RESUMEN
There are many decisions and risks associated with the design and development of new pharmaceutical agents. To help improve decision-making, and reduce the associated risks--prior to synthesis, we have developed interactive web-browser tools for: (i) tracking, searching, clustering and categorizing (by reactive moieties) chemical reactants, (ii) interactively assessing risks, either synthetic--based on prior experience, absorption following oral administration--based on rules of 5, or diversity, and (iii) a complete architecture for enumerating, analyzing, submitting and plating large combinatorial or small biased libraries. We believe the implementation of this highly interactive system has given our scientists a competitive advantage by maintaining their focus on the lowest risk, highest quality molecules throughout the research process.
Asunto(s)
Química Farmacéutica/métodos , Metodologías Computacionales , Programas Informáticos , Administración Oral , Disponibilidad Biológica , Técnicas Químicas Combinatorias , Relación Estructura-Actividad Cuantitativa , Medición de RiesgoRESUMEN
Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Regiones Determinantes de Complementariedad/inmunología , Fragmentos de Péptidos/inmunología , Somatostatina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Sitios de Unión , Sitios de Unión de Anticuerpos , Células CHO , Regiones Determinantes de Complementariedad/química , Cricetinae , Cristalografía por Rayos X , Cisteína/química , Interacciones Hidrofóbicas e Hidrofílicas , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Receptores de Somatostatina/metabolismo , Somatostatina/químicaRESUMEN
Mu opioid receptors are present throughout the central and peripheral nervous systems. Peripheral inflammation causes an increase in mu receptor levels on peripheral terminals of primary afferent neurons. Recent studies indicate that activation of peripheral mu receptors produces antihyperalgesic effects in animals and humans. Here, we describe the in vitro pharmacological and in vivo pharmacokinetic properties of a novel, highly potent, and peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). In a radioligand binding assay, DiPOA inhibited [(3)H]-diprenorphine binding to recombinant human mu receptors with a K(i) value of approximately 0.8 nM. The rank order of affinity for DiPOA binding to recombinant human opioid receptors was mu > kappa approximately ORL-1 >> delta. DiPOA showed potent agonist effects in a human mu receptor guanosine 5'-O-(3-[(35)S]thio)triphosphate functional assay, with an EC(50) value of approximately 33 nM and efficacy of approximately 85% [normalized to the mu agonist, [d-Ala2,MePhe4,Gly(ol)5]enkephalin]. Low potency agonist activity was also seen at ORL-1 and kappa receptors. DiPOA bound competitively to the opioid binding site of human mu receptors as demonstrated by a parallel rightward shift in its concentration-response curve in the presence of increasing concentrations of naltrexone. High and sustained (> or =5 h) plasma levels for DiPOA were achieved following intraperitoneal administration at 3 and 10 mg/kg; central nervous system penetration, however, was < or =4% of the plasma concentration, even at levels exceeding 1500 ng/ml. As such, DiPOA represents a systemically available, peripherally restricted small molecule mu opioid agonist that will aid in understanding the role played by mu opioid receptors in the periphery.
Asunto(s)
Acetatos/farmacocinética , Analgésicos Opioides/farmacología , Analgésicos Opioides/farmacocinética , Compuestos Aza/farmacocinética , Hiperalgesia/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Compuestos de Espiro/farmacología , Compuestos de Espiro/farmacocinética , Acetatos/farmacología , Acetatos/uso terapéutico , Analgésicos Opioides/uso terapéutico , Animales , Compuestos Aza/farmacología , Compuestos Aza/uso terapéutico , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Hiperalgesia/tratamiento farmacológico , Masculino , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/antagonistas & inhibidores , Compuestos de Espiro/uso terapéuticoRESUMEN
Mu opioid receptors are expressed throughout the central and peripheral nervous systems. Peripheral inflammation leads to an increase in mu receptor present on the peripheral terminals of primary sensory neurons. Activation of peripheral mu receptors produces potent antihyperalgesic effects in both humans and animals. Here, we describe the in vivo pharmacological properties of the structurally novel, highly potent, systemically available yet peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). DiPOA administered i.p. produced naltrexone-sensitive, dose-dependent reversal of Freund's complete adjuvant-induced inflammatory mechanical hyperalgesia (1-10 mg/kg). Maximum percent reversal (67%) was seen 1 h postadministration at 10 mg/kg (the highest dose studied). DiPOA also proved antihyperalgesic in a model of postsurgical pain with a maximum percent reversal of 85% 1 h postadministration at 30 mg/kg i.p. (the highest dose studied). DiPOA administered i.p. had no effect in the tail flick assay of acute pain (0.1-10 mg/kg), produced no ataxia as measured by latency to fall from an accelerating rotarod (3-30 mg/kg), and was not antihyperalgesic in the Seltzer model of neuropathic pain (1-10 mg/kg). This is the first report of a peripherally restricted, small-molecule mu opioid agonist that is nonsedating, antihyperalgesic, and effective against inflammatory and postsurgical pain when administered systemically.