RESUMO
G protein-coupled receptor 119 (GPR119) has been shown to be highly expressed in small intestinal L-cells and pancreatic ß-cells and mediates intracellular cAMP concentration, glucagon-like peptide (GLP-1) secretion, and glucose-stimulated insulin secretion (GSIS). This study examined the pharmacological effects of 4-(5-{(1R)-1-[4-(cyclopropylcarbonyl) phenoxy]propyl}-1,2,4-oxadiazol-3-yl)-2-fluoro-N-[(2R)-1-hydroxypropan-2-yl]benzamide (DS-8500a), a novel, orally available, selective GPR119 agonist. In in vitro studies, DS-8500a increased intracellular cAMP in a concentration-dependent manner in human, rat, and mouse GPR119-expressing Chinese hamster ovary (CHO)-K1 cells, with EC50 values of 51.5, 98.4, and 108.1 nmol/l, respectively. DS-8500a had no effect on intracellular cAMP in pcDNA3.1/CHO-K1 cells. In in vivo studies, DS-8500a augmented plasma GLP-1 concentration in Zucker fatty (ZF) rats, and enhanced GSIS and did not change plasma glucose concentration in fasted Sprague-Dawley (SD) rats. A single dose of DS-8500a showed dose-dependent glucose-lowering effects at oral glucose tolerance test (OGTT) in ZF rats. In a repeat-dosing study, DS-8500a had statistically significant glucose-lowering effects at OGTT performed at the 1st day and after 2 weeks of treatment in neonatal streptozotocin-treated (nSTZ) rats, and the efficacy levels of DS-8500a in each test were greater than those of GSK1292263 or MBX-2982, which had been clinically tested previously as GPR119 agonists. Through pharmacokinetics and pharmacodynamics assessment, the high intrinsic activity of DS-8500a was suggested to be one of the reasons for the greater glucose lowering effect in the nSTZ rats. DS-8500a is a useful compound among GPR119 agonists that can maximize the potential benefit of GPR119 in type 2 diabetes.
Assuntos
Benzamidas/farmacologia , Ciclopropanos/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/metabolismo , Secreção de Insulina/efeitos dos fármacos , Oxidiazóis/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Regulação para Cima/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Mesilatos/farmacologia , Camundongos , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Tetrazóis/farmacologia , Tiazóis/farmacologiaRESUMO
BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a key molecule in the regulation of both angiogenesis and vascular permeability. However, it is known that overproduction of VEGF induces abnormal blood vessel formation and these vessels cause several disease pathologies, such as diabetic retinopathy. The purpose of this study was to find novel vasoactive compounds which have different properties from VEGF. METHODS/RESULTS: We screened a natural product library using a co-culture angiogenic assay of endothelial cells and fibroblasts. By focusing on morphological changes of endothelial cells, we isolated the novel compounds vestaine A1 and vestaine B1 from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 63697. Vestaine A1 enhanced tube formation of endothelial cells in Matrigel and suppressed cell death induced by serum deprivation. Vestaine A1 activated both MEK1/2 and PI-3 kinase pathways independently of the VEGF pathway in a dose- and time-dependent fashion. Finally, vestaine A1 potently suppressed VEGF-induced vascular permeability both in vitro and in vivo. CONCLUSION: Vestaine A1 has the potential to exhibit both pro-angiogenic and anti-permeability properties, and would therefore be useful for therapeutic treatment for abnormal vascular permeability-related diseases.
Assuntos
Acetilcisteína/análogos & derivados , Indutores da Angiogênese/farmacologia , Produtos Biológicos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Streptomyces/química , Acetilcisteína/química , Acetilcisteína/isolamento & purificação , Acetilcisteína/farmacologia , Indutores da Angiogênese/química , Indutores da Angiogênese/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Permeabilidade Capilar/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Colágeno/química , Combinação de Medicamentos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Laminina/química , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteoglicanas/química , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/isolamento & purificação , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Streptomyces/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs.
