RESUMO
Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.
Assuntos
Anticorpos Neutralizantes/imunologia , Mapeamento de Epitopos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Cinética , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos/imunologia , Estrutura Quaternária de Proteína , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUND: The sites of mycobacterial infection in the lungs of tuberculosis (TB) patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations, resulting in compromised TB treatment response and resistance development. Quantifying lesion-specific drug uptake and pharmacokinetics (PKs) in TB patients is necessary to optimize treatment regimens at all infection sites, to identify patients at risk, to improve existing regimens, and to advance development of novel regimens. Using drug-level data in plasma and from 9 distinct pulmonary lesion types (vascular, avascular, and mixed) obtained from 15 hard-to-treat TB patients who failed TB treatments and therefore underwent lung resection surgery, we quantified the distribution and the penetration of 7 major TB drugs at these sites, and we provide novel tools for treatment optimization. METHODS AND FINDINGS: A total of 329 plasma- and 1,362 tissue-specific drug concentrations from 9 distinct lung lesion types were obtained according to optimal PK sampling schema from 15 patients (10 men, 5 women, aged 23 to 58) undergoing lung resection surgery (clinical study NCT00816426 performed in South Korea between 9 June 2010 and 24 June 2014). Seven major TB drugs (rifampin [RIF], isoniazid [INH], linezolid [LZD], moxifloxacin [MFX], clofazimine [CFZ], pyrazinamide [PZA], and kanamycin [KAN]) were quantified. We developed and evaluated a site-of-action mechanistic PK model using nonlinear mixed effects methodology. We quantified population- and patient-specific lesion/plasma ratios (RPLs), dynamics, and variability of drug uptake into each lesion for each drug. CFZ and MFX had higher drug exposures in lesions compared to plasma (median RPL 2.37, range across lesions 1.26-22.03); RIF, PZA, and LZD showed moderate yet suboptimal lesion penetration (median RPL 0.61, range 0.21-2.4), while INH and KAN showed poor tissue penetration (median RPL 0.4, range 0.03-0.73). Stochastic PK/pharmacodynamic (PD) simulations were carried out to evaluate current regimen combinations and dosing guidelines in distinct patient strata. Patients receiving standard doses of RIF and INH, who are of the lower range of exposure distribution, spent substantial periods (>12 h/d) below effective concentrations in hard-to-treat lesions, such as caseous lesions and cavities. Standard doses of INH (300 mg) and KAN (1,000 mg) did not reach therapeutic thresholds in most lesions for a majority of the population. Drugs and doses that did reach target exposure in most subjects include 400 mg MFX and 100 mg CFZ. Patients with cavitary lesions, irrespective of drug choice, have an increased likelihood of subtherapeutic concentrations, leading to a higher risk of resistance acquisition while on treatment. A limitation of this study was the small sample size of 15 patients, performed in a unique study population of TB patients who failed treatment and underwent lung resection surgery. These results still need further exploration and validation in larger and more diverse cohorts. CONCLUSIONS: Our results suggest that the ability to reach and maintain therapeutic concentrations is both lesion and drug specific, indicating that stratifying patients based on disease extent, lesion types, and individual drug-susceptibility profiles may eventually be useful for guiding the selection of patient-tailored drug regimens and may lead to improved TB treatment outcomes. We provide a web-based tool to further explore this model and results at http://saviclab.org/tb-lesion/.
Assuntos
Antituberculosos/administração & dosagem , Antituberculosos/farmacocinética , Pulmão/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/etiologia , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Técnicas de Apoio para a Decisão , Progressão da Doença , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Farmacorresistência Bacteriana Múltipla , Quimioterapia Combinada , Feminino , Humanos , Isoniazida/administração & dosagem , Isoniazida/farmacocinética , Canamicina/administração & dosagem , Canamicina/farmacocinética , Linezolida/administração & dosagem , Linezolida/farmacocinética , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Pirazinamida/administração & dosagem , Pirazinamida/farmacocinética , Estudos Retrospectivos , Rifampina/administração & dosagem , Rifampina/farmacocinética , Distribuição Tecidual , Falha de Tratamento , Tuberculose Resistente a Múltiplos Medicamentos/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/patologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologia , Adulto JovemRESUMO
Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.
