RESUMO
In 2012, the U.S. Food & Drug Administration (FDA) published an established list of 93 harmful and potentially harmful constituents (HPHCs) targeting four tobacco product types (cigarettes, cigarette tobacco, roll-your-own tobacco, smokeless tobacco). In 2016, the FDA finalized the deeming rule to regulate electronic nicotine delivery systems (ENDS). However, knowledge gaps exist regarding whether certain HPHCs are present in ENDS e-liquids and aerosols. We identified and addressed these gaps by conducting literature searches and then experimentally quantifying HPHCs in the e-liquid and aerosol of 37 ENDS brands based on gaps in the literature. The literature searches identified 66 e-liquid HPHCs and 68 aerosol HPHCs that have limited to no information regarding the quantifiability of these constituents. A contracted ISO 17025 accredited laboratory performed the HPHC quantifications. The availability of validated analytical methods in the contracted laboratory determined the HPHCs included in the study scope (63/66 for e-liquids, 64/68 for aerosols). Combining the results from the quantifications and literature searches, 36 (39%) and 34 (37%) HPHCs were found quantifiable (≥limit of quantification [LOQ]) in ENDS e-liquids and aerosols, respectively, with 25 HPHCs being quantifiable in both matrices. Quantifiability results imply potential HPHC transfers between matrices, leaching from components, or formations from aerosol generation. The study results can inform the scientific basis for manufacturers and regulators regarding regulatory requirements for HPHC reporting. The HPHC quantities can also inform evaluations of the public health impact of ENDS and public communications regarding ENDS health risks.
Assuntos
Aerossóis , Sistemas Eletrônicos de Liberação de Nicotina , Aerossóis/análise , Aerossóis/química , Humanos , Nicotina/análiseRESUMO
Adenosine suppresses antitumor immune responses via A2a and A2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5'-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A2aR/A2bR antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 µM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.
Assuntos
Antagonistas de Receptores Purinérgicos P1/administração & dosagem , Administração Oral , Adolescente , Adulto , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Método Duplo-Cego , Feminino , Interações Alimento-Droga , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Antagonistas de Receptores Purinérgicos P1/sangue , Antagonistas de Receptores Purinérgicos P1/farmacocinética , Adulto JovemRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11c+CD206+ immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c+CD206+ myeloid cells correlated with systemic insulin resistance. CD11c+CD206+ cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11b+CD11c+F4/80+ monocytes, which are functional homologs of human CD11c+CD206+ cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11c+CD206+ myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.
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Quimiotaxia , Resistência à Insulina , Fígado/metabolismo , Monócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores CCR2/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Glicemia/metabolismo , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Quimiocina CCL2/metabolismo , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/genética , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
A new tobacco filler Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST) in September 2016 with certified and reference mass fraction values for nicotine, N-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and volatiles. The constituents have been determined by multiple analytical methods with measurements at NIST and at the Centers for Disease Control and Prevention, and with confirmatory measurements by commercial laboratories. This effort highlights the development of the first SRM for reduced nicotine and reduced tobacco-specific nitrosamines with certified values for composition.
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Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Produtos do Tabaco/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Congelamento , Cromatografia Gasosa-Espectrometria de Massas/normas , Nicotina/análise , Nicotina/normas , Nitrosaminas/análise , Nitrosaminas/normas , Transição de Fase , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Produtos do Tabaco/normas , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/normasRESUMO
There are an estimated 8 million users of smokeless tobacco products (STPs) in the United States, and yet limited data on microbial populations within these products exist. To better understand the potential microbiological risks associated with STP use, a study was conducted to provide a baseline microbiological profile of STPs. A total of 90 samples, representing 15 common STPs, were purchased in metropolitan areas in Little Rock, AR, and Washington, DC, in November 2012, March 2013, and July 2013. Bacterial populations were evaluated using culture, pyrosequencing, and denaturing gradient gel electrophoresis (DGGE). Moist-snuff products exhibited higher levels of bacteria (average of 1.05 × 106 CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 × 101 CFU/g STP) and some chewing tobacco products (average of 2.54 × 105 CFU/g STP). The most common species identified by culturing were Bacillus pumilus, B. licheniformis, B. safensis, and B. subtilis, followed by members of the genera Oceanobacillus, Staphylococcus, and Tetragenococcus. Pyrosequencing analyses of the 16S rRNA genes identified the genera Tetragenococcus, Carnobacterium, Lactobacillus, Geobacillus, Bacillus, and Staphylococcus as the predominant taxa. Several species identified are of possible concern due to their potential to cause opportunistic infections and reported abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. This report provides a microbiological baseline to help fill knowledge gaps associated with microbiological risks of STPs and to inform potential regulations regarding manufacture and testing of STPs. IMPORTANCE: It is estimated that there 8 million users of smokeless tobacco products (STPs) in the United States; however, there are limited data on microbial populations that exist within these products. The current study was undertaken to better understand the potential microbiological risks associated with STP use and provide a baseline microbiological profile of STPs. Several bacterial species were identified that are of possible concern due to their potential to cause opportunistic infections. In addition, some species have abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. Overall, this report provides a microbiological baseline to help fill knowledge gaps related to the microbiological risks of STPs and to inform potential regulations regarding the manufacture and testing of STPs.
Assuntos
Bactérias/isolamento & purificação , Tabaco sem Fumaça/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Qualidade de Produtos para o Consumidor , Nitratos/metabolismo , Nitritos/metabolismo , Estados UnidosRESUMO
The aim of this study is to explore how differences in cigarette physical design parameters influence tar, nicotine, and carbon monoxide (TNCO) yields in mainstream smoke (MSS) using the International Organization of Standardization (ISO) smoking regimen. Standardized smoking methods were used to evaluate 50 U.S. domestic brand cigarettes and a reference cigarette representing a range of TNCO yields in MSS collected from linear smoking machines using a nonintense smoking regimen. Multivariate statistical methods were used to form clusters of cigarettes based on their ISO TNCO yields and then to explore the relationship between the ISO generated TNCO yields and the nine cigarette physical design parameters between and within each cluster simultaneously. The ISO generated TNCO yields in MSS are 1.1-17.0 mg tar/cigarette, 0.1-2.2 mg nicotine/cigarette, and 1.6-17.3 mg CO/cigarette. Cluster analysis divided the 51 cigarettes into five discrete clusters based on their ISO TNCO yields. No one physical parameter dominated across all clusters. Predicting ISO machine generated TNCO yields based on these nine physical design parameters is complex due to the correlation among and between the nine physical design parameters and TNCO yields. From these analyses, it is estimated that approximately 20% of the variability in the ISO generated TNCO yields comes from other parameters (e.g., filter material, filter type, inclusion of expanded or reconstituted tobacco, and tobacco blend composition, along with differences in tobacco leaf origin and stalk positions and added ingredients). A future article will examine the influence of these physical design parameters on TNCO yields under a Canadian Intense (CI) smoking regimen. Together, these papers will provide a more robust picture of the design features that contribute to TNCO exposure across the range of real world smoking patterns.
Assuntos
Modelos Estatísticos , Fumaça/análise , Produtos do Tabaco/análise , Monóxido de Carbono/análise , Monóxido de Carbono/normas , Cooperação Internacional , Análise Multivariada , Nicotina/análise , Nicotina/normas , Padrões de Referência , Alcatrões/análise , Alcatrões/normas , Produtos do Tabaco/normasRESUMO
INTRODUCTION: A significant portion of the increased risk of cancer and respiratory disease from exposure to cigarette smoke is attributed to volatile organic compounds (VOCs). In this study, 21 VOCs were quantified in mainstream cigarette smoke from 50U.S. domestic brand varieties that included high market share brands and 2 Kentucky research cigarettes (3R4F and 1R5F). METHODS: Mainstream smoke was generated under ISO 3308 and Canadian Intense (CI) smoking protocols with linear smoking machines with a gas sampling bag collection followed by solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) analysis. RESULTS: For both protocols, mainstream smoke VOC amounts among the different brand varieties were strongly correlated between the majority of the analytes. Overall, Pearson correlation (r) ranged from 0.68 to 0.99 for ISO and 0.36 to 0.95 for CI. However, monoaromatic compounds were found to increase disproportionately compared to unsaturated, nitro, and carbonyl compounds under the CI smoking protocol where filter ventilation is blocked. CONCLUSIONS: Overall, machine generated "vapor phase" amounts (µg/cigarette) are primarily attributed to smoking protocol (e.g., blocking of vent holes, puff volume, and puff duration) and filter ventilation. A possible cause for the disproportionate increase in monoaromatic compounds could be increased pyrolysis under low oxygen conditions associated with the CI protocol. IMPLICATIONS: This is the most comprehensive assessment of volatile organic compounds (VOCs) in cigarette smoke to date, encompassing 21 toxic VOCs, 50 different cigarette brand varieties, and 2 different machine smoking protocols (ISO and CI). For most analytes relative proportions remain consistent among U.S. cigarette brand varieties regardless of smoking protocol, however the CI smoking protocol did cause up to a factor of 6 increase in the proportion of monoaromatic compounds. This study serves as a basis to assess VOC exposure as cigarette smoke is a principle source of overall population-level VOC exposure in the United States.
Assuntos
Poluentes Atmosféricos/análise , Nicotiana/química , Fumar , Compostos Orgânicos Voláteis/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Kentucky , Rotulagem de Produtos , Padrões de Referência , Fumaça/análise , Estados UnidosRESUMO
While it has long been established that the chemokine receptor CCR9 and its ligand CCL25 are essential for the movement of leukocytes into the small intestine and the development of small-intestinal inflammation, the role of this chemokine-receptor pair in colonic inflammation is not clear. Toward this end, we compared colonic CCL25 protein levels in healthy individuals to those in patients with ulcerative colitis. In addition, we determined the effect of CCR9 pharmacological inhibition in the mdr1a(-/-) mouse model of ulcerative colitis. Colon samples from patients with ulcerative colitis had significantly higher levels of CCL25 protein compared to healthy controls, a finding mirrored in the mdr1a(-/-) mice. In the mdr1a(-/-) mice, CCR9 antagonists significantly decreased the extent of wasting and colonic remodeling and reduced the levels of inflammatory cytokines in the colon. These findings indicate that the CCR9:CCL25 pair plays a causative role in ulcerative colitis and suggest that CCR9 antagonists will provide a therapeutic benefit in patients with colonic inflammation.
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Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Receptores CCR/antagonistas & inibidores , Receptores CCR/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colite Ulcerativa/genética , Feminino , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Sulfonamidas/uso terapêuticoRESUMO
The concentration of CXCL12/SDF-1 in the bloodstream is tightly regulated, given its central role in leucocyte and stem/progenitor cell egress from bone marrow and recruitment to sites of inflammation or injury. The mechanism responsible for this regulation is unknown. Here we show that both genetic deletion and pharmacological inhibition of CXCR7, a high-affinity CXCL12 receptor, caused pronounced increases in plasma CXCL12 levels. The rise in plasma CXCL12 levels was associated with an impairment in the ability of leucocytes to migrate to a local source of CXCL12. Using a set of complementary and highly sensitive techniques, we found that CXCR7 protein is expressed at low levels in multiple organs in both humans and mice. In humans, CXCR7 was detected primarily on venule endothelium and arteriole smooth muscle cells. CXCR7 expression on venule endothelium was also documented in immunodeficient mice and CXCR7(+/lacZ) mice. The vascular expression of CXCR7 therefore gives it immediate access to circulating CXCL12. These studies suggest that endothelial CXCR7 regulates circulating CXCL12 levels and that CXCR7 inhibitors might be used to block CXCL12-mediated cell migration for therapeutic purposes.
