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OBJECTIVES: This study aimed to compare antibody trajectories among individuals with SARS-CoV-2 hybrid and vaccine-induced immunity. METHODS: Danish adults receiving three doses of BTN162b2 or mRNA-1237 were included prior to first vaccination (Day 0). SARS-CoV-2 anti-spike IgG levels were assessed before each vaccine dose, at Day 90, Day 180, 28 days after 3rd vaccination (Day 251), Day 365, and prior to 4th vaccination (Day 535). SARS-CoV-2 PCR results were extracted from the national microbiology database. Mixed-effect multivariable linear regression investigated the impact of hybrid-immunity (stratified into 4 groups: no hybrid immunity, PCR+ prior to 3rd dose, PCR+ after 3rd dose and before Day 365, PCR+ after Day 365) on anti-spike IgG trajectories. RESULTS: A total of 4,936 individuals were included, 47% developed hybrid-immunity. Anti-spike IgG increases were observed in all groups at Day 251, with the highest levels in those PCR+ prior to 3rd dose (Geometric Mean; 535,647AU/mL vs. 374,665AU/mL with no hybrid-immunity, P<0.0001). Further increases were observed in participants who developed hybrid immunity after their 3rd dose. Anti-spike IgG levels declined from Day 251-535 in individuals without hybrid-immunity and in those who developed hybrid-immunity prior to their 3rd dose, with lower rate of decline in those with hybrid-immunity. CONCLUSION: Hybrid-immunity results in higher and more durable antibody trajectories in vaccinated individuals.
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Anticorpos Antivirais , Vacina BNT162 , Vacinas contra COVID-19 , COVID-19 , Imunoglobulina G , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Anticorpos Antivirais/sangue , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Masculino , Feminino , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pessoa de Meia-Idade , Adulto , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacina BNT162/imunologia , Vacina BNT162/administração & dosagem , Glicoproteína da Espícula de Coronavírus/imunologia , Dinamarca , Idoso , Vacinação , Adulto JovemRESUMO
BACKGROUND: Within a year of the SARS-CoV-2 pandemic, vaccines inducing a robust humoral and cellular immune response were implemented worldwide. However, emergence of novel variants and waning vaccine induced immunity led to implementation of additional vaccine boosters. METHODS: This prospective study evaluated the temporal profile of cellular and serological responses in a cohort of 639 SARS-CoV-2 vaccinated participants, of whom a large proportion experienced a SARS-CoV-2 infection. All participants were infection naïve at the time of their first vaccine dose. Proportions of SARS-CoV-2 Spike-specific T cells were determined after each vaccine dose using the Activation Induced Markers (AIM) assay, while levels of circulating SARS-CoV-2 antibodies were determined by the Meso Scale serology assay. RESULTS: We found a significant increase in SARS-CoV-2 Spike-specific CD4+ and CD8+ T cell responses following the third dose of a SARS-CoV-2 mRNA vaccine as well as enhanced CD8+ T cell responses after the fourth dose. Further, increased age was associated with a poorer response. Finally, we observed that SARS-CoV-2 infection boosts both the cellular and humoral immune response, relative to vaccine-induced immunity alone. CONCLUSION: Our findings highlight the boosting effect on T cell immunity of repeated vaccine administration. The combination of multiple vaccine doses and SARS-CoV-2 infections maintains population T cell immunity although with reduced levels in the elderly.
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PURPOSE OF REVIEW: Advancements in antiretroviral therapy (ART) have positively impacted the life expectancy and possibility of living a normal life for people with HIV-1. However, lifelong daily medication is necessary to prevent disease progression. To this end, immunotherapeutic strategies are being tested with the aim of developing a functional cure in which the immune system effectively controls HIV-1 in the absence of ART. RECENT FINDINGS: The most promising advances in achieving sustained HIV-1 remission or cure include broadly neutralizing antibodies (bNAbs) that are administered alone or in combination with other agents. Newer and more innovative approaches redirecting T cells or natural killer cells to kill HIV-1 infected cells have also shown promising results. Finally, multiple ongoing trials focus on combining bNAbs with other immune-directed therapies to enhance both innate and adaptive immunity. SUMMARY: While immunotherapies as an alternative to conventional ART have generally proven to be well tolerated, these therapeutic approaches have largely been unsuccessful in inducing ART-free control of HIV-1. However, promising results from recent trials involving bNAbs that have reported durable HIV-1 control among a subset of participants, provide reason for cautious optimism that we with further optimization of these treatment strategies may be able to achieve functional cure for HIV-1.
