RESUMEN
A rapid-acting insulin lispro and long-acting insulin glargine are commonly used for the treatment of diabetes. Clinical cases have described the formation of injectable amyloidosis with these insulin analogues, but their amyloid core regions of fibrils were unknown. To reveal these regions, we have analysed the hydrolyzates of insulin fibrils and its analogues using high-performance liquid chromatography and mass spectrometry methods and found that insulin and its analogues have almost identical amyloid core regions that intersect with the predicted amyloidogenic regions. The obtained results can be used to create new insulin analogues with a low ability to form fibrils. ABBREVIATIONS: a.a., amino acid residues; HPLC-MS, high-performance liquid chromatography/mass spectrometry; m/z, mass-to-charge ratio; TEM, transmission electron microscopy.
Asunto(s)
Amiloide/química , Insulina/análogos & derivados , Amiloide/metabolismo , Amiloide/ultraestructura , Humanos , Hidrólisis , Insulina Glargina/química , Insulina Lispro/química , Espectrometría de Masas , Proteolisis , Programas InformáticosRESUMEN
Two capillary electrophoresis (CE) methods have been developed for the separation of charge and mass variants of human insulin and its recombinant analogue lispro. Since the capillary zone (CZE) and Capillary gel electrophoresis (CGE) are based on different principles of separation, they can be used to detect different impurities of insulin and its analogues. Application of CZE enabled a separation of compounds with different m/z ratio, therefore CZE is a suitable method for the separation of deamidation products of insulin. After the optimization, this method is validated according ICH requirements. CGE method was used for the separation of higher molecular weight transformation products. Experimental data have shown that CZE and CGE are simple, fast and robust methods which could be used as a routine analysis for quality control of insulin formulations.
Asunto(s)
Electroforesis Capilar/métodos , Hipoglucemiantes/análisis , Insulina Lispro/química , Insulina/análisis , Humanos , Hipoglucemiantes/química , Insulina/química , Peso Molecular , Control de CalidadRESUMEN
Adequacy of insulin concentration in commercially available insulin formulations has recently been challenged. We therefore repeatedly evaluated insulin content and stability of 58 insulin vials containing 5 different insulin formulations (human insulin, standard/faster-acting insulin aspart, insulin lispro, and insulin glargine) over a period of 85 days. High-resolution mass spectrometry was used to quantify intact monomeric insulin in glass vials and plastic pump cartridges exposed to three different temperatures (4°C, 22°C, 37°C), simulating real-life conditions. In all cases, measured insulin concentration was in accordance with FDA and European Medicines Agency (EMA) requirements without evidence of chemical instability.
Asunto(s)
Composición de Medicamentos , Hipoglucemiantes/química , Insulina/análisis , Insulinas/química , Espectrometría de Masas , Humanos , Insulina Aspart/química , Insulina Glargina/química , Insulina Lispro/química , Insulina Regular Humana/químicaRESUMEN
Formulations that can increase the dissociation of insulin oligomers into monomers/dimers are important considerations in the development of ultra-rapid-acting insulins with faster onset and shorter duration of actions. Here we present a novel strategy to characterize the oligomeric states of insulin in solution that leverages the ability of nuclear magnetic resonance spectroscopy to assess higher-order structure of proteins in solution. The oligomeric structures and solution behaviors of 2 fast-acting insulins, aspart and lispro, with varying excipient concentrations were studied using 1D and diffusion profiling methods. These methods can provide insight on the structural differences and distributions of the molecular association states in different insulin formulations, which is consistent with other orthogonal biophysical characterization tools. In addition, these methods also highlight their sensitivity to subtle changes in solution behaviors in response to excipient that are difficult to monitor with other tools. This work introduces the utility of 1D and diffusion profiling methods to characterize the oligomeric assembly of fast-acting insulins, suggesting promising applications in compound screening, excipient selection, and formulation development of fast-acting insulins as well as other peptide or protein therapeutics.
