Biocompatibility study of different hyaluronan products for intra-articular treatment of knee osteoarthritis.

BACKGROUND: Intra-articular (IA) injection of hyaluronic acid (HA) (IA-HA) is a well-recognized treatment option for pain associated with symptomatic knee osteoarthritis (OA). IA-HA products differ in their HA content, molecular weight, cross-linking, and source of HA. These differences are assumed to affect the biocompatibility of the IA-HA products once injected inside the knee joint. METHODS: In the present study, we investigated the biocompatibility of three multiple-injection IA-HA products available in the global market. These included SUPARTZ FX™, a medium range molecular weight HA derived from rooster comb (Avian-HA); ORTHOVISC®, a high range molecular weight HA obtained through biological fermentation (Bio-HA); and SYNVISC®, a high molecular weight cross-linked hyaluronan derived from rooster comb (Avian-CL-HA). Rabbit knee joint tissues were histologically and biochemically examined after IA injection of the products. Furthermore, we compared the amounts of impurities in the IA-HA products. RESULTS: IA injection of Avian-CL-HA into rabbit knee joints induced the aggregation of inflammatory cells, infiltration of eosinophils, and an increase in the number of cells in the synovial fluid. However, these effects were not seen in the Avian-HA and Bio-HA groups. The residual protein content and the contaminant levels of bacterial endotoxins were below the limit of quantitation in all HA products. Avian-CL-HA contained relatively a large amount of (1 → 3)-ß-D-glucan, but this was below the lower limit of quantification in the other HA products. CONCLUSIONS: The present results clearly demonstrate that the biocompatibility of Avian-HA is comparable to that of Bio-HA, and they were both considered to have a favorable safety profile for the treatment of symptomatic OA of the knee. However, immunostimulatory activity was observed after injection of Avian-CL-HA: this might be a result of its unique cross-linking structure and/or the considerable amount of (1 → 3)-ß-D-glucan impurity present in the formulation.

Antitumor activity of kinetochore-associated protein 2 siRNA against lung cancer patient-derived tumor xenografts.

It has been widely reported that patient-derived tumor xenografts (PDXs) are more similar to tumor tissues than conventional cancer cell lines. Kinetochore-associated protein 2 (KNTC2) is known to be upregulated specifically in tumor tissues of cancer patients and is recognized as a potential target for cancer therapy. Previously, in vivo antitumor activities of KNTC2 short interfering RNA encapsulated into a lipid nanoparticle (KNTC2-LNP) were reported in orthotopic hepatocellular carcinoma mouse models. However, it remains unclear whether KNTC2-LNP exhibits antitumor activities against lung cancer PDXs. In the present study, the antitumor activities of KNTC2-LNP were clarified in a three-dimensional culture system and a subcutaneous tumor model of lung cancer PDX, LC-60, which was resistant to erlotinib. Growth inhibitory activities of KNTC2-LNP were associated with knockdown activities. Furthermore, KNTC2-LNP also exhibited in vivo antitumor activity against another lung cancer PDX, LC-45, which was sensitive to erlotinib. These results suggest that KNTC2 is a promising target for patients with lung cancer.

Anti-tumor activity of KNTC2 siRNA in orthotopic tumor model mice of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is still one of the major causes of cancer-related death. Kinetochore-associated protein 2 (KNTC2) is specifically upregulated in tumor tissues of HCC patients and recognized as a potential candidate target for the treatment of HCC. However, the relationship between KNTC2 and in vivo tumor growth of HCC is not yet fully understood. Here we encapsulated KNTC2 siRNAs into a lipid nanoparticle (LNP) and investigated their knockdown activity, target engagement marker, anti-tumor activity and hepatotoxicity in an orthotopic HCC model mice of Hep3B-luc cells. Single i.v. administration of KNTC2 siRNA-LNP specifically suppressed the expression levels of both human KNTC2 mRNA and mouse Kntc2 mRNA in tumor tissues. Phosphorylation levels of histone H3 (HH3) at serine 10 in tumor tissues were increased by KNTC2 siRNA-LNP. Repeated administration of KNTC2 siRNA-LNP (twice a week) specifically inhibited the growth of tumor tissues without increasing the plasma AST and ALT levels. Their growth inhibitory activities were consistent with knockdown activities. These data strongly indicated that KNTC2 is a promising target for the treatment of HCC and that phosphorylated HH3 at serine 10 is one of the target engagement markers for KNTC2.

Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform activity in response to convulsant drugs. Our study indicates the possibility that co-culture system of hiPSC-derived neurons with hiPSC-derived astrocytes could be useful in the risk assessment of drug-induced seizure.

Fluid shear stress stimulates MATE2-K expression via Nrf2 pathway activation.

Accurate prediction of drug-induced renal toxicity is necessary for development of safer drugs for patients. Cellular assay systems that recapitulate physiologically relevant microenvironments have been proposed for correct estimation of drug responses in the human body. However, establishment of such assay systems for accurate prediction of renal toxicity is challenging because of the lack of readily available in vitro assay systems. In this study, we investigated the cellular response to fluid shear stress, which is a characteristic of the environment in the kidney proximal tubules, using microfluidic devices. The global gene expression profiles of human primary proximal tubule cells under the fluidic conditions revealed upregulation of MATE2-K and activation of Nrf2 signaling in response to fluid shear stress. Network and cell biological analysis additionally showed that expression of MATE2-K is regulated by Nrf2 signaling. These results strongly suggest that fluid shear stress is involved in the expression and maintenance of function of tissue-specific drug transporters in the proximal tubule, where the cells are exposed to continuous shear stress by primary urine. Furthermore, the microfluidic culture of human proximal tubules was demonstrated to be a useful system to analyze the regulatory mechanisms of gene expression in physiologically relevant cell conditions.

Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex.

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.

Neurochemical properties of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist.

Ramelteon (TAK-375) is a novel melatonin receptor agonist currently under investigation for the treatment of insomnia. This study describes the neurochemical and receptor binding characteristics of ramelteon in vitro. Ramelteon showed very high affinity for human MT1 (Mel1a) and MT2 (Mel1b) receptors (expressed in Chinese hamster ovary [CHO] cells), and chick forebrain melatonin receptors (consisting of Mel1a and Mel1c receptors) with Ki values of 14.0, 112, and 23.1 pM, respectively, making the affinities of ramelteon for these receptors 3-16 times higher than those of melatonin. The affinity of ramelteon for hamster brain MT3 binding sites was extremely weak (Ki: 2.65 microM) compared to melatonin's affinity for the MT3 binding site (Ki: 24.1 nM). In addition, ramelteon showed no measurable affinity for a large number of ligand binding sites (including benzodiazepine receptors, dopamine receptors, opiate receptors, ion channels, and transporters) and no effect on the activity of various enzymes. Ramelteon inhibited forskolin-stimulated cAMP production in the CHO cells that express the human MT1 or MT2 receptors. Taken together, these results indicate that ramelteon is a potent and highly selective agonist of MT1/MT2 melatonin receptors.

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic beta cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

Molecular properties of endogenous RFamide-related peptide-3 and its interaction with receptors.

Based on database searches of DNA sequences, we previously reported a gene encoding peptides possessing Arg-Phe-NH(2) (RFamide) at their C termini. This gene, RFamide-related peptide (RFRP), was expected to encode several different peptides (i.e., RFRP-1, -2, and -3). In the present study, we purified endogenous RFRP-3 from bovine hypothalamus, and demonstrated that it consisted of 28 amino acid residues. After constructing a sandwich enzyme immunoassay for RFRP-3, we analyzed the tissue distribution of endogenous RFRP-3 in rats and found its concentration to be highest in the hypothalamus. In binding assays, [125I]-labeled RFRP-3 bound to OT7T022 with high affinity, but its binding affinity to HLWAR77 was low. On the other hand, [125I]-labeled neuropeptide FF (NPFF) bound to both OT7T022 and HLWAR77 with high affinity. By serial deletion in the N-terminal portions of RFRP-3 and NPFF, we found that four C-terminal amino acid residues (i.e., PQRFamide), which were common between the two peptides, comprised a core sequence responsible for binding with the receptors, whereas three amino acid residues (i.e., PNL in RFRP-3 and LFQ in NPFF) added to the N terminus of PQRFamide played crucial roles in the agonistic activities of RFRP-3 and NPFF for OT7T022 and HLWAR77, respectively.

A G protein-coupled receptor responsive to bile acids.

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.

Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7.

We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 (O'Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995) Genomics 28, 84-91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O'Dowd, B. F. (1999) Mol. Brain Res. 71, 96-103). The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor.