RESUMEN
SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFN{gamma}-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite Fc{gamma}R binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development. One Sentence SummaryAnti-SARS-CoV-2 IgG1 antibody SC31 controls infection in vivo by blocking SP:ACE2 binding and triggering a Fc-mediated anti-viral response.
RESUMEN
SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed > 99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 µM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans. In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution ( > 0.6 L/kg), oral bioavailability of 24%, â¼ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate.
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Antineoplásicos/farmacocinética , Hepatocitos/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Microsomas Hepáticos/metabolismo , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Disponibilidad Biológica , Células CACO-2 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Femenino , Hepatocitos/enzimología , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Concentración 50 Inhibidora , Janus Quinasa 2/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratas , Ratas Wistar , Especificidad de la Especie , Distribución Tisular , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
A series of 3-(1,2-disubstituted-1H-benzimidazol-5-yl)-N-hydroxyacrylamides (1) were designed and synthesized as HDAC inhibitors. Extensive SARs have been established for in vitro potency (HDAC1 enzyme and COLO 205 cellular IC(50)), liver microsomal stability (t(1/2)), cytochrome P450 inhibitory (3A4 IC(50)), and clogP, among others. These parameters were fine-tuned by carefully adjusting the substituents at positions 1 and 2 of the benzimidazole ring. After comprehensive in vitro and in vivo profiling of the selected compounds, SB939 (3) was identified as a preclinical development candidate. 3 is a potent pan-HDAC inhibitor with excellent druglike properties, is highly efficacious in in vivo tumor models (HCT-116, PC-3, A2780, MV4-11, Ramos), and has high and dose-proportional oral exposures and very good ADME, safety, and pharmaceutical properties. When orally dosed to tumor-bearing mice, 3 is enriched in tumor tissue which may contribute to its potent antitumor activity and prolonged duration of action. 3 is currently being tested in phase I and phase II clinical trials.
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Antineoplásicos/síntesis química , Bencimidazoles/síntesis química , Inhibidores de Histona Desacetilasas/síntesis química , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Bencimidazoles/farmacocinética , Bencimidazoles/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Perros , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Histona Desacetilasa 1/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacocinética , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Trasplante de Neoplasias , Relación Estructura-Actividad Cuantitativa , Ratas , Ratas Wistar , EstereoisomerismoRESUMEN
In vitro liver microsomal stability, permeability, pharmacokinetics (PK) and oral bioavailability of SB639, a novel HDACi (Histone Deacetylase inhibitor), were determined. The in vitro metabolism was examined in mouse, rat, dog and human liver microsomes. The permeability and efflux potential of SB639 were determined using Caco-2 cell monolayers. To determine pharmacokinetics and oral bioavailability, blood samples were drawn at pre-determined intervals up to 24 h post-dose after single intravenous (i.v.) or oral (p.o.) administration of SB639 to mouse or rat. The concentrations of SB639 in plasma samples were determined by a validated LC-MS/MS method. In vitro liver microsomal stability data revealed that SB639 was stable in human and dog liver microsomes, unstable in mouse and rat liver microsomes. The Caco-2 data has shown that SB639 is highly permeable with an apparent permeability of 3.01.10(-6) cm/s at 10 microM. After oral administration, maximum concentrations of SB639 were achieved within 0.5 h of post dose. Following i.v. administration, the concentration of SB639 declined in a bi-exponential fashion with terminal elimination half-life of 1.67 h for mice and 1.12 h for rats. The systemic clearance and volume of distribution of SB639 in mice were 15.8 l/h/kg and 38 l/kg, respectively, while the respective values in rats were 3.84 l/h/kg and 3.67 l/kg. Elimination half-life in rats ranged between 1.12-2.26 h. Absolute oral bioavailability of SB639 in mouse and rat was 13% and 10%, respectively. In conclusion, the superior potency, physicochemical and PK properties of SB639 compared to the recently FDA approved drug Zolinza (Suberoylanilide hydroxamic acid or Vorinostat) in the preclinical setting makes it a potential clinical candidate.
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Bencimidazoles/farmacocinética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/farmacocinética , Inhibidores de Histona Desacetilasas , Microsomas Hepáticos/efectos de los fármacos , Pirroles/farmacocinética , Administración Oral , Animales , Bencimidazoles/sangre , Disponibilidad Biológica , Células CACO-2 , Perros , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/sangre , Femenino , Semivida , Humanos , Ácidos Hidroxámicos/sangre , Ácidos Hidroxámicos/farmacocinética , Masculino , Fase I de la Desintoxicación Metabólica , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Pirroles/sangre , Pirrolidinas , Ratas , Ratas Wistar , VorinostatRESUMEN
A liquid chromatography/tandem mass spectrometric method for the quantification of 6-(3-benzoyl-ureido)-hexanoic acid hydroxyamide (EX-2), a novel histone deacetylase (HDAC) inhibitor, in mouse plasma was developed to support in-house pharmacokinetic (PK) studies in the lead optimization stage. In order to determine the PK parameters for EX-2 in comparison to other HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), PXD-101 and LBH-589, which are currently in different stages of clinical trials, research-grade bio-analytical method validations were carried out for EX-2 and these reference HDAC inhibitors, which were synthesized by in-house medicinal chemists. The components of validation consisted of specificity, extraction efficiency, signal-response of calibration standards, lower limit of quantification, autosampler stability and accuracy and precision of quality control samples. The validated LC/MS/MS methods were accurate and precise. The calibration curve ranged from 1 to 1600 ng/mL for all the analytes. The methods developed were used to quantify EX-2 and other HDAC inhibitors in mouse plasma obtained from pharmacokinetic studies. The results suggest that EX-2 has better PK parameters compared with the reference drugs and is a promising drug development candidate.