndrome and performed gene analysis of the FBN1 gene, which encodes fibrillin-1. Mutational analysis showed the missence mutation, p. Ile 2585 Thr, present in exon 62 of the FBN1 gene. To investigate the genesis of microscopic and dismorphic hematuria, we performed a renal biopsy. Light microscopic analysis revealed the absence of any apparent histological changes in the glomerulus, small artery and arteriole. Electron microscopic analysis revealed the glomerular basement membrane to be irregularly thickened, however, there was neither any electron dense deposition nor fibrillar material. Marfan syndrome is an inherited disorder of connective tissue based on abnormality of the FBN1 gene. Fibfibrllin-1 acts not only as a component of microfibrils, but also as the regulator of transforming growth factor -beta signal transduction. From these points of view, we speculated that irregular formation of the glomerular basement membrane of our patient was induced by an imbalance in production of the extracellular matrix as the consequence of abnormal fibrillin-1--TGF-beta signaling.

Jervell and Lange-Nielsen syndrome with novel KCNQ1 and additional gene mutations.

We encountered a boy with Jervell and Lange-Nielsen syndrome (JLNS) with compound heterozygous KCNQ1 mutations, maternal Trp248Phe and a novel paternal mutation, Leu347Arg. His father showed long QT (LQT) and arrhythmia. His mother was asymptomatic with no ECG abnormalities. The proband and his father had an additional mutation (SNTA1 Thr372Met), which is reportedly related to SIDS. These results suggest that multiple gene mutations influence the phenotype of KCNQ1 mutation-related arrhythmia.

Human PZP and common marmoset A2ML1 as pregnancy related proteins.

While pregnancy-related proteins (PRP) are known to contribute to immunotolerance during pregnancy, their significance to development of invasive placenta is unclear. We compared PRP expression in humans and the common marmoset (Callithrix jacchus), a new-world monkey. Invasive placenta was observed at the maternal-foetal interface of marmoset placenta from green fluorescent protein (GFP)-expressing foetus and wild type mother. The pregnancy zone protein (PZP) and alpha-2 macroglobulin-like 1 (A2ML1) proteins exhibited the most prominent increase in expression during the second trimester in humans and marmoset, respectively. In humans, PZP accumulated at the maternal-foetal interface and A2ML1 accumulated in the amnion. Similarly, A2ML1 mRNA was detected in marmoset placenta. These proteins belong to the A2M family of protease inhibitors, and both PZP and A2ML1 share around 90% homology between human and marmoset and have highly conserved structures. However, the protease-reacting bait regions of the proteins had lower homology (56.8-60.7% in proteins) relative to the rest of the sequence. Notably, the cleavage site of a proinflammatory proline-endopeptidase was preserved in human PZP and marmoset A2ML1. These proteins contain multiple sites that are cleaved by proteases involving proline-endopeptidase. Systemic regulation of these A2M family proteins may be important in animals with invasive placenta.

Intracellular metabolism of favipiravir (T-705) in uninfected and influenza A (H5N1) virus-infected cells.

OBJECTIVES: To determine the metabolism of favipiravir (T-705, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) to its ribosylated, triphosphorylated form (T-705 RTP) in uninfected and influenza A/Duck/MN/1525/81 (H5N1) virus-infected cells. Effects of treatment on intracellular guanosine triphosphate (GTP) pools and influenza virus-inhibitory activity were also assessed. METHODS: A strong anion exchange HPLC separation method with UV detection was used to quantify T-705 RTP and GTP levels in Madin-Darby canine kidney cells. Antiviral activity was determined by virus yield reduction assay. RESULTS: Accumulation of T-705 RTP in uninfected cells increased linearly from 3 to 320 pmol/10(6) cells in cells exposed to 1-1000 microM extracellular T-705 for 24 h, approaching maximum levels by 9 h. Virus infection did not result in greater T-705 RTP accumulation compared with uninfected cells. Catabolism of T-705 RTP occurred after removal of T-705 from the extracellular medium, with a half-life of decay of 5.6 +/- 0.6 h. Based upon these results, short-term incubation of T-705 with H5N1 virus-infected cells was predicted to provide an antiviral benefit. Indeed, 4-8 h 10-100 microM T-705 treatment of cells resulted in virus yield reductions, but less than continuous exposure. A 100-fold higher extracellular concentration of T-705 was required to inhibit intracellular GTP levels compared with ribavirin, which helps explain ribavirin's greater toxicity. CONCLUSIONS: The favourable intracellular metabolic properties of T-705 combined with its reduced cell-inhibitory properties make this compound an attractive candidate for treating human influenza virus infections.