Asunto(s)
Excipientes/química , Insulina Aspart/química , Insulina Lispro/química , Espectroscopía de Protones por Resonancia Magnética , Difusión , Composición de Medicamentos , Conformación Proteica , SolubilidadRESUMEN
Direct qualitative methods that allow the rapid screening and identification of insulin products during early stages of the drug development process and those already in the market can be of great utility for manufacturers and regulatory agencies and the recent scientific literature describes several methods. Herein, a qualitative proteomic method is presented for the identification of recombinant human insulin and all marketed biosynthetic analogues -insulin lispro, aspart, glulisine, glargine, detemir and degludec- via tryptic digestion and identification of proteotypic peptides for each insulin. Individual insulins were first denatured under reducing conditions and the cysteine residues blocked by iodoacetamide. The proteins were then digested with trypsin and the peptide products separated by reversed phase liquid chromatography on an Ascentis® Express ES-C18 column and detected by positive polarity ESI-MS/MS. The digestion peptides were characterized using a multiplexed MRM approach that monitors the fragmentation of the doubly charged unlabeled precursor ion of each peptide into a collection of signature y and b ions. The MRM transitions for the individual peptides were optimized to allow maximal ionization on a standard triple quadrupole mass spectrometer. All products of the digestion procedure for all insulins were detected with adequate signal intensity except for the C-terminal B30Thr whenever it was present and cleaved and the tryptic B1-3 tripeptide of insulin glulisine. The unique proteotypic peptides identified for each of the insulin analogues coupled with their signature y and b ions permitted the unambiguous verification of all sequence variations and chemical modifications. The elution of the A polypeptide chain for all insulins and the tryptic peptides of the B chain, with the exception of a very few, occurred around the same time point. This underscores the close similarity in the physicochemical properties between the digestion peptides and is consistent with the subtle variations in amino acid sequence among the various insulins. Therefore, the identification and distinction of the different types of insulin based solely on the chromatographic retention time of their respective proteolytic products can be deceptive without proper mass spectrometric analysis and may result in false positives.
Asunto(s)
Insulina/química , Péptidos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Humanos , Insulina/análogos & derivados , Insulina Aspart/química , Insulina Detemir/química , Insulina Glargina/química , Insulina Lispro/química , Insulina de Acción Prolongada/química , Fragmentos de Péptidos/química , Proteolisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
The present study was aimed to establish the First National Reference Standard (NRS) for Insulin lispro to allow stakeholders including manufacturer's laboratories, drug testing laboratories, drug regulatory authorities and academic institutions to demonstrate accuracy of the test results and to enable comparison and validation of analytical methods. The candidate standard for Insulin lispro was evaluated in a collaborative study to assign the vial content in order to serve it as NRS to support the Indian Pharmacopoeia (IP) monograph. The candidate standard was calibrated against the Ph. Eur. Insulin lispro reference standard by each of six participant laboratories in India using HPLC assay method as per the requirements of IP monograph. The results indicate that the candidate standard has an average content of 5.79â¯mg per vial with purity of 99.87%. Based on the study results the candidate standard was judged suitable to serve as the first NRS for Insulin lispro.
Asunto(s)
Insulina Lispro/química , Insulina Lispro/normas , Europa (Continente) , Humanos , India , Estándares de ReferenciaRESUMEN
Since the discovery of amylin its use has been discouraged by the inadequacy of the protocol involving multiple injections in addition to insulin. We aimed here to develop a combined fixed-dose formulation of pramlintide with fast-acting insulin. We have investigated the compatibility of regular and fast-acting insulin analogues (Aspart, AspB28, and LisPro, LysB28ProB29) with the amylin analogue pramlintide by using electrospray ionization - ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), kinetic aggregation assays monitored by thioflavin T, and transmission electron microscopy (TEM) in the evaluation of the aggregation product. Insulin interacts with pramlintide, forming heterodimers as probed by ESI-IMS-MS. While their interaction is likely to delay the amyloid aggregation of pramlintide in phosphate-buffered solution pH 7.0, they do not prevent aggregation at this condition. At acidic sodium acetate solution pH 5.0, combination of pramlintide and the fast-acting insulin analogues become stable against amyloid aggregation. The co-formulated product at high concentration of both pramlintide (600⯵g/mL,150⯵M) and LisPro insulin (50â¯IU/mL, 300⯵M) showed also stability against amyloid aggregation. These data indicate the physico-chemical short-term stability of the co-formulated preparation of LisPro insulin with pramlintide, which could bring benefits for the combined therapy.