Assuntos
Quimiocina CXCL12/imunologia , Endotélio Vascular/imunologia , Regulação da Expressão Gênica/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Receptores CXCR/imunologia , Animais , Movimento Celular/imunologia , Quimiocina CXCL12/sangue , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos , Especificidade de Órgãos/imunologia , Receptores CXCR/biossínteseRESUMO
T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) is an inhibitory receptor on immune cells that outcompetes an activating receptor, CD226, for shared ligands. Tumor-infiltrating lymphocytes express TIGIT and CD226 on regulatory T cells (Treg) and on CD8+ T cells with tumor-reactive or exhausted phenotypes, supporting the potential of therapeutically targeting TIGIT to enhance antitumor immunity. To optimize the efficacy of therapeutic antibodies against TIGIT, it is necessary to understand IgG Fc (Fcγ) receptor binding for therapeutic benefit. In this study, we showed that combining Fc-enabled (Fce) or Fc-silent (Fcs) anti-TIGIT with antiprogrammed cell death protein 1 in mice resulted in enhanced control of tumors by differential mechanisms: Fce anti-TIGIT promoted the depletion of intratumoral Treg, whereas Fcs anti-TIGIT did not. Despite leaving Treg numbers intact, Fcs anti-TIGIT potentiated the activation of tumor-specific exhausted CD8+ populations in a lymph node-dependent manner. Fce anti-TIGIT induced antibody-dependent cell-mediated cytotoxicity against human Treg in vitro, and significant decreases in Treg were measured in the peripheral blood of patients with phase I solid tumor cancer treated with Fce anti-TIGIT. In contrast, Fcs anti-TIGIT did not deplete human Treg in vitro and was associated with anecdotal objective clinical responses in two patients with phase I solid tumor cancer whose peripheral Treg frequencies remained stable on treatment. Collectively, these data provide evidence for pharmacologic activity and antitumor efficacy of anti-TIGIT antibodies lacking the ability to engage Fcγ receptor. SIGNIFICANCE: Fcs-silent anti-TIGIT antibodies enhance the activation of tumor-specific pre-exhausted T cells and promote antitumor efficacy without depleting T regulatory cells.
Assuntos
Receptores Imunológicos , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Camundongos , Receptores Imunológicos/imunologia , Receptores Imunológicos/antagonistas & inibidores , Humanos , Linfócitos do Interstício Tumoral/imunologia , Feminino , Linfócitos T CD8-Positivos/imunologia , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Neoplasias/imunologia , Neoplasias/tratamento farmacológicoRESUMO
AB598 is a CD39 inhibitory antibody being pursued for the treatment of solid tumors in combination with chemotherapy and immunotherapy. CD39 metabolizes extracellular ATP (eATP), an alarmin capable of promoting anti-tumor immune responses, into adenosine, an immuno-inhibitory metabolite. By inhibiting CD39, the consumption of eATP is reduced, resulting in a pro-inflammatory milieu in which eATP can activate myeloid cells to promote anti-tumor immunity. The preclinical characterization of AB598 provides a mechanistic rationale for combining AB598 with chemotherapy in the clinic. Chemotherapy can induce ATP release from tumor cells and, when preserved by AB598, both chemotherapy-induced eATP and exogenously added ATP promote the function of monocyte-derived dendritic cells via P2Y11 signaling. Inhibition of CD39 in the presence of ATP can promote inflammasome activation in in vitro-derived macrophages, an effect mediated by P2X7. In a MOLP8 murine xenograft model, AB598 results in full inhibition of intratumoral enzymatic activity, an increase in intratumoral ATP, a decrease of extracellular CD39 on tumor cells, and ultimately, control of tumor growth. In cynomolgus monkeys, systemically dosed AB598 results in effective enzymatic inhibition in tissues, full peripheral and tissue target engagement, and a reduction in cell surface CD39 both in tissues and in the periphery. Taken together, these data support a promising therapeutic strategy of harnessing the eATP generated by standard-of-care chemotherapies to prime the tumor microenvironment for a productive anti-tumor immune response.
