RESUMO
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.
Assuntos
Composição de Medicamentos , Hipoglicemiantes/química , Insulina/análise , Insulinas/química , Espectrometria de Massas , Humanos , Insulina Aspart/química , Insulina Glargina/química , Insulina Lispro/química , Insulina Regular Humana/químicaRESUMO
Background: This study compared the efficacy, safety, and immunogenicity of insulin aspart biosimilar/follow-on biologic product SAR341402 (SAR-Asp) with originator insulin aspart-NovoLog®/NovoRapid® (NN-Asp) in people with type 1 diabetes (T1D) or type 2 diabetes (T2D) treated with multiple daily injections in combination with insulin glargine (Lantus®; Gla-100). Materials and Methods: This 6-month, randomized, open-label, phase 3 study (NCT03211858) enrolled 597 people with T1D (n = 497) or T2D (n = 100). Participants were randomized 1:1 to mealtime SAR-Asp (n = 301) or NN-Asp (n = 296) in combination with Gla-100. The primary objective was to demonstrate noninferiority (by 0.3% margin in the intent-to-treat population) of SAR-Asp versus NN-Asp in HbA1c change from baseline to week 26. Immunogenicity was also assessed in terms of anti-insulin aspart antibody (AIA) status (positive/negative) and titers during the study. Results: HbA1c was similarly improved in both treatment groups (SAR-Asp -0.38%; NN-Asp -0.30%); the least squares mean difference at week 26 for SAR-Asp minus NN-Asp was -0.08% (95% confidence interval: -0.192 to 0.039), thus meeting the criteria for noninferiority between SAR-Asp and NN-Asp and inverse noninferiority of NN-Asp versus SAR-Asp. Changes in fasting plasma glucose and seven-point self-monitored plasma glucose profile, including postprandial glucose excursions, and insulin dosages were similar in both groups at week 26. Safety and tolerability, including AIA responses (incidence, prevalence), hypoglycemia, and adverse events (including hypersensitivity events and injection site reactions), were similar between groups. Conclusions: SAR-Asp demonstrated effective glycemic control with a similar safety and immunogenicity profile to NN-Asp in people with diabetes treated for 26 weeks.
Assuntos
Medicamentos Biossimilares/administração & dosagem , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Insulina Aspart/administração & dosagem , Insulina Glargina/administração & dosagem , Adulto , Medicamentos Biossimilares/química , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Quimioterapia Combinada , Feminino , Hemoglobinas Glicadas/efeitos dos fármacos , Humanos , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/química , Injeções Subcutâneas , Anticorpos Anti-Insulina/sangue , Insulina Aspart/química , Masculino , Refeições , Pessoa de Meia-Idade , Período Pós-Prandial , Resultado do TratamentoRESUMO
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.
Assuntos
Excipientes/química , Insulina Aspart/química , Insulina Lispro/química , Espectroscopia de Prótons por Ressonância Magnética , Difusão , Composição de Medicamentos , Conformação Proteica , SolubilidadeRESUMO
Batch-level inference-based quality control is the standard practice for drug products. However, rare drug product defects may be missed by batch-level statistical sampling, where a subset of vials in a batch is tested quantitatively but destructively. In 2013, a suspension insulin product, NovoLog® Mix 70/30 was recalled due to a manufacturing error, which resulted in insulin strength deviations up to 50% from the labeled value. This study analyzed currently marketed FlexPen® devices by the water proton transverse relaxation rate using a benchtop nuclear magnetic resonance relaxometer. The water proton transverse relaxation rate was found to be sensitive to detecting concentration changes of the FlexPen® product. These findings support the development of vial-level verification-based quality control for drug products where every vial in a batch is inspected quantitatively but nondestructively.
Assuntos
Insulinas Bifásicas/análise , Insulina Aspart/análise , Insulina Isófana/análise , Espectroscopia de Ressonância Magnética/métodos , Insulinas Bifásicas/química , Insulinas Bifásicas/normas , Insulina Aspart/química , Insulina Aspart/normas , Insulina Isófana/química , Insulina Isófana/normas , Prótons , Controle de Qualidade , Água/químicaRESUMO
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.