Asunto(s)
Composición de Medicamentos/métodos , Hipoglucemiantes/química , Insulina Lispro/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Benzotiazoles , Diabetes Mellitus/tratamiento farmacológico , Combinación de Medicamentos , Estabilidad de Medicamentos , Humanos , Hipoglucemiantes/farmacología , Insulina Aspart/química , Insulina Aspart/farmacología , Insulina Lispro/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Microscopía Electrónica de Transmisión , Agregación Patológica de Proteínas/prevención & control , Espectrometría de Masa por Ionización de Electrospray , Tiazoles/químicaRESUMEN
The tendency of peptides to adsorb to surfaces can raise a concern in variety of analytical fields where the qualitative/quantitative measurement of low concentration analytes (ng/mL-pg/mL) is required. To demonstrate the importance of using the optimal glassware/plasticware, four doping relevant model peptides (GHRP 5, TB-500, Insulin Lispro, Synachten) were chosen and their recovery from various surfaces were evaluated. Our experiments showed that choosing expensive consumables with low-bind characteristics is not beneficial in all cases. A careful selection of the consumables based on the evaluation of the physico/chemical features of the peptide is recommended.
Asunto(s)
Cosintropina/química , Doping en los Deportes , Insulina Lispro/química , Oligopéptidos/química , Adsorción , Animales , Cromatografía Líquida de Alta Presión , Cosintropina/sangre , Vidrio/química , Humanos , Insulina Lispro/sangre , Oligopéptidos/sangre , Polipropilenos/químicaRESUMEN
PURPOSE: Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. METHODS: Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. RESULTS: Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. CONCLUSION: Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
Asunto(s)
Hipoglucemiantes/química , Insulina Regular Humana/química , Humanos , Inyecciones Subcutáneas , Insulina/análogos & derivados , Insulina/química , Insulina Aspart/química , Insulina Lispro/química , Insulina de Acción Corta , Cinética , Peso Molecular , Fenoles/química , Agregado de Proteínas , Estabilidad Proteica , Relación Estructura-Actividad , Zinc/químicaRESUMEN
BACKGROUND: SAR342434 is a biosimilar follow-on of insulin lispro-Humalog®. This study aimed to show similar efficacy, safety, and immunogenicity of SAR342434 (SAR-Lis) versus insulin lispro-Humalog (Ly-Lis) in adult patients with type 1 diabetes (T1DM) treated with multiple daily injections while using basal insulin glargine (Lantus®; GLA-100). MATERIALS AND METHODS: SORELLA-1 was a randomized, open-label phase 3 study (NCT02273180). Patients completing the 6-month main study continued on SAR-Lis or Ly-Lis, as randomized, for a 6-month safety extension. Assessments included change in HbA1c, fasting plasma glucose (FPG), seven-point self-monitored plasma glucose (SMPG) profiles, hypoglycemic events, treatment-emergent adverse events (TEAEs), and anti-insulin antibodies (AIAs). RESULTS: Five hundred seven patients were randomized (SAR-Lis n = 253; Ly-Lis n = 254). Least square (LS) mean (SEM) change in glycosylated hemoglobin (HbA1c) (baseline to week 26; primary endpoint) was similar in both treatment groups (SAR-Lis: -0.42% [0.051]; Ly-Lis: -0.47% [0.050]). Noninferiority at prespecified 0.3% noninferiority margin and inverse noninferiority were demonstrated (LS mean difference of SAR-Lis vs. Ly-Lis: 0.06% [95% confidence interval: -0.084 to 0.197]). At week 52 (end of extension period) versus week 26, a small HbA1c increase was observed in both groups. FPG and seven-point SMPG profile changes, including postprandial glucose excursions, were similar between groups. At week 52, similar changes in mean daily mealtime and basal insulin doses were observed. Hypoglycemia, TEAEs, and AIAs (incidence, prevalence) did not differ between groups. CONCLUSIONS: Results from this controlled study in patients with T1DM also using GLA-100 support similar efficacy and long-term safety (including immunogenicity) of SAR-Lis and Ly-Lis.