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INTRODUCTION: Chemokines may directly participate in the pathogenesis of neonatal chronic hypoxia-induced pulmonary hypertension (PH). Although stromal-derived factor-1 (SDF-1) has been shown to be involved in PH, the role of its most recently discovered receptor, chemokine receptor type 7 (CXCR7), remains unclear. We sought to determine whether antagonism of the CXCR7 receptor would decrease pulmonary vascular remodeling in newborn mice exposed to chronic hypoxia by decreasing pulmonary vascular cell proliferation. METHODS: Neonatal mice were exposed to hypoxia (fractional inspired oxygen concentration = 0.12) or room air (RA) for 2 wk. After 1 wk of exposure, mice received daily injections of placebo or a CXCR7 antagonist (CCX771) from postnatal day 7 (P7) to P14. Right ventricular systolic pressure (RVSP), the ratio of the weight of the right ventricle to left ventricle + septum (RV/LV + S), and pulmonary vascular cell proliferation and remodeling were determined at P14. RESULTS: As compared with mice exposed to RA, hypoxia placebo mice had a significant increase in the lung protein expression of CXCR7. Although hypoxic placebo-treated mice had a significant increase in RVSP, RV/LV+S, and pulmonary vascular cell proliferation and remodeling, the administration of CCX771 markedly decreased these changes. DISCUSSION: These results indicate that antagonism of CXCR7 may be a potent strategy to decrease PH and vascular remodeling.
Assuntos
Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/prevenção & controle , Hipóxia/complicações , Receptores CXCR/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Artérias/efeitos dos fármacos , Artérias/patologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Hipertensão Pulmonar/patologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos , Modelos Animais , Gravidez , Receptores CXCR/efeitos dos fármacos , Receptores CXCR/metabolismoRESUMO
The U.S. Food and Drug Administration established a list of 93 harmful and potentially harmful constituents (HPHCs) in tobacco products. While HPHCs are required to be submitted for tobacco products, knowledge gaps exist regarding which tobacco-containing tobacco product (TCTP, i.e., tobacco products that contain tobacco(s) as a component) types (cigarettes, cigars, roll-your-own tobaccos [RYOs], pipe tobaccos [pipes], smokeless tobacco products [STPs], waterpipe tobaccos [waterpipes]) and matrices (filler, smoke) contain which HPHCs. This study identified and addressed such gaps by conducting literature searches and measuring the amount of HPHCs in TCTP types and matrices. First, literature searches, performed for cigarettes, RYOs, and STPs for publications up to 2014 and for cigars, pipes, and waterpipes for publications up to 2016, identified knowledge gaps for the 93 HPHCs (or 119 HPHCs if cresols [o-, m-, p-cresol] are counted as 3 and chlorinated dioxins/furans as 25) across TCTP types and matrices. Then, three ISO 17025 accredited laboratories including two subcontracted laboratories performed the HPHC quantifications. Inclusion of the HPHCs, TCTP types, and matrices in the study scope was also determined by the availability of validated analytical methods in each laboratory. Eleven (9%) HPHCs are quantifiable in all brands for all TCTP types and matrices, 33 (28%) HPHCs are not quantifiable in any brands of any TCTP type and matrix, and 74 (63%) HPHCs are quantifiable only in some brands across TCTP types and matrices examined. Understanding the quantifiability of HPHCs in each TCTP type and matrix can inform the scientific basis for manufacturers regarding the regulatory requirements for reporting HPHCs. The quantity of HPHCs observed can also inform the evaluation of the public health impact of HPHCs and public communications regarding the health risks of tobacco products.
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T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell-mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti-PD-1, to improve cancer patient outcomes.
Assuntos
Melanoma , Receptor de Morte Celular Programada 1 , Adenosina/metabolismo , Adenosina/farmacologia , Adenosina/uso terapêutico , Animais , Humanos , Inibidores de Checkpoint Imunológico , Melanoma/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Microambiente TumoralRESUMO
Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.