Assuntos
Insulina/química , Peptídeos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Humanos , Insulina/análogos & derivados , Insulina Aspart/química , Insulina Detemir/química , Insulina Glargina/química , Insulina Lispro/química , Insulina de Ação Prolongada/química , Fragmentos de Peptídeos/química , Proteólise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Insulin is a frequently prescribed drug in hospitals and is usually administered by syringe pumps with an extension line which can be made of various materials. Two insulin solutions were studied: an insulin analogue, Novorapid® which contains insulin aspart and two phenolic preservatives (e.g. phenol and metacresol) and Umuline rapide® with human insulin and metacresol as preservative. Some studies have indicated interactions between insulin, polyvinyl chloride (PVC) and polyethylene (PE). The aim of this work was to study such interactions between Novorapid® or Umuline rapide® and infusion extension line materials (PVC, PE and coextruded (PE/PVC)). Insulin solution at 1 IU/mL was infused at 2 mL/h over 24 hours with 16 different extension lines (8 in PVC, 3 in PE and 5 in PE/PVC). Ultra-Fast Liquid Chromatography with diode array detection (UFLC-DAD) was performed to quantify insulin (human and aspart) and preservatives (metacresol and phenol). Limited human insulin sorption was observed thirty minutes after the onset of infusion: 24.3 ± 12.9%, 3.1 ± 1.6% and 18.6 ± 10.0% for PVC, PE and PE/PVC respectively. With insulin aspart, sorption of about 5% was observed at the onset of infusion for all materials. However, there were interactions between phenol and especially metacresol with PVC, but no interactions with PE and PE/PVC. This study shows that insulin interacts with PVC, PE and PE/PVC at the onset of infusion. It also demonstrates that insulin preservatives interact with PVC, which may result in problems of insulin conservation and conformation. Some more studies are required to understand the clinical impact of the latter during infusion.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Insulina Aspart/química , Insulina Regular Humana/química , Administração Intravenosa , Cromatografia Líquida , Humanos , Técnicas In Vitro , Insulina Aspart/administração & dosagem , Polietileno/química , Cloreto de Polivinila/química , SeringasRESUMO
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.
Assuntos
Composição de Medicamentos/métodos , Hipoglicemiantes/química , Insulina Lispro/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Benzotiazóis , Diabetes Mellitus/tratamento farmacológico , Combinação de Medicamentos , Estabilidade de Medicamentos , Humanos , Hipoglicemiantes/farmacologia , Insulina Aspart/química , Insulina Aspart/farmacologia , Insulina Lispro/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Microscopia Eletrônica de Transmissão , Agregação Patológica de Proteínas/prevenção & controle , Espectrometria de Massas por Ionização por Electrospray , Tiazóis/químicaAssuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Insulina Aspart/administração & dosagem , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Composição de Medicamentos , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/química , Hipoglicemiantes/farmacocinética , Insulina Aspart/efeitos adversos , Insulina Aspart/química , Insulina Aspart/farmacocinética , Resultado do TratamentoRESUMO
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.
Assuntos
Hipoglicemiantes/química , Insulina Regular Humana/química , Humanos , Injeções Subcutâneas , Insulina/análogos & derivados , Insulina/química , Insulina Aspart/química , Insulina Lispro/química , Insulina de Ação Curta , Cinética , Peso Molecular , Fenóis/química , Agregados Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade , Zinco/químicaRESUMO
BACKGROUND: Due to population aging, an increasing number of elderly patients with diabetes use insulin. It is therefore important to investigate the characteristics of new insulins in this population. Faster-acting insulin aspart (faster aspart) is insulin aspart (IAsp) in a new formulation with faster absorption. This study investigated the pharmacological properties of faster aspart in elderly subjects with type 1 diabetes mellitus (T1DM). METHODS: In a randomised, double-blind, two-period crossover trial, 30 elderly (≥65 years) and 37 younger adults (18-35 years) with T1DM received single subcutaneous faster aspart or IAsp dosing (0.2 U/kg) and underwent an euglycaemic clamp (target 5.5 mmol/L) for up to 12 h. RESULTS: The pharmacokinetic and pharmacodynamic time profiles were left-shifted for faster aspart versus IAsp. In each age group, onset of appearance occurred approximately twice as fast (~3 min earlier) and early exposure (area under the concentration-time curve [AUC] for serum IAsp from time zero to 30 min [AUCIAsp,0-30 min]) was greater (by 86% in elderly