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Infecções por HIV , HIV-1 , Imunoterapia , Humanos , Infecções por HIV/imunologia , Infecções por HIV/terapia , HIV-1/imunologia , Imunoterapia/métodos , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêuticoRESUMO
BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in inflammatory bowel disease [IBD]. METHODS: We used dynamic adhesion and transmigration assays, as well as a humanised in vivo model of intestinal cell trafficking, to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD, cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD, and GPR15 also promoted T cell recruitment to the colon in humanised mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data, as well as observations in a cohort of patients treated with vedolizumab, suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective, impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.
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Adesão Celular , Doenças Inflamatórias Intestinais , Mucoproteínas , Receptores Acoplados a Proteínas G , Linfócitos T , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Camundongos , Linfócitos T/metabolismo , Linfócitos T/imunologia , Mucoproteínas/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Integrinas/metabolismo , Movimento Celular , Moléculas de Adesão Celular/metabolismo , Colo/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Integrina alfa4beta1/metabolismo , Imunoglobulinas/metabolismo , Feminino , Receptores de PeptídeosRESUMO
SARS-CoV-2 Omicron quickly spread globally, also in regions with high vaccination coverage, emphasizing the importance of exploring the immunological requirements for protection against Omicron breakthrough infection. The test-negative matched case-control study (N = 964) characterized Omicron breakthrough infections in triple-vaccinated individuals from the ENFORCE cohort. Within 60 days before a PCR test spike-specific IgG levels were significantly lower in cases compared to controls (GMR [95% CI] for BA.2: 0.83 [0.73-0.95], p = 0.006). Multivariable logistic regression showed significant associations between high antibody levels and lower odds of infection (aOR [95% CI] for BA.2 spike-specific IgG: 0.65 [0.48-0.88], p = 0.006 and BA.2 ACE2-blocking antibodies: 0.46 [0.30-0.69], p = 0.0002). A sex-stratified analysis showed more pronounced associations for females than males. High levels of vaccine-induced antibodies provide partial protection against Omicron breakthrough infections. This is important knowledge to further characterize a threshold for protection against new variants and to estimate the necessity and timing of booster vaccination.
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BACKGROUND: Continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outpaces monovalent vaccine cross-protection to new viral variants. Consequently, bivalent coronavirus disease 2019 (COVID-19) vaccines including Omicron antigens were developed. The contrasting immunogenicity of the bivalent vaccines and the impact of prior antigenic exposure on new immune imprinting remains to be clarified. METHODS: In the large prospective ENFORCE cohort, we quantified spike-specific antibodies to 5 Omicron variants (BA.1 to BA.5) before and after BA.1 or BA.4/5 bivalent booster vaccination to compare Omicron variant-specific antibody inductions. We evaluated the impact of previous infection and characterized the dominant antibody responses. RESULTS: Prior to the bivalent fourth vaccine, all participants (N = 1697) had high levels of Omicron-specific antibodies. Antibody levels were significantly higher in individuals with a previous polymerase chain reaction positive (PCR+) infection, particularly for BA.2-specific antibodies (geometric mean ratio [GMR] 6.79, 95% confidence interval [CI] 6.05-7.62). Antibody levels were further significantly boosted in all individuals by receiving either of the bivalent vaccines, but greater fold inductions to all Omicron variants were observed in individuals with no prior infection. The BA.1 bivalent vaccine generated a dominant response toward BA.1 (adjusted GMR 1.31, 95% CI 1.09-1.57) and BA.3 (1.32, 1.09-1.59) antigens in individuals with no prior infection, whereas the BA.4/5 bivalent vaccine generated a dominant response toward BA.2 (0.87, 0.76-0.98), BA.4 (0.85, 0.75-0.97), and BA.5 (0.87, 0.76-0.99) antigens in individuals with a prior infection. CONCLUSIONS: Vaccination and previous infection leave a clear serological imprint that is focused on the variant-specific antigen. Importantly, both bivalent vaccines induce high levels of Omicron variant-specific antibodies, suggesting broad cross-protection of Omicron variants.
