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The objective of this work was to evaluate the safety and efficacy of a recombinant, subunit SARS-CoV-2 animal vaccine in cats against virulent SARS-CoV-2 challenge. Two groups of cats were immunized with two doses of either a recombinant SARS-CoV-2 spike protein vaccine or a placebo, administered three weeks apart. Seven weeks after the second vaccination, both groups of cats were challenged with SARS-CoV-2 via the intranasal and oral routes simultaneously. Animals were monitored for 14 days post-infection for clinical signs and viral shedding before being humanely euthanized and evaluated for macroscopic and microscopic lesions. The recombinant SARS-CoV-2 spike protein subunit vaccine induced strong serologic responses post-vaccination and significantly increased neutralizing antibody responses post-challenge. A significant difference in nasal and oral viral shedding, with significantly reduced virus load (detected using RT-qPCR) was observed in vaccinates compared to mock-vaccinated controls. Duration of nasal, oral, and rectal viral shedding was also significantly reduced in vaccinates compared to controls. No differences in histopathological lesion scores were noted between the two groups. Our findings support the safety and efficacy of the recombinant spike protein-based SARS-CoV-2 vaccine which induced high levels of neutralizing antibodies and reduced nasal, oral, and rectal viral shedding, indicating that this vaccine will be efficacious as a COVID-19 vaccine for domestic cats.
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Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.
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Aciltransferasas/antagonistas & inhibidores , Isoindoles/química , Sulfonamidas/química , Aciltransferasas/genética , Animales , Células Cultivadas , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Perros , Humanos , Isoindoles/farmacocinética , Isoindoles/farmacología , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Triglicéridos/biosíntesisRESUMEN
The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Receptor TIE-2/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Estructura Molecular , Neoplasias/enzimología , Neoplasias/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/síntesis química , Pirimidinas/química , Pirroles/síntesis química , Pirroles/química , Ratas , Ratas Sprague-Dawley , Receptor TIE-2/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibitors of the Hedgehog signaling pathway have generated a great deal of interest in the oncology area due to the mounting evidence of their potential to provide promising therapeutic options for patients. Herein, we describe the discovery strategy to overcome the issues inherent in lead structure 1 that resulted in the identification of Smoothened inhibitor 1-((2R,4R)-2-(1H-benzo[d]imidazol-2-yl)-1-methylpiperidin-4-yl)-3-(4-cyanophenyl)urea (PF-04449913, 26), which has been advanced to human clinical studies.
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The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.
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Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Infecciones/inmunología , Quinolinas/farmacología , Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Unión Proteica/efectos de los fármacos , Resonancia por Plasmón de SuperficieRESUMEN
PURPOSE: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor. EXPERIMENTAL DESIGN: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (K(d) = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties. RESULTS: This antibody blocks binding of IGF-1 to its receptor (IC(50) 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC(50) 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. CONCLUSION: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.
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Anticuerpos Monoclonales/inmunología , Neoplasias Colorrectales/patología , Mieloma Múltiple/patología , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/fisiología , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Modelos Animales de Enfermedad , Regulación hacia Abajo , Inmunoglobulina G/inmunología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Macaca fascicularis , Ratones , Fosforilación , Receptor IGF Tipo 1/biosíntesis , Transducción de Señal , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Bencimidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Becaplermina , Procesos de Crecimiento Celular/efectos de los fármacos , Femenino , Glioblastoma/irrigación sanguínea , Glioblastoma/tratamiento farmacológico , Glioblastoma/enzimología , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/enzimología , Paclitaxel/administración & dosificación , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-sis , Ratas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have previously described osteoblast/osteocyte factor 45 (OF45), a novel bone-specific extracellular matrix protein, and demonstrated that its expression is tightly linked to mineralization and bone formation. In this report, we have cloned and characterized the mouse OF45 cDNA and genomic region. Mouse OF45 (also called MEPE) was similar to its rat orthologue in that its expression was increased during mineralization in osteoblast cultures and the protein was highly expressed within the osteocytes that are imbedded within bone. To further determine the role of OF45 in bone metabolism, we generated a targeted mouse line deficient in this protein. Ablation of OF45 resulted in increased bone mass. In fact, disruption of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the bone histomorphometric results, we also determined that OF45 knockout osteoblasts produced significantly more mineralized nodules in ex vivo cell cultures than did wild type osteoblasts. Osteoclastogenesis and bone resorption in ex vivo cultures was unaffected by OF45 mutation. We conclude that OF45 plays an inhibitory role in bone formation in mouse.