RESUMEN
As metabolism impacts the efficacy and safety of therapeutic peptides and proteins (TPPs), understanding of the metabolic fate of TPPs is critical for their preclinical and clinical development. Despite the continued increase of new TPPs entering clinical trials, the metabolite identification (MetID) of these emerging modalities remains challenging. In the present study, we report an analytical workflow for MetID of TPPs. Using insulin detemir as an example, we demonstrated that top-down differential mass spectrometry (dMS) was able to distinguish and discover metabolites from complex biological matrices. For structural interpretation, we developed an algorithm to generate a complete and nonredundant theoretical metabolite database for a TPP of any topology (e.g., branched, multicyclic, etc.). Candidate structures of a metabolite were obtained by matching the monoisotopic mass of a dMS feature to the theoretical metabolite database. Finally, the MS/MS sequence tags enabled unambiguous characterization of metabolite structures when isobaric/isomeric candidates were present. This platform is widely applicable to TPPs with complex structures and will ultimately guide the structural optimization of TPPs in pharmaceutical development.
Asunto(s)
Bases de Datos de Proteínas , Hepatocitos/química , Insulina Detemir/química , Riñón/química , Proteínas/análisis , Animales , Cromatografía Liquida , Hepatocitos/metabolismo , Humanos , Insulina Detemir/metabolismo , Riñón/metabolismo , Proteínas/metabolismo , Ratas , Ratas Wistar , Espectrometría de Masas en TándemRESUMEN
Peripheral insulin resistance is a key component of metabolic syndrome associated with obesity, dyslipidemia, hypertension, and type 2 diabetes. While the impact of insulin resistance is well recognized in the periphery, it is also becoming apparent in the brain. Recent studies suggest that insulin resistance may be a factor in brain aging and Alzheimer's disease (AD) whereby intranasal insulin therapy, which delivers insulin to the brain, improves cognition and memory in AD patients. Here, we tested a clinically relevant delivery method to determine the impact of two forms of insulin, short-acting insulin lispro (Humalog) or long-acting insulin detemir (Levemir), on cognitive functions in aged F344 rats. We also explored insulin effects on the Ca(2+)-dependent hippocampal afterhyperpolarization (AHP), a well-characterized neurophysiological marker of aging which is increased in the aged, memory impaired animal. Low-dose intranasal insulin improved memory recall in aged animals such that their performance was similar to that seen in younger animals. Further, because ex vivo insulin also reduced the AHP, our results suggest that the AHP may be a novel cellular target of insulin in the brain, and improved cognitive performance following intranasal insulin therapy may be the result of insulin actions on the AHP.
Asunto(s)
Envejecimiento , Encéfalo , Senescencia Celular/fisiología , Cognición , Insulina Detemir , Insulina Lispro , Administración Intranasal , Envejecimiento/metabolismo , Envejecimiento/psicología , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Senescencia Celular/efectos de los fármacos , Cognición/efectos de los fármacos , Cognición/fisiología , Trastornos del Conocimiento/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/metabolismo , Insulina Detemir/administración & dosificación , Insulina Detemir/metabolismo , Insulina Lispro/administración & dosificación , Insulina Lispro/metabolismo , Resistencia a la Insulina , Memoria/efectos de los fármacos , Ratas , Resultado del TratamientoRESUMEN
AIMS: To find an explanation for the lower potency of insulin detemir observed in humans compared with unmodified human insulin by investigating insulin detemir and human insulin concentrations directly at the level of peripheral insulin-sensitive tissues in humans in vivo. METHODS: Euglycaemic-hyperinsulinaemic clamp experiments were performed in healthy volunteers. Human insulin was administered i.v. at 6 pmol/kg/min and insulin detemir at 60 pmol/kg/min, achieving a comparable steady-state pharmacodynamic action. In addition, insulin detemir was doubled to 120 pmol/kg/min. Minimally invasive open-flow microperfusion (OFM) sampling methodology was combined with inulin calibration to quantify human insulin and insulin detemir in the interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissue. RESULTS: The human insulin concentration in the ISF was â¼115 pmol/l or â¼30% of the serum concentration, whereas the insulin detemir concentration in the ISF was â¼680 pmol/l or â¼2% of the serum concentration. The molar insulin detemir interstitial concentration was five to six times higher than the human insulin interstitial concentration and metabolic clearance of insulin detemir from serum was substantially reduced compared with human insulin. CONCLUSIONS: OFM proved useful for target tissue measurements of human insulin and the analogue insulin detemir. Our tissue data confirm a highly effective retention of insulin detemir in the vascular compartment. The higher insulin detemir relative to human insulin tissue concentrations at comparable pharmacodynamics, however, indicate that the lower potency of insulin detemir in humans is attributable to a reduced effect in peripheral insulin-sensitive tissues and is consistent with the reduced in vitro receptor affinity.