RESUMEN
To evaluate the relationship between the in vitro and in vivo potency of sodium-glucose cotransporter (SGLT) inhibitors, a pharmacokinetic and pharmacodynamic (PK-PD) study was performed using normal rats. A highly selective SGLT2 inhibitor, tofogliflozin, and four other inhibitors with different in vitro inhibition potency to SGLT2 and selectivity toward SGLT2, versus SGLT1 were used as test compounds, and the time courses for urinary glucose excretion (UGE) and the plasma glucose and compound concentrations were monitored after administration of the compounds. A PK-PD analysis of the UGE caused by SGLT inhibition was performed on the basis of a nonlinear parallel tube model that took into consideration the consecutive reabsorption by different glucose transport properties of SGLT2 and SGLT1. The model adequately captured the time course of cumulative UGE caused by SGLT inhibition; then, the in vivo inhibition constants (Ki) of inhibitors for both SGLT1 and SGLT2 were estimated. The in vivo selectivity toward SGLT2 showed a good correlation with the in vitro data (r = 0.985; P < 0.05), with in vivo Ki values for SGLT2 in the range of 0.3-3.4-fold the in vitro data. This suggests that in vitro inhibition potency to both SGLT2 and SGLT1 is reflected in vivo. Furthermore, the complementary role of SGLT1 to SGLT2 and how selectivity toward SGLT2 affects the inhibitory potency for renal glucose reabsorption were discussed using the PK-PD model.
Asunto(s)
Hipoglucemiantes , Modelos Biológicos , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Transporte Biológico , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Estructura Molecular , Unión Proteica , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Transportador 2 de Sodio-Glucosa , Espectrometría de Masas en TándemRESUMEN
BACKGROUND AND PURPOSE: P-glycoprotein (P-gp) exhibits a broad substrate specificity and affects pharmacokinetics, especially intestinal absorption. However, prediction, in vivo, of P-gp-mediated drug-drug interaction (DDI) and non-linear absorption at the preclinical stage, is challenging. Here we evaluate the use of human MDR1 mouse artificial chromosome (hMDR1-MAC) mice carrying human P-gp and lacking their own murine P-gp to quantitatively predict human P-gp-mediated DDI and non-linear absorption. EXPERIMENTAL APPROACH: The P-gp substrates (aliskiren, betrixaban, celiprolol, digoxin, fexofenadine and talinolol) were administered orally to wild-type, Mdr1a/b-knockout (KO) and hMDR1-MAC mice, and their plasma concentrations were measured. We calculated the ratio of area under the curve (AUCR) in mice (AUCMdr1a/b-KO /AUCwild-type or AUCMdr1a/b-KO /AUChMDR1-MAC ) estimated as attributable to complete P-gp inhibition and the human AUCR with and without P-gp inhibitor administration. The correlations of AUCRhuman with AUCRwild-type and AUCRhMDR1-MAC were investigated. For aliskiren, betrixaban and celiprolol, the Km and Vmax values for P-gp in hMDR1-MAC mice and humans were optimized from different dosing studies using GastroPlus. The correlations of Km and Vmax for P-gp between human and hMDR1-MAC mice were investigated. KEY RESULTS: A better correlation between AUCRhuman and AUCRhMDR1-MAC (R2 = 0.88) was observed. Moreover, good relationships of Km (R2 = 1.00) and Vmax (R2 = 0.98) for P-gp between humans and hMDR1-MAC mice were observed. CONCLUSIONS AND IMPLICATIONS: These results suggest that P-gp-mediated DDI and non-linear absorption can be predicted using hMDR1-MAC mice. These mice are a useful in vivo tool for quantitatively predicting P-gp-mediated disposition in drug discovery and development.
Asunto(s)
Absorción Intestinal , Preparaciones Farmacéuticas , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Interacciones Farmacológicas , RatonesRESUMEN
The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Anticuerpos/metabolismo , Espacio Extracelular/metabolismo , Animales , Humanos , Ratones , Microambiente TumoralRESUMEN
A versatile immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify the total concentration of a protein-based antigen in non-clinical pharmacokinetics (PK) studies of a human monoclonal antibody drug. The method combines using magnetic beads that have been coated with a commercial anti-human Fc region antibody to capture an immune complex of the antigen and antibody drug, with subsequent digestion and quantification of the antigen-derived tryptic peptide via LC-MS/MS. Although a typical immunoassay or an immunoaffinity LC-MS/MS assay requires an antigen-specific antibody that uses a different epitope from the antibody drug, this method requires only a commercial anti-human Fc region antibody. The method was applied to quantify total receptor activator of nuclear factor-κB ligand (RANKL) in the presence of denosumab, a humanized monoclonal antibody (mAb) specific to RANKL. The assay was validated as fit-for-purpose and found to be accurate (<115% interbatch accuracies) and precise (<15%, interbatch coefficient of variation) across a range of 3.13-200ng/mL RANKL. Commercial enzyme-linked immunosorbent assay (ELISA) kit was not able to determine the total RANKL because interference by denosumab decreased recovery. In contrast, the antibody drug had less effect on the LC-MS/MS method. The method now provides a bioanalytical platform for developing other protein-based antigen assays in the early drug stage.