Assuntos
Autofagia/fisiologia , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático/fisiologia , Flavonoides/farmacologia , Piperidinas/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Autofagia/genética , Técnicas de Cultura de Células , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Flavonoides/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Inanição/patologia , Células Tumorais Cultivadas , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
The potentiating action of the flavonolignan, (-)-hydnocarpin, in combination with vincristine was evaluated in the 697 acute lymphoblastic leukemia cell line and a P-gp-expressing variant, 697-R. Vincristine at 3 nM caused nearly complete growth inhibition in 697 cells versus a 17% growth inhibition in 697-R cells. When combined with (-)-hydnocarpin at concentrations of 10 and 5 µM, vincristine-mediated growth inhibition in the 697-R cells increased significantly over the sum of the individual agents to 72% (p ≤ 0.0001) and 41% (p = 0.0256), respectively. Vincristine at 1.5 nM (66% growth inhibition) and 0.75 nM (39% growth inhibition) combined with (-)-hydnocarpin at 10 µM (42% growth inhibition) in the 697 cells caused a significant increase in growth inhibition to 83% (p = 0.03) and to 61% (p < 0.0001), respectively, when compared to vincristine treatment as a single agent. To investigate the mechanism for the vincristine re-sensitization caused by (-)-hydnocarpin, the P-gp inhibitory effect of (-)-hydnocarpin was evaluated.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Flavonolignanos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Vincristina/farmacologia , Linhagem Celular Tumoral , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
BACKGROUND AND OBJECTIVE: VIR-2218 is an investigational N-acetylgalactosamine-conjugated RNA interference therapeutic in development for chronic hepatitis B virus (HBV) infection. VIR-2218 was designed to silence HBV transcripts across all genotypes and uses Enhanced Stabilization Chemistry Plus (ESC+) technology. This study was designed to evaluate the single-dose pharmacokinetics of VIR-2218 in preclinical species and healthy volunteers. METHODS: Preclinically, a single subcutaneous dose of VIR-2218 (10 mg/kg) was administered to rats and nonhuman primates (NHPs), and the pharmacokinetics were assessed in plasma, urine, and liver using standard noncompartmental analysis (NCA) methods. Clinically, healthy volunteers were randomized (6:2 active:placebo) to receive a single subcutaneous dose of VIR-2218 (50-900 mg) or placebo. Pharmacokinetics were similarly assessed within human plasma and urine using NCA methods. RESULTS: In rats and NHPs, VIR-2218 was stable in plasma and was converted to AS(N-1)3'VIR-2218, the most prominent circulating metabolite, at < 10% plasma exposure compared with parent. VIR-2218 rapidly distributed to the liver, reaching peak liver concentrations within 7 and 24 h in rats and NHPs, respectively. In humans, VIR-2218 was rapidly absorbed, with a median time to peak plasma concentration (tmax) of 4-7 h, and had a short median plasma half-life of 2-5 h. Plasma exposures for area under the plasma concentration-time curve up to 12 h (AUC0-12) and mean maximum concentrations (Cmax) increased in a slightly greater-than-dose-proportional manner across the dose range studied. Interindividual pharmacokinetic variability was low to moderate, with a percent coefficient of variation of < 32% for AUC and < 43% for Cmax. A portion of VIR-2218 was converted to an active metabolite, AS(N-1)3'VIR-2218, with a median tmax of 6-10 h, both of which declined below the lower limit of quantification in plasma within 48 h. The pharmacokinetic profile of AS(N-1)3'VIR-2218 was similar to that of VIR-2218, with plasma AUC0-12 and Cmax values ≤ 12% of VIR-2218. VIR-2218 and AS(N-1)3'VIR-2218 were detectable in urine through the last measured time point, with approximately 17-48% of the administered dose recovered in urine as unchanged VIR-2218 over 0-24 h postdose. Based on pharmacokinetics in preclinical species, VIR-2218 localizes to the liver and likely exhibits prolonged hepatic exposure. Overall, no severe or serious adverse events or discontinuations due to adverse events were observed within the dose range evaluated for VIR-2218 in healthy volunteers (Vir Biotechnology, Inc., unpublished data). CONCLUSIONS: VIR-2218 showed favorable pharmacokinetics in healthy volunteers supportive of subcutaneous dosing and continued development in patients with chronic HBV infection. CLINICAL TRIAL REGISTRATION NO: NCT03672188, September 14, 2018.
Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Animais , Área Sob a Curva , Humanos , Interferência de RNA , Terapêutica com RNAi , RatosRESUMO
Nanowire (NW) arrays offer opportunities for parallel, nondestructive intracellular access for biomolecule delivery, intracellular recording, and sensing. Spontaneous cell membrane penetration by vertical nanowires is essential for these applications, yet the time- and geometry-dependent penetration process is still poorly understood. In this work, the dynamic NW-cell interface during cell spreading was examined through experimental cell penetration measurements combined with two mechanical models based on substrate adhesion force or cell traction forces. Penetration was determined by comparing the induced tension at a series of given membrane configurations to the critical membrane failure tension. The adhesion model predicts that penetration occurs within a finite window shortly after initial cell contact and adhesion, while the traction model predicts increasing penetration over a longer period. NW penetration rates determined from a cobalt ion delivery assay are compared to the predicted results from the two models. In addition, the effects of NW geometry and cell properties are systematically evaluated to identify the key factors for penetration.
Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Nanofios/química , Animais , Células CHO , Adesão Celular/fisiologia , Cricetulus , Microscopia Eletrônica de VarreduraRESUMO
PURPOSE: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. EXPERIMENTAL DESIGN: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line. RESULTS: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. CONCLUSIONS: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease.
Assuntos
Antineoplásicos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , NF-kappa B/metabolismo , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Compostos de Benzil/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrocarbonetos Fluorados/farmacologia , Indolizinas , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Compostos de Piridínio/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Resistencia a Medicamentos Antineoplásicos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Triterpenos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Interpretação Estatística de Dados , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras , RNA Interferente Pequeno/farmacologia , Regulação para CimaRESUMO
A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the plant natural product silvestrol in mice, using ansamitocin P-3 as the internal standard. The method was validated in plasma with a lower limit of quantification of 1 ng/mL, accuracy ranging from 87 to 114%, and precision (coefficient of variation) below 15%. The validated method was used to characterize pharmacokinetics in C57BL/6 mice and metabolism in mouse, human and rat plasma, and liver microsomes. Mice were dosed with silvestrol formulated in hydroxypropyl-ß-cyclodextrin via intravenous, intraperitoneal, and oral routes followed by blood sampling up to 24 h. Intraperitoneal systemic availability was 100%, but oral administration resulted in only 1.7% bioavailability. Gradual degradation of silvestrol was observed in mouse and human plasma, with approximately 60% of the parent drug remaining after 6 h. In rat plasma, however, silvestrol was completely converted to silvestric acid (SA) within 10 min. Evaluation in microsomes provided further evidence that the main metabolite formed was SA, which subsequently showed no cytotoxic or cytostatic activity in a silvestrol-sensitive lymphoblastic cell line. The ability of the analytical assay to measure tissue levels of silvestrol was evaluated in liver, brain, kidney, and spleen. Results indicated the method was capable of accurately measuring tissue levels of silvestrol and suggested it has a relatively low distribution to brain. Together, these data suggest an overall favorable pharmacokinetic profile of silvestrol in mice and provide crucial information for its continued development toward potential clinical testing.