A novel model of chronic limb ischemia to therapeutically evaluate the angiogenic effects of drug candidates.

Critical limb ischemia (CLI) is a severe state of peripheral artery disease with high unmet clinical needs. Further, there are no effective treatment options for patients with CLI. Based on preclinical study results, predicting the clinical efficacy of CLI treatments is typically difficult because conventional hindlimb ischemia (HLI) rodent models display spontaneous recovery from ischemia, which is not observed in patients with CLI. Therefore, we aimed to develop a novel chronic and severe HLI model to properly evaluate the therapeutic effects of drug candidates for CLI. Severe HLI mice (Type-N) were generated by increasing the excised area of blood vessels in a hindlimb of NOG mice. Immunohistochemistry and gene expression analysis at 9 wk after the Type-N operation revealed that the ischemic limb was in a steady state with impaired angiogenesis, like that observed in patients with CLI. We did selection of chronic Type-N mice based on the number of necrotic nails and blood flow rate at 2 wk after surgery because some Type-N mice showed mild symptoms. Therapeutic treatment with cilostazol, which is used for intermittent claudication, did not restore blood flow in chronic Type-N mice. In contrast, therapeutic transplantation of pericytes and vascular endothelial cells, which can form new blood vessels in vivo, significantly improved blood flow in a subset of Type-N mice. These findings suggest that this novel chronic and severe HLI model may be a valuable standard animal model for therapeutic evaluation of the angiogenic effects of CLI drug candidates.NEW & NOTEWORTHY We developed a chronic and severe hindlimb ischemia (HLI) mouse model for preclinical research on critical limb ischemia (CLI). This model partially reflects human CLI pathology in that it does not show spontaneous restoration of blood flow or expression of angiogenic genes in the ischemic limb. This novel model may be valuable for therapeutic evaluation of the angiogenic effects of CLI drug candidates.

Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.

A novel anti-NGF PEGylated Fab' provides analgesia with lower risk of adverse effects.

Nerve growth factor (NGF) has emerged as a key driver of pain perception in several chronic pain conditions, including osteoarthritis (OA), and plays an important role in the generation and survival of neurons. Although anti-NGF antibodies improve pain control and physical function in patients with clinical chronic pain conditions, anti-NGF IgGs are associated with safety concerns such as effects on fetal and postnatal development and the risk of rapidly progressive osteoarthritis. To overcome these drawbacks, we generated a novel anti-NGF PEGylated Fab' antibody. The anti-NGF PEGylated Fab' showed specific binding to and biological inhibitory activity against NGF, and analgesic effects in adjuvant-induced arthritis model mice in a similar manner to an anti-NGF IgG. In collagen-induced arthritis model mice, the anti-NGF PEGylated Fab' showed higher accumulation in inflamed foot pads than the anti-NGF IgG. In pregnant rats and non-human primates, the anti-NGF PEGylated Fab' was undetectable in fetuses, while the anti-NGF IgG was detected and caused abnormal postnatal development. The PEGylated Fab' and IgG also differed in their ability to form immune complexes in vitro. Additionally, while both PEGylated Fab' and IgG showed analgesic effects in sodium monoiodoacetate-induced arthritic model rats, their effects on edema were surprisingly quite different. While the anti-NGF IgG promoted edema over time, the anti-NGF PEGylated Fab' did not. The anti-NGF PEGylated Fab' (ASP6294) may thus be a potential therapeutic candidate with lower risk of adverse effects for various diseases in which NGF is involved such as OA and chronic back pain.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1.

Activation of the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor by angiopoietin-1 (Ang1) is critical for vascular stabilization: it promotes survival signal transduction via auto-phosphorylation and reduces vascular permeability by strengthening tight junctions between endothelial cells. Thus, Tie2/Ang1 signaling is a promising therapeutic target for vascular diseases. However, in vivo use of existing Tie2 signaling modulators, such as recombinant Ang1, is restricted by limitations in manufacturability and stability. Here, we present a novel engineered tetra-valent agonistic antibody, ASP4021, which can specifically and fully activate the Tie2 receptor in an equivalent manner to Ang1. ASP4021 induced Tie2 self-phosphorylation and inhibited apoptosis in a human primary endothelial cell line. Additionally, single administration of ASP4021 significantly suppressed mustard-oil-induced vascular permeability in rats. ASP4021 may thus be a potential therapeutic candidate for diseases associated with vascular weakness such as diabetic retinopathy, diabetic macular edema and critical limb ischemia.

