RESUMEN
Antibody-drug conjugates (ADCs) containing pyrrolobenzodiazepine (PBD) dimers are currently being evaluated in human oncology clinical trials with encouraging results. To further improve the therapeutic window, next-generation PBD drug-linker design has focused on the inclusion of additional tumor-selective triggers and use of lower-potency PBDs. ß-Glucuronidase is a well-known target for discovery prodrugs due to increased presence in tumor cells and microenvironment. In this study, a ß-glucuronidase cleavable cap was investigated at the PBD N10-position and compared with corresponding free imine ADCs. SG3600 (glucuronide) ADCs showed in vitro and in vivo efficacy/tolerability comparable to SG3400 (imine) ADCs, and good 50% inhibitory concentration differentials were observed in vitro between control non-antigen-targeted ADCs and targeted ADCs. Dependence on ß-glucuronidase for SG3600 activity was demonstrated through CRISPRCas9 knockdown studies and addition of exogenous ß-glucuronidase. SG3600 showed better serum stability, improved conjugation efficiency and was able to reach high drug-to-antibody ratio without aggregation.
Asunto(s)
Benzodiazepinas/farmacología , Dipéptidos/farmacología , Glucurónidos/farmacología , Inmunoconjugados/farmacología , Pirroles/farmacología , Benzodiazepinas/síntesis química , Benzodiazepinas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dipéptidos/química , Relación Dosis-Respuesta a Droga , Glucurónidos/química , Humanos , Inmunoconjugados/química , Estructura Molecular , Pirroles/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic ß-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant ß-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Péptido 1 Similar al Glucagón/agonistas , Islotes Pancreáticos/citología , Receptores de la Hormona Gastrointestinal/agonistas , Animales , Línea Celular , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Secreción de Insulina , Cariotipificación , Ratones , Ratones Noqueados , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
Identifying in vivo models that are naturally predictive for particular areas of study in humans can be challenging due to the divergence that has occurred during speciation. One solution to this challenge that is gaining increasing traction is the use of genetic engineering to introduce human genes into mice to generate superior models for predicting human responses. This review describes the state-of-the-art for generating such models, provides an overview of the types of genetically humanized mouse models described to date and their applications in basic research, drug discovery and development and to understand clinical drug toxicity. We discuss limitations and explore promising future directions for the use of genetically humanized mice to further improve translational research.
Asunto(s)
Diseño de Fármacos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Ingeniería Genética/métodos , Animales , Animales Modificados Genéticamente , Descubrimiento de Drogas/métodos , Humanos , Ratones , Ratones Transgénicos , Modelos Animales , Investigación Biomédica Traslacional/métodosRESUMEN
Melanin-concentrating hormone (MCH) plays an important role in energy balance. The current studies were carried out on a new line of mice lacking the rodent MCH receptor (MCHR1(-/-) mice). These mice confirmed the previously reported lean phenotype characterized by increased energy expenditure and modestly increased caloric intake. Because MCH is expressed in the lateral hypothalamic area, which also has an important role in the regulation of the autonomic nervous system, heart rate and blood pressure were measured by a telemetric method to investigate whether the increased energy expenditure in these mice might be due to altered autonomic nervous system activity. Male MCHR1(-/-) mice demonstrated a significantly increased heart rate [24-h period: wild type 495 +/- 4 vs. MCHR1(-/-) 561 +/- 8 beats/min (P < 0.001); dark phase: wild type 506 +/- 8 vs. MCHR1(-/-) 582 +/- 9 beats/min (P < 0.001); light phase: wild type 484 +/- 13 vs. MCHR1(-/-) 539 +/- 9 beats/min (P < 0.005)] with no significant difference in mean arterial pressure [wild type 110 +/- 0.3 vs. MCHR1(-/-) 113 +/- 0.4 mmHg (P > 0.05)]. Locomotor activity and core body temperature were higher in the MCHR1(-/-) mice during the dark phase only and thus temporally dissociated from heart rate differences. On fasting, wild-type animals rapidly downregulated body temperature and heart rate. MCHR1(-/-) mice displayed a distinct delay in the onset of this downregulation. To investigate the mechanism underlying these differences, autonomic blockade experiments were carried out. Administration of the adrenergic antagonist metoprolol completely reversed the tachycardia seen in MCHR1(-/-) mice, suggesting an increased sympathetic tone.