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COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2/genética , Estudos de Coortes , Estudos Prospectivos , Vacinação , Vacinas contra COVID-19 , Vacinas Combinadas , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Diabetes mellitus (DM) is an independent risk factor for micro- and macrovascular complications such as nephropathy and atherosclerosis respectively, which are the major causes of premature morbidity and mortality in Type 1 and Type 2 diabetic patients. Endothelial dysfunction is the critical first step of vascular disease and is characterized by reduced bioavailability of the essential endothelial vasodilator, nitric oxide (NO), coupled with an elevation in inflammation and oxidative stress. A novel pathway to bolster NO activity is to upregulate soluble guanylate cyclase (sGC), an enzyme responsible for mediating the protective actions of NO. Two classes of sGC modulators exist, activators and stimulators, with differing sensitivity to oxidative stress. In this study, we investigated the therapeutic effects of the sGC stimulator BAY 41-2272 (Bay 41) and the sGC activator BAY 60-2770 (Bay 60) on endpoints of atherosclerosis and renal disease as well as inflammation and oxidative stress in diabetic Apolipoprotein E knockout (ApoE-/-) mice. We hypothesized that under oxidative conditions known to accompany diabetes, sGC activation might be more efficacious than sGC stimulation in limiting diabetic vascular complications. We demonstrate that Bay 60 not only significantly decreased nitrotyrosine staining (P < 0.01) and F4/80 positive cells by 75% (P < 0.05), but it also significantly reduced total plaque area (P < 0.05) and improved endothelial function (P < 0.01). Our data suggest an important anti-atherogenic role for Bay 60 accompanied by reduced oxidative stress and inflammation under diabetic settings. Treatment with the stimulator Bay 41, on the other hand, had minimal effects or caused no changes with respect to cardiovascular or renal pathology. In the kidneys, treatment with Bay 60 significantly lessened urinary albuminuria, mesangial expansion and nitrotyrosine staining under diabetic conditions. In summary, our head-to-head comparator is the first preclinical study to show that a sGC activator is more efficacious than a sGC stimulator for the treatment of diabetes-associated vascular and renal complications.
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BACKGROUND: Older age and chronic disease are important risk factors for developing severe COVID-19. At population level, vaccine-induced immunity substantially reduces the risk of severe COVID-19 disease and hospitalization. However, the relative impact of humoral and cellular immunity on protection from breakthrough infection and severe disease is not fully understood. METHODS: In a study cohort of 655 primarily older study participants (median of 63 years (IQR: 51-72)), we determined serum levels of Spike IgG antibodies using a Multiantigen Serological Assay and quantified the frequency of SARS-CoV-2 Spike-specific CD4 + and CD8 + T cells using activation induced marker assay. This enabled characterization of suboptimal vaccine-induced cellular immunity. The risk factors of being a cellular hypo responder were assessed using logistic regression. Further follow-up of study participants allowed for an evaluation of the impact of T cell immunity on breakthrough infections. RESULTS: We show reduced serological immunity and frequency of CD4 + Spike-specific T cells in the oldest age group (≥75 years) and higher Charlson Comorbidity Index (CCI) categories. Male sex, age group ≥75 years, and CCI > 0 is associated with an increased likelihood of being a cellular hypo-responder while vaccine type is a significant risk factor. Assessing breakthrough infections, no protective effect of T cell immunity is identified. CONCLUSIONS: SARS-CoV-2 Spike-specific immune responses in both the cellular and serological compartment of the adaptive immune system increase with each vaccine dose and are progressively lower with older age and higher prevalence of comorbidities. The findings contribute to the understanding of the vaccine response in individuals with increased risk of severe COVID-19 disease and hospitalization.
