RESUMEN
A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific antitumor antibody streptavidin fusion protein (scFv4-SA) in vivo, to improve tumor-to-normal tissue concentration ratios for multistep targeted (MST) radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with an average molecular weight of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogues (e.g., CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of (124)I-labeled-scFv4-SA clearance and dual-label biodistribution studies following MST directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a scFv4-SA specific for an antitumor-associated glycoprotein (TAG-72); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of the (124)I-labeled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e., biotin linked with an extended spacer, referred to herein as long-chain (LC)) can impact the binding to the antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor-to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin.
Asunto(s)
Neoplasias del Colon/diagnóstico , Neoplasias del Colon/radioterapia , Complejos de Coordinación , Dextranos , Imagen Molecular , Radioinmunoterapia , Radioisótopos/química , Animales , Secuencia de Carbohidratos , Línea Celular Tumoral , Complejos de Coordinación/química , Complejos de Coordinación/uso terapéutico , Dextranos/química , Dextranos/uso terapéutico , Sistemas de Liberación de Medicamentos , Estabilidad de Medicamentos , Xenoinjertos , Humanos , Ratones , Estructura Molecular , Tomografía de Emisión de PositronesRESUMEN
Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model (131)I-labeled single chain variable fragment antibody-streptavidin ((131)I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism.
Asunto(s)
Acetilgalactosamina/metabolismo , Dendrímeros/metabolismo , Hígado/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Acetilgalactosamina/farmacocinética , Animales , Dendrímeros/farmacocinética , Femenino , Radioisótopos de Yodo , Ratones , Proteínas Recombinantes de Fusión/sangre , Anticuerpos de Cadena Única/sangre , Estreptavidina/genéticaRESUMEN
Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Tiohidantoínas/uso terapéutico , Antagonistas de Andrógenos/farmacocinética , Anilidas/farmacocinética , Anilidas/uso terapéutico , Animales , Antineoplásicos Hormonales/sangre , Antineoplásicos Hormonales/farmacocinética , Benzamidas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Nitrilos/farmacocinética , Nitrilos/uso terapéutico , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/sangre , Feniltiohidantoína/farmacocinética , Feniltiohidantoína/uso terapéutico , Ratas , Receptores Androgénicos/efectos de los fármacos , Tiohidantoínas/sangre , Tiohidantoínas/síntesis química , Tiohidantoínas/farmacocinética , Compuestos de Tosilo/farmacocinética , Compuestos de Tosilo/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
[reaction: see text] beta-C-galacto-Pyranosides with CHF and CF2 substitutes for the glycosidic oxygen were prepared through a four-step sequence starting from a central 1-thio-1,2-O-isopropylidene acetal alcohol and different alpha-fluoro- and alpha,alpha-difluoro acids. The key step in the synthesis is the oxocarbenium cyclization of an intermediate enol ether-thioacetal to a C1-substituted glycal.
Asunto(s)
Flúor/química , Galactosa/química , Ácidos/química , Galactosa/síntesis química , Glicósidos/química , Estructura MolecularRESUMEN
Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-L-idopyranuronate 6beta/6alpha, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key synthon in heparin/heparan sulfate synthesis. The 1H and 13C NMR spectra of the furanose-pyranose mixture of 4, after dissolution and equilibration in d(4)-methanol, were fully assigned allowing to expect that 4 could crystallise in the beta-pyranose form. New acetylation conditions able to trap this form were subsequently devised, allowing the isolation of 83% of pure 6beta by simple crystallisation, along with 9% of the 6beta/6alpha mixture. This represents a major advantage over the previously published procedure, especially on multigram scales.