Evaluation of FGFR inhibitor ASP5878 as a drug candidate for achondroplasia.

Achondroplasia is caused by gain-of-function mutations in FGFR3 gene and leads to short-limb dwarfism. A stabilized analogue of C-type natriuretic peptide (CNP) is known to elongate bone by interacting with FGFR3 signals and thus is a promising drug candidate. However, it needs daily administration by percutaneous injection. FGFR inhibitor compounds are other drug candidates for achondroplasia because they directly fix the mutant protein malfunction. Although FGFR inhibitors elongate the bone of model mice, their adverse effects are not well studied. In this study, we found that a new FGFR inhibitor, ASP5878, which was originally developed as an anti-cancer drug, elongated the bone of achondroplasia model male mice at the dose of 300 µg/kg, which confers an AUC of 275 ng·h/ml in juvenile mice. Although ASP5878 was less effective in bone elongation than a CNP analogue, it is advantageous in that ASP5878 can be administered orally. The AUC at which minimal adverse effects were observed (very slight atrophy of the corneal epithelium) was 459 ng·h/ml in juvenile rats. The positive discrepancy between AUCs that brought efficacy and minimal adverse effect suggests the applicability of ASP5878 to achondroplasia in the clinical setting. We also analyzed effects of ASP5878 in a patient-specific induced pluripotent stem cell (iPSC) model for achondroplasia and found the effects on patient chondrocyte equivalents. Nevertheless, cautious consideration is needed when referring to safety data obtained from its application to adult patients with cancer in clinical tests.

cDNA cloning and characterization of porcine histamine H4 receptor.

The cDNA encoding histamine H4 receptor was cloned from the porcine spleen cDNA library. Porcine H4 receptor, which shares 72% homology with its human counterpart, bound to histamine in receptor-expressing mammalian cells. Isolation of the porcine H4 receptor, which is important for understanding of the pharmacology, will aid in better interpretation of physiological role of this subtype of histamine receptor.

Molecular cloning and characterization of prokineticin receptors.

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.

Molecular modeling of vasopressin receptor and in silico screening of V1b receptor antagonists.

INTRODUCTION: G protein-coupled receptors (GPCRs) are integral membrane proteins which contain seven-transmembrane-spanning alpha-helices. GPCR-mediated signaling has been associated with various human diseases, positioning GPCRs as attractive targets in the drug discovery field. Recently, through advances in protein engineering and crystallography, the number of resolved GPCR structures has increased dramatically. This growing availability of GPCR structures has greatly accelerated structure-based drug design (SBDD) and in silico screening for GPCR-targeted drug discovery. AREAS COVERED: The authors introduce the current status of X-ray crystallography of GPCRs and what has been revealed from the resolved crystal structures. They also review the recent advances in SBDD and in silico screening for GPCR-targeted drug discovery and discuss a docking study, using homology modeling, with the discovery of potent antagonists of the vasopressin 1b receptor. EXPERT OPINION: Several innovative protein engineering techniques and crystallographic methods have greatly accelerated SBDD, not only for already-resolved GPCRs but also for those structures which remain unclear. These technological advances are expected to enable the determination of GPCR-fragment complexes, making it practical to perform fragment-based drug discovery. This paves the way for a new era of GPCR-targeted drug discovery.

Both V(1A) and V(1B) vasopressin receptors deficiency result in impaired glucose tolerance.