Vaccination has proven very effective in protecting against severe disease and hospitalization of people with COVID-19, the disease caused by SARS-CoV-2. It is still unclear, however, how the different components of the immune system respond to SARS-CoV-2 vaccination and protect from infection and severe disease. Two of the most predominant components of the immune system are specialized proteins and cells. The proteins circulate in the blood and help clear the virus by binding to it, while the cells either kill the virus or help other cells to produce more antibodies. Here, we examined the response of these two components to the SARS-CoV-2 vaccine in 655 Danish citizens. The response of both components was lower in people over 75 years old and with other diseases. These findings help in understanding the immune responses following SARS-CoV-2 vaccination in people at increased risk of severe symptoms of COVID-19.
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The SARS-CoV-2 pandemic has, as of July 2022, infected more than 550 million people and caused over 6 million deaths across the world. COVID-19 vaccines were quickly developed to protect against severe disease, hospitalization and death. In the present study, we performed a direct comparative analysis of four COVID-19 vaccines: BNT162b2 (Pfizer/BioNTech), mRNA-1273 (Moderna), ChAdOx1 (Oxford/AstraZeneca) and Ad26.COV2.S (Johnson & Johnson/Janssen), following primary and booster vaccination. We focused on the vaccine-induced antibody-mediated immune response against multiple SARS-CoV-2 variants: wildtype, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta) and B.1.1.529 (Omicron). The analysis included the quantification of total IgG levels against SARS-CoV-2 Spike, as well as the quantification of antibody neutralization titers. Furthermore, the study assessed the high-throughput ACE2 competition assay as a surrogate for the traditional pseudovirus neutralization assay. The results demonstrated marked differences in antibody-mediated immune responses. The lowest Spike-specific IgG levels and antibody neutralization titers were induced by one dose of the Ad26.COV2.S vaccine, intermediate levels by two doses of the BNT162b2 vaccine, and the highest levels by two doses of the mRNA-1273 vaccine or heterologous vaccination of one dose of the ChAdOx1 vaccine and a subsequent mRNA vaccine. The study also demonstrated that accumulation of SARS-CoV-2 Spike protein mutations was accompanied by a marked decline in antibody neutralization capacity, especially for B.1.1.529. Administration of a booster dose was shown to significantly increase Spike-specific IgG levels and antibody neutralization titers, erasing the differences between the vaccine-induced antibody-mediated immune response between the four vaccines. The findings of this study highlight the importance of booster vaccines and the potential inclusion of future heterologous vaccination strategies for broad protection against current and emerging SARS-CoV-2 variants.
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BACKGROUND: Oxidative stress associated with severe cardiopulmonary diseases leads to impairment in the nitric oxide/soluble guanylate cyclase signaling pathway, shifting native soluble guanylate cyclase toward heme-free apo-soluble guanylate cyclase. Here we describe a new inhaled soluble guanylate cyclase activator to target apo-soluble guanylate cyclase and outline its therapeutic potential. METHODS: We aimed to generate a novel soluble guanylate cyclase activator, specifically designed for local inhaled application in the lung. We report the discovery and in vitro and in vivo characterization of the soluble guanylate cyclase activator mosliciguat (BAY 1237592). RESULTS: Mosliciguat specifically activates apo-soluble guanylate cyclase leading to improved cardiopulmonary circulation. Lung-selective effects, e.g., reduced pulmonary artery pressure without reduced systemic artery pressure, were seen after inhaled but not after intravenous administration in a thromboxane-induced pulmonary hypertension minipig model. These effects were observed over a broad dose range with a long duration of action and were further enhanced under experimental oxidative stress conditions. In a unilateral broncho-occlusion minipig model, inhaled mosliciguat decreased pulmonary arterial pressure without ventilation/perfusion mismatch. With respect to airway resistance, mosliciguat showed additional beneficial bronchodilatory effects in an acetylcholine-induced rat model. CONCLUSION: Inhaled mosliciguat may overcome treatment limitations in patients with pulmonary hypertension by improving pulmonary circulation and airway resistance without systemic exposure or ventilation/perfusion mismatch. Mosliciguat has the potential to become a new therapeutic paradigm, exhibiting a unique mode of action and route of application, and is currently under clinical development in phase Ib for pulmonary hypertension.