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hiperglucemia/prevención & control , Hipoglucemia/prevención & control , Hipoglucemiantes/uso terapéutico , Insulina Lispro/uso terapéutico , Adulto , Autoanticuerpos/análisis , Glucemia/análisis , Automonitorización de la Glucosa Sanguínea , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Esquema de Medicación , Hipersensibilidad a las Drogas/epidemiología , Hipersensibilidad a las Drogas/etiología , Quimioterapia Combinada/efectos adversos , Estudios de Equivalencia como Asunto , Hemoglobina Glucada/análisis , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/antagonistas & inhibidores , Incidencia , Inyecciones Subcutáneas , Insulina Glargina/efectos adversos , Insulina Glargina/química , Insulina Glargina/uso terapéutico , Insulina Lispro/administración & dosificación , Insulina Lispro/efectos adversos , Insulina Lispro/química , Análisis de Intención de Tratar , Pacientes Desistentes del Tratamiento , PrevalenciaRESUMEN
There are different insulin analogues with various pharmacokinetic characteristics, such as, rapid-acting, long-acting, or intermediate-acting analogues. Since insulin tends to form amyloid aggregates, it is of particular interest to measure characteristic times of formation of amyloid aggregates and compare those to action times for insulin and its analogues. For the study we have chosen one of the insulin analogues - insulin Lispro, which is a fast acting insulin analog. It is usually thought of amyloid aggregation as a nucleation-dependent process. We have estimated the size of the primary nucleus to be one monomer and the size of the secondary nucleus to be around zero in both insulin and Lispro insulin aggregation processes. The main structural element of insulin and Lispro insulin amyloid fibrils is a rounded ring oligomer of about 6-7 nm in diameter, about 2-3 nm in height and about 2 nm in diameter of the hole. Fibrils of several µm in length are produced due to interaction of such oligomers. The packing of ring oligomers in fibrils differs because of the difference in their orderliness. Though the initial stages of fibril formation (monomer, oligomer) are similar, the further process depends on the unique sequence of each peptide. Namely the sequence affects the final morphology of mature amyloids. These observations allow us to conclude that formation of fibrils by short peptides occurs via and by means of oligomer ring structures. Such an important issue as the nature of polymorphism of insulin amyloid fibrils has been settled by us. The role of early oligomeric aggregates in such processes as nucleation and aggregation of amyloid fibrils has been examined.
Asunto(s)
Insulina Lispro/química , Insulina Lispro/metabolismo , Insulina/química , Insulina/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestructura , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , Proteínas Amiloidogénicas/ultraestructura , Animales , Humanos , Insulina/genética , Insulina/farmacocinética , Insulina Lispro/farmacocinética , Proteínas Mutantes , Agregado de Proteínas , Agregación Patológica de Proteínas , Multimerización de ProteínaRESUMEN
We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-ß-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.