Assuntos
Antígenos de Diferenciação , Antígeno CD47 , Imunoconjugados , Receptores Imunológicos , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/imunologia , Animais , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Imunológicos/imunologia , Humanos , Camundongos , Imunoconjugados/farmacologia , Antígenos de Diferenciação/imunologia , Linhagem Celular Tumoral , Feminino , Trastuzumab/farmacologia , Inibidores da Topoisomerase I/farmacologia , Imunoterapia/métodos , Camundongos Endogâmicos BALB CRESUMO
G-protein-coupled receptor class 5 member D (GPRC5D) is detected in malignant plasma cells in approximately 90% of patients diagnosed with multiple myeloma (MM). Here, we constructed BsAb5003, a novel humanized bispecific monoclonal antibody targeting CD3 and GPRC5D, and evaluated its therapeutic impact on MM. BsAb5003 induced specific cytotoxicity of GPRC5D-positive MM cells with concomitant T cell activation and cytokine release. The efficacy of BsAb5003 was associated with GPRC5D expression levels in MM cell lines. Flow cytometry analysis of bone marrow mononuclear cells (BMMNCs) from 49 MM patients revealed that GPRC5D was expressed in a wide population of MM patients, including heavily treated and high-risk patients. In ex vivo assays using BMMNCs, BsAb5003 induced potent efficacy against CD138 + MM cells in both newly diagnosed and relapsed/refractory patient samples in a GPRC5D expression-dependent manner. BsAb5003 significantly enhanced T cell activation and cytokine production in combination with immunomodulatory drugs (IMiDs) against MM cell lines. BsAb5003 also demonstrated significant inhibition of in vivo tumor growth by recruiting T cells. Taken together, these results suggest that T cell-redirecting bispecific antibody targeting GPRC5D as monotherapy and combination therapy with IMiDs could be a highly potent and effective treatment approach for a wide population of MM patients.
Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Humanos , Anticorpos Biespecíficos/uso terapêutico , Citocinas/metabolismo , Agentes de Imunomodulação , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Receptores Acoplados a Proteínas G , Linfócitos TRESUMO
Taurine (2-aminoethanesulfonic acid), a sulfur-containing ß-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. ß-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. ß-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by ß-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine.
Assuntos
Proteínas de Transporte/genética , Expressão Gênica/efeitos dos fármacos , Taurina/farmacologia , Região 5'-Flanqueadora/efeitos dos fármacos , Aminoácidos/farmacologia , Células CACO-2 , Perfilação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , Transcrição Gênica/efeitos dos fármacos , Regulação para CimaRESUMO
We analyzed changes in gene expression in Caco-2 cells cocultured with THP-1 cells over time periods of 0, 1, 3, 6, 24, and 48 h using a DNA microarray. Differentially expressed genes extracted with maSigPro indicated that an early defense response and cell death occured at the same time, causing an inflammatory condition.
Assuntos
Apoptose/genética , DNA/genética , Imunidade/genética , Monócitos/citologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células CACO-2 , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Monócitos/metabolismo , Regulação para Cima/genéticaRESUMO
Effects of dietary taurine on the experimental colitis induced by dextran sulfate sodium (DSS) were studied. C57BL/6 mice administrated taurine or placebo for 5 days were given 3% DSS to induce acute. The colitis was as-sessed using indices such as diarrhea/bleeding scores, colon length change, histological score and tissue myeloperoxidase (MPO) activity. Further, tissue mRNA levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2, were determined by real-time PCR. Taurine supplementation significantly attenuated the severity of diarrhea, colon shortening, histological score, MPO activity elevation and abnormal MIP-2 gene expression, indicating that taurine prevents DSS-induced colitis. Taurine also inhibited the TNF-alpha-induced secretion of IL-8 (a human homologue of MIP-2) from human intestinal epithelial Caco-2 cells. Inhibition of chemokine secretion from intestinal cells may be involved in the mechanisms underlying the cytoprotective function of taurine in the intestinal epithelium.
