RESUMO
We developed a focused series of original phenyl-glycinamide derivatives which showed potent activity across in vivo mouse seizure models, namely, maximal electroshock (MES) and 6 Hz (using both 32 and 44 mA current intensities) seizure models. Following intraperitoneal (i.p.) administration, compound (R)-32, which was identified as a lead molecule, demonstrated potent protection against all seizure models with ED50 values of 73.9 mg/kg (MES test), 18.8 mg/kg (6 Hz, 32 mA test), and 26.5 mg/kg (6 Hz, 44 mA test). Furthermore, (R)-32 demonstrated efficacy in both the PTZ-induced kindling paradigm and the ivPTZ seizure threshold test. The expression of neurotrophic factors, such as mature brain-derived neurotrophic factor (mBDNF) and nerve growth factor (NGF), in the hippocampus and/or cortex of mice, and the levels of glutamate and GABA were normalized after PTZ-induced kindling by (R)-32. Importantly, besides antiseizure activity, (R)-32 demonstrated potent antinociceptive efficacy in formalin-induced pain, capsaicin-induced pain, as well as oxaliplatin- and streptozotocin-induced peripheral neuropathy in mice (i.p.). No influence on muscular strength and body temperature in mice was observed. Pharmacokinetic studies and in vitro ADME-Tox data (i.e., high metabolic stability in human liver microsomes, a weak influence on CYPs, no hepatotoxicity, satisfactory passive transport, etc.) proved favorable drug-like properties of (R)-32. Thermal stability of (R)-32 shown in thermogravimetry and differential scanning calorimetry gives the opportunity to develop innovative oral solid dosage forms loaded with this compound. The in vitro binding and functional assays indicated its multimodal mechanism of action. (R)-32, beyond TRPV1 antagonism, inhibited calcium and sodium currents at a concentration of 10 µM. Therefore, the data obtained in the current studies justify a more detailed preclinical development of (R)-32 for epilepsy and pain indications.
Assuntos
Analgésicos , Anticonvulsivantes , Convulsões , Animais , Analgésicos/farmacologia , Camundongos , Convulsões/tratamento farmacológico , Anticonvulsivantes/farmacologia , Anticonvulsivantes/química , Masculino , Glicina/farmacologia , Glicina/análogos & derivados , Glicina/química , Modelos Animais de Doenças , Eletrochoque , Humanos , Excitação Neurológica/efeitos dos fármacos , Pentilenotetrazol , Dor/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Descoberta de DrogasRESUMO
Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Pulmão/imunologia , Megacariócitos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , RNA-Seq , Análise de Célula ÚnicaRESUMO
The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.
Assuntos
Envelhecimento/fisiologia , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Animais , Medula Óssea/patologia , Caspase 1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos , Fagocitose , Fenótipo , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Receptor Tirosina Quinase AxlRESUMO
Lysophosphatidic acid (LPA) is produced by tumor cells and is present in the ascites fluid of ovarian cancer patients. To determine the role of endogenous LPA in the ovarian cancer cell line SKOV3, we treated cells with the LPA receptor antagonist VPC32183 and found that it inhibited cell growth and induced apoptosis. Exogenous LPA further stimulated ERK and Akt phosphorylation and NF-κB activity. To determine if reactive oxygen species (ROS), which have been implicated as second messengers in cell signaling, were also involved in LPA signaling, we treated cells with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), and antioxidants N-acetyl cysteine, EUK-134 and curcumin, and showed that all blocked LPA-dependent NF-κB activity and cell proliferation. DPI and EUK-134 also inhibited Akt and ERK phosphorylation. LPA was shown to stimulate dichlorofluorescein fluorescence, though not in the presence of DPI, apocynin (an inhibitor of NADPH oxidase), VPC32183, or PEG-catalase. Akt phosphorylation was also inhibited by PEG-catalase and apocynin. These data indicate that NADPH oxidase is a major source of ROS and H(2)O(2) is critical for LPA-mediated signaling. Thus, LPA acts as a growth factor and prevents apoptosis in SKOV3 cells by signaling through redox-dependent activation of ERK, Akt, and NF-κB-dependent signaling pathways.