Asunto(s)
Hipoglucemiantes/síntesis química , Insulina Lispro/síntesis química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Disulfuros/química , Fluorenos/química , Hipoglucemiantes/química , Insulina Lispro/química , Pliegue de Proteína , Estructura Terciaria de Proteína , Técnicas de Síntesis en Fase SólidaRESUMEN
BACKGROUND: Premixed insulins are recommended starter insulins in Chinese patients after oral antihyperglycemic medication (OAM) failure. In the present study, we compared the efficacy and safety of insulin lispro mix 25 (LM25) twice daily (b.i.d.) and insulin lispro mix 50 (LM50) b.i.d. as a starter insulin regimen in Chinese patients with type 2 diabetes mellitus (T2DM) who had inadequate glycemic control with OAMs. METHODS: The primary efficacy outcome in the present open-label parallel randomized clinical trial was change in HbA1c from baseline to 26 weeks. Patients were randomized in a ratio of 1: 1 to LM25 (n = 80) or LM50 (n = 76). A mixed-effects model with repeated measures was used to analyze continuous variables. The Cochran-Mantel-Haenszel test with stratification factor was used to analyze categorical variables. RESULTS: At the end of the study, LM50 was more efficacious than LM25 in reducing mean HbA1c levels (least-squares [LS] mean difference 0.48; 95 % confidence interval [CI] 0.22, 0.74; P < 0.001). More subjects in the LM50 than LM25 group achieved HbA1c targets of <7.0 % (72.4 % vs 45.0 %; P = 0.001) or ≤6.5 % (52.6 % vs 20.0 %; P < 0.001). Furthermore, LM50 was more effective than LM25 at reducing HbA1c in patients with baseline HbA1c, blood glucose excursion, and postprandial glucose greater than or equal to median levels (P ≤ 0.001). The rate and incidence of hypoglycemic episodes and increase in weight at the end of the study were similar between treatment groups. CONCLUSIONS: In Chinese patients with T2DM, LM50 was more efficacious than LM25 as a starter insulin.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hemoglobina Glucada/metabolismo , Insulina Lispro/uso terapéutico , Anciano , Pueblo Asiatico , Glucemia/metabolismo , China , Diabetes Mellitus Tipo 2/etnología , Femenino , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Insulina Lispro/química , Masculino , Persona de Mediana Edad , Periodo Posprandial , Factores de Tiempo , Resultado del TratamientoRESUMEN
PURPOSE: Determine the pharmacokinetics of insulin peglispro (BIL) in 5/6-nephrectomized rats and study the absorption in lymph duct cannulated (LDC) sheep. METHODS: BIL is insulin lispro modified with 20-kDa linear PEG at lysine B28 increasing the hydrodynamic size to 4-fold larger than insulin lispro. Pharmacokinetics of BIL and insulin lispro after IV administration were compared in 5/6-nephrectomized and sham rats. BIL was administered IV or SC into the interdigital space of the hind leg, and peripheral lymph and/or serum samples were collected from both LDC and non-LDC sheep to determine pharmacokinetics and absorption route of BIL. RESULTS: The clearance of BIL was similar in 5/6-nephrectomized and sham rats, while the clearance of insulin lispro was 3.3-fold slower in 5/6-nephrectomized rats than in the sham rats. In non-LDC sheep, the terminal half-life after SC was about twice as long vs IV suggesting flip-flop pharmacokinetics. In LDC sheep, bioavailability decreased to <2%; most of the dose was absorbed via the lymphatic system, with 88% ± 19% of the dose collected in the lymph after SC administration. CONCLUSION: This work demonstrates that increasing the hydrodynamic size of insulin lispro through PEGylation can impact both absorption and clearance to prolong drug action.
Asunto(s)
Hipoglucemiantes/química , Insulina Lispro/química , Linfa/efectos de los fármacos , Polietilenglicoles/química , Animales , Disponibilidad Biológica , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Semivida , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Inyecciones Intravenosas , Inyecciones Subcutáneas , Insulina Lispro/administración & dosificación , Insulina Lispro/farmacocinética , Cinética , Masculino , Peso Molecular , Ratas Sprague-Dawley , OvinosRESUMEN
The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor-dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.
Asunto(s)
Insulina Lispro/química , Insulina Lispro/farmacología , Polietilenglicoles/química , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Humanos , Insulina Lispro/metabolismo , Insulina Lispro/farmacocinética , Lipogénesis/efectos de los fármacos , Masculino , Ratones , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance.