Assuntos
Sulfato de Dextrana/antagonistas & inibidores , Dieta , Doenças Inflamatórias Intestinais/prevenção & controle , Taurina/farmacologia , Animais , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/enzimologia , Interleucina-8/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Peroxidase/metabolismo , Taurina/administração & dosagemRESUMO
In the course of our screening program for vasoactive compounds using co-culture assay of endothelial cells and fibroblast cells, potent activity was detected in the cultured broth of Incrucipulum sp. SANK 10414. Two active compounds, F-36316 A and B, and a non-active homolog, F-36316 C, were isolated from the broth. The structures of F-36316 A, B and C were elucidated by physicochemical data and spectral analyses, and found to be new 3-acylated tetronic acid homologs. F-36316 A and B induced morphological changes of endothelial cells different from vascular endothelial growth factor (VEGF) or vestaines in the assay with EC50 values of 1.8 and 11.7 µM, respectively. Furthermore, F-36316 A and B suppressed VEGF-induced vascular permeability induction in mice.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Produtos Biológicos/isolamento & purificação , Permeabilidade Capilar/efeitos dos fármacos , Meios de Cultura/química , Células Endoteliais/efeitos dos fármacos , Animais , Produtos Biológicos/química , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Análise EspectralRESUMO
We conducted a screening program for vasoactive compounds and detected a potent activity in the cultured broth of Streptomyces sp. SANK 63697. From the cultured broth, two active compounds, vestaine A1 and B1, were isolated. The structures of these compounds were elucidated by physicochemical data and spectral analyses, and found to be new compounds.
Assuntos
Acetilcisteína/análogos & derivados , Compostos de Amônio Quaternário/metabolismo , Streptomyces/metabolismo , Acetilcisteína/química , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/metabolismo , Fármacos Cardiovasculares/farmacologia , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Estrutura Molecular , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologiaRESUMO
We have reported previously that apoptosis of intestinal epithelial Caco-2 cells is induced by co-culturing with human macrophage-like THP-1 cells, mainly via the action of TNFα (tumour necrosis factor α) secreted from THP-1 cells [Satsu, Ishimoto, Nakano, Mochizuki, Iwanaga and Shimizu (2006) Exp. Cell Res. 312, 3909-3919]. Our recent DNA microarray analysis of co-cultured Caco-2 cells showed that IEX-1 (immediate early-response gene X-1) is the most significantly increased gene during co-culture [Ishimoto, Nakai, Satsu, Totsuka and Shimizu (2010) Biosci. Biotechnol. Biochem. 74, 437-439]. Hence, we investigated the role of IEX-1 in the co-culture-induced damage of Caco-2 cells. We showed that IEX-1 expression induced in Caco-2 cells was suppressed by anti-TNFα antibody treatment. Experiments using IEX-1-overexpressing and -knockdown Caco-2 cells suggested that IEX-1 was involved in the suppression of Caco-2 cell damage. Increases in caspase 3 activity and TNFR1 (TNF receptor 1) mRNA expression were shown in IEX-1-knockdown Caco-2 cells, suggesting that IEX-1 plays a role in the suppression of apoptosis and protects cells by controlling sensitivity to TNFα under both normal and inflammatory conditions.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Citoproteção , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/fisiologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Células CACO-2 , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citoproteção/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Mucosa Intestinal/patologia , Macrófagos/patologia , Proteínas de Membrana/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Intestinal epithelial cells interact with immune cells located in the intestinal epithelium via soluble factors. An in vitro model system using coculture was constructed to analyze the effect of macrophages on intestinal epithelial cells, and human intestinal epithelial-like Caco-2 monolayers and activated macrophage-like THP-1 cells were used in this study. Coculturing with THP-1 cells resulted in an increase of lactate dehydrogenase release from Caco-2 and a decrease in the transepithelial electrical resistance of the monolayers, showing that coculturing with THP-1 induced cell damage to Caco-2 cells. This disruption was significantly suppressed by adding anti-TNF-alpha antibody and etanercept, strongly suggesting that TNF-alpha secreted from THP-1 had caused cell damage to Caco-2 monolayers. The disrupted Caco-2 monolayers showed both apoptotic and necrotic characteristics by morphological and biochemical analyses. TNFRI and NF-kappaB seem to have been involved in this regulation. It is suggested that this phenomenon is similar in some respects to that observed with IBD and that this in vitro coculture system could be a good model for searching for the drugs or food substances that can be used to treat or prevent IBD.