KRN951, a highly potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, has antitumor activities and affects functional vascular properties.

Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor (VEGFR) tyrosine kinases. Therefore, VEGFRs are an attractive therapeutic target for cancer treatment. In the present study, we show that a quinoline-urea derivative, KRN951, is a novel tyrosine kinase inhibitor for VEGFRs with antitumor angiogenesis and antigrowth activities. KRN951 potently inhibited VEGF-induced VEGFR-2 phosphorylation in endothelial cells at in vitro subnanomolar IC50 values (IC50 = 0.16 nmol/L). It also inhibited ligand-induced phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-beta) and c-Kit (IC50 = 1.72 and 1.63 nmol/L, respectively). KRN951 blocked VEGF-dependent, but not VEGF-independent, activation of mitogen-activated protein kinases and proliferation of endothelial cells. In addition, it inhibited VEGF-mediated migration of human umbilical vein endothelial cells. Following p.o. administration to athymic rats, KRN951 decreased the microvessel density within tumor xenografts and attenuated VEGFR-2 phosphorylation levels in tumor endothelium. It also displayed antitumor activity against a wide variety of human tumor xenografts, including lung, breast, colon, ovarian, pancreas, and prostate cancer. Furthermore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis revealed that a significant reduction in tumor vascular hyperpermeability was closely associated with the antitumor activity of KRN951. These findings suggest that KRN951 is a highly potent, p.o. active antiangiogenesis and antitumor agent and that DCE-MRI would be useful in detecting early responses to KRN951 in a clinical setting. KRN951 is currently in phase I clinical development for the treatment of patients with advanced cancer.

Exome and copy number variation analyses of Mayer-Rokitansky-Küster- Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital absence of the vagina and uterus. We conducted genome-wide SNP analyses and exome sequencing to detect the causes of MRKH syndrome. We identified de novo variants of MYCBP2, NAV3, and PTPN3 in three families and a variant of MYCBP2 in a sporadic case. Here, we demonstrated the partial genetic makeup of Japanese MRKH syndrome.

Neuronal activity of histaminergic tuberomammillary neurons during wake-sleep states in the mouse.

Using extracellular single-unit recordings alone and in combination with neurobiotin juxtacellular labeling and histamine immunohistochemistry, we have identified, for the first time in nonanesthetized, head-restrained mice, histamine neurons in the tuberomammillary nuclei of the posterior hypothalamus. They are all characterized by triphasic broad action potentials. They are active only during wakefulness, and their activity is related to a high level of vigilance. During waking states, they display a slow (<10 Hz) tonic, repetitive, irregular firing pattern. Their activity varies in the different waking states, being lowest during quiet waking, moderate during active waking, and highest during attentive waking. They cease firing during quiet waking before the onset of EEG synchronization, the EEG sign of sleep (drowsy state), and remain silent during slow-wave sleep and paradoxical (or rapid eye movement) sleep. They exhibit a pronounced delay in firing during transitions from sleep to wakefulness or remain quiescent during the same transitions if the animals are not fully alert. They either respond with a long delay, or do not respond, to an arousing stimulus if the stimulus does not elicit an overt alert state. These data support the view that the activity of histaminergic tuberomammillary neurons plays an important role, not in the induction of wakefulness per se, but in the maintenance of the high level of vigilance necessary for cognitive processes. Conversely, cessation of their activity may play an important role in both the initiation and maintenance of sleep.

Requirement of tryptophan hydroxylase during development for maturation of sensorimotor gating.

Deficits in sensorimotor gating, a function to focus on the most salient stimulus, could lead to a breakdown of cognitive integrity, and could reflect the "flooding" by sensory overload and cognitive fragmentation seen in schizophrenia. Sensorimotor gating emerges at infancy, and matures during childhood. The mechanisms that underlie its development are largely unclear. Here, we screened the mouse genome, and found that tryptophan hydroxylase (TPH) is implicated in the maturation of sensorimotor gating. TPH, an enzyme involved in the biosynthesis of serotonin, proved to be required only during the weaning period for maturation of sensorimotor gating, but was dispensable for its emergence. Proper serotonin levels during development underlie the mature functional architecture for sensorimotor gating via appropriate actin polymerization. Thus, maintaining proper serotonin levels during childhood may be important for mature sensorimotor gating in adulthood.