Asunto(s)
Cromatografía en Gel , Insulina Lispro/química , Tecnología Farmacéutica/métodos , Quelantes/química , Estabilidad de Medicamentos , Ácido Edético/química , Cromatografía de Gases y Espectrometría de Masas , Luz , Espectroscopía de Resonancia Magnética , Proteolisis , Dispersión de Radiación , Zinc/químicaRESUMEN
The stability of three commercial "fast-acting" insulin analogs, insulin lispro, insulin aspart, and insulin glulisine, was studied at various concentrations of phenolic preservatives (phenol and/or meta-cresol) during 9 days of incubation at 37 °C. The analysis by both size-exclusion and reversed-phase chromatography showed degradation of lispro and aspart that was inversely dependent on the concentration of phenolic preservatives. Insulin glulisine was much more stable than the other analogs and showed minimal degradation even in the absence of phenolic preservatives. With sedimentation velocity ultracentrifugation, we determined the preservatives' effect on the insulins' self-assembly. When depleted of preservatives, insulin glulisine dissociates from higher molecular weight species into a number of intermediate molecular weight species, in between monomer and hexamer, whereas insulin aspart and insulin lispro dissociate into monomers and dimers. Decreased stability of insulin lispro and insulin aspart seems to be because of the extent of dissociation when depleted of preservative. Insulin glulisine's dissociation to intermediate molecular weight species appears to help minimize its degradation during incubation at 37 °C.
Asunto(s)
Cresoles/química , Excipientes/química , Hipoglucemiantes/química , Insulina Aspart/química , Insulina Lispro/química , Insulina/análogos & derivados , Fenol/química , Estabilidad de Medicamentos , Insulina/química , Agregado de Proteínas , TemperaturaRESUMEN
The insulin hexamer is resistant to degradation and fibrillation, which makes it an important quaternary structure for its in vivo storage in Zn(2+)- and Ca(2+)-rich vesicles in the pancreas and for pharmaceutical formulations. In addition to the two Zn(2+) ions that are required for its formation, three other species, Zn-coordinating anions (e.g., Cl(-)), Ca(2+), and phenols (e.g., resorcinol), bind to the hexamer and affect the subunit conformation and stability. The contributions of these four species to the thermodynamics of insulin unfolding have been quantified by differential scanning calorimetry and thermal unfolding measurements to determine the extent and nature of their stabilization of the insulin hexamer. Both Zn(2+) and resorcinol make a significant enthalpic contribution, while Ca(2+) primarily affects the protein heat capacity (solvation) by its interactions in the central cation-binding cavity, which is modulated by the surrounding subunit conformations. Coordinating anions have a negligible effect on the stability of the hexamer, even though subunits shift to an alternate conformation when these anions bind to the Zn(2+) ions. Finally, Zn(2+) in excess of the two that are required to form the hexamer further stabilizes the protein by additional enthalpic contributions.
Asunto(s)
Insulina/química , Termodinámica , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Cristalografía por Rayos X , Humanos , Insulina Lispro/química , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Desplegamiento Proteico , PorcinosRESUMEN
Insulin is a commonly used protein for studies of amyloidogenesis. There are a few insulin analogues with different pharmacokinetic characteristics, in particular the onset and duration of action. One of them is LysPro insulin. The behavior of LysPro insulin in the process of amyloid formation has not been studied in detail yet. To quantitatively investigate the differences between insulin and LysPro insulin in the aggregation reaction, we used thioflavin T fluorescence assay, electron microscopy, X-ray diffraction methods, and theoretical modeling. Kinetic experimental data for both insulin samples demonstrated the increase of the lag-time for LysPro insulin at low concentrations of monomers, particularly at 2 and 4 mg/mL, which corresponds to the pharmaceutical concentration. However, the morphology of insulin and LysPro insulin fibrils and their X-ray diffraction patterns is identical. Mature fibrils reach 10-12 µm in length and about 3-4 nm in diameter. The obtained analytical solution allow us to determine the sizes of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the ratio of the lag-time duration to the time of growth of amyloid protofibrils. In the case of insulin and LysPro insulin, we have exponential growth of amyloid protofibrils following the "bifurcation + lateral growth" scenario. In accord with the developed theory and the experimental data, we obtained that the size of the primary nucleus is equal to one monomer and the size of the secondary nucleus is zero in both insulin and LysPro insulin.
Asunto(s)
Amiloide/química , Insulina/química , Amiloide/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Insulina Lispro/química , Insulina Lispro/genética , Insulina Lispro/metabolismo , Cinética , Modelos Moleculares , Tamaño de la Partícula , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3-6 and B22-24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3-6, whereas they increased in B22-24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.