Assuntos
Amidas/química , Classe Ib de Fosfatidilinositol 3-Quinase/química , Desenho de Fármacos , Descoberta de Drogas , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirimidinas/química , Animais , Humanos , Masculino , Simulação de Acoplamento Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. Naproxen exhibits gastrointestinal toxicity, but its cardiovascular toxicity may be reduced compared with other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis, and x-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition, and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH(2) for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and x-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 Å resolution, respectively. The combination of mutagenesis, structure analysis, and x-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2.
Assuntos
Anti-Inflamatórios não Esteroides/química , Ciclo-Oxigenase 2/química , Naproxeno/química , Substituição de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação ProteicaRESUMO
The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.
Assuntos
Aldeído Liases/genética , Aldeído Liases/metabolismo , Caprilatos/metabolismo , Engenharia de Proteínas/métodos , Aldeído Liases/química , Catálise , DNA Bacteriano/química , DNA Bacteriano/genética , Evolução Molecular Direcionada/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Reação em Cadeia da PolimeraseRESUMO
The chemokine system represents a diverse group of G protein-coupled receptors responsible for orchestrating cell recruitment under both homeostatic and inflammatory conditions. Chemokine receptor 9 (CCR9) is a chemokine receptor known to be central for migration of immune cells into the intestine. Its only ligand, CCL25, is expressed at the mucosal surface of the intestine and is known to be elevated in intestinal inflammation. To date, there are no reports of small-molecule antagonists targeting CCR9. We report, for the first time, the discovery of a small molecule, CCX282-B, which is an orally bioavailable, selective, and potent antagonist of human CCR9. CCX282-B inhibited CCR9-mediated Ca(2+) mobilization and chemotaxis on Molt-4 cells with IC(50) values of 5.4 and 3.4 nM, respectively. In the presence of 100% human serum, CCX282-B inhibited CCR9-mediated chemotaxis with an IC(50) of 33 nM, and the addition of α1-acid glycoprotein did not affect its potency. CCX282-B inhibited chemotaxis of primary CCR9-expressing cells to CCL25 with an IC(50) of 6.8 nM. CCX282-B was an equipotent inhibitor of CCL25-directed chemotaxis of both splice forms of CCR9 (CCR9A and CCR9B) with IC(50) values of 2.8 and 2.6 nM, respectively. CCX282-B also inhibited mouse and rat CCR9-mediated chemotaxis. Inhibition of CCR9 with CCX282-B results in normalization of Crohn's disease such as histopathology associated with the TNF(ΔARE) mice. Analysis of the plasma level of drug associated with this improvement provides an understanding of the pharmacokinetic/pharmacodynamic relationship for CCR9 antagonists in the treatment of intestinal inflammation.
Assuntos
Fármacos Gastrointestinais/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Receptores CCR/antagonistas & inibidores , Sulfonamidas/farmacologia , Administração Oral , Animais , Cálcio/metabolismo , Linhagem Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Fármacos Gastrointestinais/farmacocinética , Humanos , Ileíte/induzido quimicamente , Ileíte/tratamento farmacológico , Ileíte/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ensaio Radioligante , Ratos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The successful application of immunotherapy in the treatment of cancer relies on effective engagement of immune cells in the tumor microenvironment. Phosphoinositide 3-kinase γ (PI3Kγ) is highly expressed in tumor-associated macrophages, and its expression levels are associated with tumor immunosuppression and growth. Selective inhibition of PI3Kγ offers a promising strategy in immuno-oncology, which has led to the development of numerous potent PI3Kγ inhibitors with variable selectivity profiles. To facilitate further investigation of the therapeutic potential of PI3Kγ inhibition, we required a potent and PI3Kγ-selective tool compound with sufficient metabolic stability for use in future in vivo studies. Herein, we describe some of our efforts to realize this goal through the systematic study of SARs within a series of 7-azaindole-based PI3Kγ inhibitors. The large volume of data generated from this study helped guide our subsequent lead optimization efforts and will inform further development of PI3Kγ-selective inhibitors for use in immunomodulation.
RESUMO
The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 µM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.