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Hipertensão Pulmonar , Acetilcolina , Animais , Guanilato Ciclase/metabolismo , Guanilato Ciclase/uso terapêutico , Óxido Nítrico/metabolismo , Ratos , Guanilil Ciclase Solúvel/metabolismo , Guanilil Ciclase Solúvel/uso terapêutico , Suínos , Porco Miniatura/metabolismo , Tromboxanos/uso terapêutico , VasodilatadoresRESUMO
In this manuscript, the European Bioanalysis Forum reports back on their discussions on practical and scientific considerations related to bioanalytical applications of quantitative polymerase chain reaction. This publication follows an earlier publication in which the European Bioanalysis Forum recommends to consider principles of context of use when defining assay acceptance criteria for method validation criteria and sample analysis.
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Bioensaio , Laboratórios , Bioensaio/métodos , Reação em Cadeia da Polimerase , Projetos de Pesquisa , Relatório de PesquisaRESUMO
Antibody-based immunotherapy targeting the interaction between programmed cell death 1 (PD-1) and its ligand PD-L1 has shown impressive clinical outcomes in various cancer types, including nonsmall cell lung cancer (NSCLC). However, regulatory mechanisms in this immune checkpoint pathway still needs clarification. PD-L2 is structurally homologous to PD-L1 and is a second PD-1 ligand. Alternative mRNA splicing from the CD274 and PDCD1LG2 genes holds the potential to generate PD-L1 and PD-L2 isoforms, respectively, with novel functionality in regulation of the PD-1 immune checkpoint pathway. Here, we describe alternative splicing in NSCLC cells potentially generating eight different PD-L2 isoforms from the PDCD1LG2 gene. Extension of exon 6 by four nucleotides is the most prominent alternative splicing event and results in PD-L2 isoform V with a cytoplasmic domain containing a 10 amino acid extension. On average 13% of the PDCD1LG2 transcripts in NSCLC cell lines and 22% of the transcripts in NSCLC tumor biopsies encode PD-L2 isoform V. PD-L2 isoform V localizes to the cell surface membrane but less efficiently than the canonical PD-L2 isoform I. The cytoplasmic domains of PD-1 ligands can affect immune checkpoint pathways by conferring membrane localization and protein stability and thereby represent alternative targets for immunotherapy. In addition, cytoplasmic domains are involved in intracellular signalling cascades in cancer cells. The presented observations of different cytoplasmic domains of PD-L2 will be important in the future delineation of the PD-1 immune checkpoint pathway.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Processamento Alternativo , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoácidos/uso terapêutico , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Citoplasma/metabolismo , Humanos , Ligantes , Neoplasias Pulmonares/tratamento farmacológico , Nucleotídeos/metabolismo , Nucleotídeos/uso terapêutico , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA MensageiroRESUMO
Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrateâto our knowledge for the first timeâthe generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.
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Antineoplásicos , Imunoconjugados , Neoplasias , Nicotinamida Fosforribosiltransferase , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antígenos B7 , Linhagem Celular Tumoral , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Soluble guanylate cyclase (sGC) requires a heme-group bound in order to produce cGMP, a second messenger involved in memory formation, while heme-free sGC is inactive. Two compound classes can increase sGC activity: sGC stimulators acting on heme-bound sGC, and sGC activators acting on heme-free sGC. In this rodent study, we investigated the potential of the novel brain-penetrant sGC stimulator BAY-747 and sGC activator runcaciguat to enhance long-term memory and attenuate short-term memory deficits induced by the NOS-inhibitor L-NAME. Furthermore, hippocampal plasticity mechanisms were investigated. In vivo, oral administration of BAY-747 and runcaciguat to male Wistar rats enhanced memory acquisition in the object location task (OLT), while only BAY-747 reversed L-NAME induced memory impairments in the OLT. Ex vivo, both BAY-747 and runcaciguat enhanced hippocampal GluA1-containing AMPA receptor (AMPAR) trafficking in a chemical LTP model for memory acquisition using acute mouse hippocampal slices. In vivo only runcaciguat acted on the glutamatergic AMPAR system in hippocampal memory acquisition processes, while for BAY-747 the effects on the neurotrophic system were more pronounced as measured in male mice using western blot. Altogether this study shows that sGC stimulators and activators have potential as cognition enhancers, while the underlying plasticity mechanisms may determine disease-specific effectiveness.