[Arg(8)]-vasopressin (AVP) is involved in the regulation of glucose homeostasis via vasopressin V(1A) and vasopressin V(1B) receptor. Our previous studies have demonstrated that vasopressin V(1A) receptor deficient (V(1A)R(-/-)) mice exhibited hyperglycemia, vasopressin V(1B) receptor deficient (V(1B)R(-/-)) mice, in contrast, exhibited hypoglycemia with hypoinsulinemia. These findings indicate that vasopressin V(1A) receptor deficiency results in decreased insulin sensitivity, whereas vasopressin V(1B) receptor deficiency results in increased insulin sensitivity. In our previous and present studies, we used the glucose tolerance test to investigate glucose tolerance in mutant mice, lacking either the vasopressin V(1A) receptor, the vasopressin V(1B) receptor, or both receptors, that were kept on a high-fat diet. Glucose and insulin levels were lower in V(1B)R(-/-) mice than in wild type (WT) mice when both groups were fed the high-fat diet, which indicates that the insulin sensitivity of the V(1B)R(-/-) mice was enhanced. V(1A)R(-/-) mice on the high-fat diet, on the other hand, exhibited overt obesity, along with an impaired glucose tolerance, while WT mice on the high-fat diet did not. Next, in order to assess the effect of vasopressin V(1B) receptor deficiency on the development of glucose intolerance caused by vasopressin V(1A) receptor deficiency, we generated mice that were deficient for both vasopressin V(1A) receptor and vasopressin V(1B) receptor (V(1AB)R(-/-)), fed them a high-fat diet, and examined their glucose tolerances using the glucose tolerance test. Glucose tolerance was impaired in V(1AB)R(-/-) mice, suggesting that the effects of vasopressin V(1B) receptor deficiency could not influence the development of hyperglycemia promoted by vasopressin V(1A) receptor deficiency, and that blockade of both receptors could lead to impaired glucose tolerance.

Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

Molecular cloning of monkey histamine H4 receptor.

Histamine H(4) receptor is considered as a novel therapeutic target for allergic diseases. To enhance the knowledge about species difference, which is essential for drug discovery research, monkey H(4) receptor was identified. Monkey H(4) receptor was characterized to have comparable similarity with its human counterpart. Discovery of monkey H(4) receptor will contribute to a better interpretation of effective drug discovery.

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides.

Proadrenomedullin N-terminal 20 peptide (PAMP[1-20]/PAMP-20) and its truncated analog, PAMP[9-20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was "PAMP-12> or =cortistatin>PAMP-20". These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca(2+) mobilization, and [(35)S]guanosine 5'-(gamma-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G(alpha q) but also G(alpha i), consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor.

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.

Comparative studies of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, and abciximab on in vitro antiplatelet effect and binding properties.

The effects of YM337, the Fab fragment of a humanized anti-glycoprotein IIb/IIIa (GPIIb/IIIa) monoclonal antibody C4G1, on in vitro platelet function and binding properties were compared with those of abciximab, the Fab fragment of the human/murine chimeric anti-GPIIb/IIIa monoclonal antibody 7E3. Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin. Further, both inhibited human platelet adhesion to von Willebrand factor, fibrinogen, fibronectin and subendothelial matrix with similar potency. Fibrinogen binding to washed platelets was dose-dependently inhibited by both agents. In binding assay using 125I-YM337 and 125I-abciximab, Kd values determined with platelet-rich plasma were 6.74 +/- 0.56 nM for YM337 and 6.65 +/- 1.45 nM for abciximab, and the number of binding sites were 42,700 +/- 3,000 for YM337 and 76,000 +/- 5,400 for abciximab. GPIIb/IIIa was precipitated from the solubilized fraction of platelets by both agents. In contrast, integrin alphavbeta3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337. Fibrinogen binding to purified GPIIb/IIIa was dose-dependently inhibited by both agents. In contrast, vitronectin binding to purified integrin alphavbeta3 was dose-dependently inhibited by abciximab but not by YM337, supporting the idea that abciximab reacts to integrin alphavbeta3. Therefore, YM337 was suggested to bind to a different epitope of GPIIb/IIIa from abciximab. These results suggest that YM337 specifically acts on platelet GPIIb/IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab.

Molecular identification of nicotinic acid receptor.

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.