Improvement by solid dispersion of the bioavailability of KRN633, a selective inhibitor of VEGF receptor-2 tyrosine kinase, and identification of its potential therapeutic window.

KRN633 is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases. However, it is poorly water-soluble; consequently, relatively high doses are required to achieve substantial in vivo tumor growth suppression after oral administration. We subjected KRN633 to the solid dispersion technique to improve its solubility, absorption, and antitumor efficacy after oral administration. This technique transformed the drug into an amorphous state and dramatically improved its dissolution rate. It also enhanced the bioavailability of the drug in rats by approximately 7.5-fold. The solid dispersion form of KRN633 also dramatically inhibited human tumor growth in murine and rat xenograft models: similar rates of tumor growth inhibition were obtained with 10- to 25-fold lower doses of the solid dispersion preparation relative to the pure drug in its crystalline state. Histologic analysis of tumors treated with the solid dispersion preparation revealed a significant reduction in microvessel density at much lower doses when compared with the crystalline form preparation. In addition, a dose-finding study using the solid dispersion form in a rat xenograft model revealed that there was a substantial range of doses at which KRN633 in the solid dispersion form showed significant antitumor activity but did not induce weight loss or elevate total urinary protein levels. These data suggest that the solid dispersion technique is an effective approach for developing KRN633 drug products and that KRN633 in the solid dispersion form may be a highly potent, orally available drug with a wide therapeutic window for diseases associated with abnormal angiogenesis.

Laparoscopic Davydov Procedure for the Creation of a Neovagina in Patients with Mayer-Rokitansky-Kuster-Hauser Syndrome: Analysis of 7 Cases.

OBJECTIVE: Several surgical techniques have been described for creating a neovagina in patients with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, however as yet there is no standardized treatment. The aim of this report is to assess anatomic and functional outcomes after the laparoscopic Davydov procedure for the creation of a neovagina in patients with MRKH syndrome. METHODS: Seven patients with MRKH syndrome underwent the laparoscopic Davydov technique from January 2005 to August 2010. The anatomic and functional results were evaluated after 3, 6, 12, 24, 36, 48, and 60 months. RESULTS: The surgical procedure was performed with no major complications except in one case in which an intraoperative bladder injury occurred and was successfully corrected. The mean duration of surgery was 162.9 minutes (range, 120-230 min). Mean lengths/widths (cm) of the neovagina were 6.4/2.6, 6.5/2.5, 6.5/2.8, 6.4/2.8, 7.1/2.8, and 7.2/2.8 at 3, 6,12, 24, 36, 48, and 60 postoperative months, respectively. CONCLUSION: The laparoscopic Davydov procedure seems to be a safe and effective surgical treatment for patients with MRKH syndrome if postoperative intermittent self dilation was done.

Novel potent orally active selective VEGFR-2 tyrosine kinase inhibitors: synthesis, structure-activity relationships, and antitumor activities of N-phenyl-N'-{4-(4-quinolyloxy)phenyl}ureas.

N-Phenyl-N'-{4-(4-quinolyloxy)phenyl}ureas were found to be a novel class of potent inhibitors for the vascular endothelial growth factor receptor 2 (VEGFR-2) tyrosine kinase through synthetic modifications of a lead compound and structure-activity relationship studies. A representative compound 6ab, termed Ki8751, inhibited VEGFR-2 phosphorylation at an IC(50) value of 0.90 nM, and also inhibited the PDGFR family members such as PDGFRalpha and c-Kit at 67 nM and 40 nM, respectively. However, 6ab did not have any inhibitory activity against other kinases such as EGFR, HGFR, InsulinR and others even at 10000 nM. 6ab suppressed the growth of the VEGF-stimulated human umbilical vein endothelial cell (HUVEC) on a nanomolar level. 6ab showed significant antitumor activity against five human tumor xenografts such as GL07 (glioma), St-4 (stomach carcinoma), LC6 (lung carcinoma), DLD-1 (colon carcinoma) and A375 (melanoma) in nude mice and also showed complete tumor growth inhibition with the LC-6 xenograft in nude rats following oral administration once a day for 14 days at 5 mg/kg without any body weight loss.

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus.

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.