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GMP Cíclico , Guanilato Ciclase , Animais , Masculino , Camundongos , Ratos , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Heme/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Ratos Wistar , Guanilil Ciclase Solúvel/metabolismo , VasodilatadoresRESUMO
Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr-/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr-/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.
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Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to a wide array of basic research purposes. COVID-19 infection testing has brought the three-letter PCR abbreviation into the vocabulary of billions of people, making it likely the most well-known laboratory test worldwide. With new modalities and translational medicine gaining importance in pharmaceutical research and development, PCR or more specifically, quantitative PCR (qPCR) is now becoming a standard tool in the (regulated) bioanalytical laboratory, driving the bioanalytical community to define best practices for method development, characterization and validation. In absence of specific guidance from health authorities, qPCR may be vulnerable to scope creep from pharmacokinetics (PK) assay validation as defined in bioanalytical method validation guidance/guidelines. In this manuscript, the European Bioanalysis Forum builds a rationale for applying context of use principles when defining requirements for qPCR assay performance and validation criteria.
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Bioensaio/métodos , Reação em Cadeia da Polimerase/métodos , Europa (Continente) , Humanos , Projetos de PesquisaRESUMO
Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.
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Ativadores de Enzimas/farmacologia , Músculo Esquelético/enzimologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Guanilil Ciclase Solúvel/metabolismo , Animais , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/enzimologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologiaRESUMO
Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45, BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.
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Desenho de Fármacos , Ativadores de Enzimas/química , Guanilil Ciclase Solúvel/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Cães , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Simulação de Dinâmica Molecular , Ratos , Ratos Endogâmicos SHR , Solubilidade , Guanilil Ciclase Solúvel/metabolismo , Relação Estrutura-AtividadeRESUMO
IL3RA (CD123) is the alpha subunit of the interleukin 3 (IL-3) receptor, which regulates the proliferation, survival, and differentiation of hematopoietic cells. IL3RA is frequently expressed in acute myeloid leukemia (AML) and classical Hodgkin lymphoma (HL), presenting an opportunity to treat AML and HL with an IL3RA-directed antibody-drug conjugate (ADC). Here, we describe BAY-943 (IL3RA-ADC), a novel IL3RA-targeting ADC consisting of a humanized anti-IL3RA antibody conjugated to a potent proprietary kinesin spindle protein inhibitor (KSPi). In vitro, IL3RA-ADC showed potent and selective antiproliferative efficacy in a panel of IL3RA-expressing AML and HL cell lines. In vivo, IL3RA-ADC improved survival and reduced tumor burden in IL3RA-positive human AML cell line-derived (MOLM-13 and MV-4-11) as well as in patient-derived xenograft (PDX) models (AM7577 and AML11655) in mice. Furthermore, IL3RA-ADC induced complete tumor remission in 12 out of 13 mice in an IL3RA-positive HL cell line-derived xenograft model (HDLM-2). IL3RA-ADC was well-tolerated and showed no signs of thrombocytopenia, neutropenia, or liver toxicity in rats, or in cynomolgus monkeys when dosed up to 20 mg/kg. Overall, the preclinical results support the further development of BAY-943 as an innovative approach for the treatment of IL3RA-positive hematologic malignancies.
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Idiopathic pulmonary fibrosis (IPF) is a rare and devastating chronic lung disease of unknown etiology. Despite the approved treatment options nintedanib and pirfenidone, the medical need for a safe and well-tolerated antifibrotic treatment of IPF remains high. The human prostaglandin F receptor (hFP-R) is widely expressed in the lung tissue and constitutes an attractive target for the treatment of fibrotic lung diseases. Herein, we present our research toward novel quinoline-based hFP-R antagonists, including synthesis and detailed structure-activity relationship (SAR). Starting from a high-throughput screening (HTS) hit of our corporate compound library, multiple parameter improvements-including increase of the relative oral bioavailability Frel from 3 to ≥100%-led to a highly potent and selective hFP-R antagonist with complete oral absorption from suspension. BAY-6672 (46) represents-to the best of our knowledge-the first reported FP-R antagonist to demonstrate in vivo efficacy in a preclinical animal model of lung fibrosis, thus paving the way for a new treatment option in IPF.