and 67% in younger adults) for faster aspart than for IAsp. Likewise, onset of action occurred 10 min faster in the elderly and 9 min faster in younger adults, and early glucose-lowering effect (AUC for the glucose infusion rate [GIR] from time zero to 30 min [AUCGIR,0-30 min]) was greater (by 109%) for faster aspart than for IAsp in both age groups. Total exposure (AUCIAsp,0-t) and the maximum concentration (C max) for faster aspart were greater (by 30 and 28%, respectively) in elderly than in younger adults. No age group differences were seen for the total (AUCGIR,0-t) or maximum (GIRmax) glucose-lowering effect. CONCLUSION: This study demonstrated that the ultra-fast pharmacological properties of faster aspart are similar in elderly subjects and younger adults with T1DM. ClinicalTrials.gov Identifier: NCT02003677.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Insulina Aspart/farmacocinética , Insulina Aspart/uso terapêutico , Adolescente , Adulto , Idoso , Envelhecimento/sangue , Envelhecimento/efeitos dos fármacos , Glicemia/análise , Química Farmacêutica , Estudos Cross-Over , Diabetes Mellitus Tipo 1/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Técnica Clamp de Glucose , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/química , Insulina Aspart/administração & dosagem , Insulina Aspart/química , Insulina de Ação Curta/administração & dosagem , Insulina de Ação Curta/química , Insulina de Ação Curta/farmacocinética , Insulina de Ação Curta/uso terapêutico , Masculino , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Localized insulin-derived amyloid masses occasionally form at the site of repeated insulin injections in patients with insulin-dependent diabetes and cause subcutaneous insulin resistance. Various kinds of insulin including porcine insulin, human insulin, and insulin analogues reportedly formed amyloid fibrils in vitro and in vivo, but the impact of the amino acid replacement in insulin molecules on amyloidogenicity is largely unknown. In the present study, we demonstrated the difference in amyloid fibril formation kinetics of human insulin and insulin analogues, which suggests an important role of the C-terminal domain of the insulin B chain in nuclear formation of amyloid fibrils. Furthermore, we determined that cyclodextrins, which are widely used as drug carriers in the pharmaceutical field, had an inhibitory effect on the nuclear formation of insulin amyloid fibrils. These findings have significant implications for the mechanism underlying insulin amyloid fibril formation and for developing optimal additives to prevent this subcutaneous adverse effect.
Assuntos
Amiloide/antagonistas & inibidores , Ciclodextrinas/química , Insulina Aspart/química , Insulina Detemir/química , Insulina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/química , Benzotiazóis , Corantes Fluorescentes , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Soluções , Espectrometria de Fluorescência , TiazóisRESUMO
A thermo-responsive random copolymer, POEGMA (poly(oligoethylene glycol) methacrylate) grafted on cationized agarose adsorbent was used for size selective protein adsorption. The effects of OEGMA300 ((oligoethylene glycol) methyl ether methacrylate, Mn=300g/mol) content and temperature on the adsorption of bovine serum albumin (BSA) were evaluated. Increasing the content of OEGMA300 resulted a reduction in BSA adsorption due to the enhanced shielding effect of OEGMA300 chains. Grafting of POEGMA chains onto cationized agarose adsorbent reduced the BSA adsorption by more than 95% at 26.5°C, which is below the LCST (lower critical solution temperature) of POEGMA. The BSA adsorption capacities for adsorbents grafted with 10 and 20mol% of OEGMA300 decreased by 48% and 46% respectively at 38°C, a temperature higher than their LCSTs. The temperature-dependent adsorption of BSA on the adsorbents was attributed to changes in the polymer conformation. The thermal transition of grafted POEGMA conformation exposed the ligand when the temperature was increased. Myoglobin (Myo), which was smaller than BSA, its adsorption behavior was less dependent on the polymer conformation. The adsorption of myoglobin onto the adsorbent with and without POEGMA showed similar percentage of reduction whereas the adsorption of BSA onto the adsorbent with POEGMA decreased by 7.6 times compared to the one without POEGMA. The packed bed of POEGMA grafted adsorbent was used for flow through separation of a protein mixture consisted of virus-like particle, Hepatitis B virus-like particle (HBVLP), BSA and insulin aspart. The recovery of HBVLP in 20mol% of OEGMA300 grafted adsorbent was increased by 19% compared to ungrafted adsorbent. The flow through of BSA can be reduced by increasing the operating temperature above LCST of 20mol% of OEGMA300 while the smaller protein, insulin aspart, remained adsorbed onto the cationized surface. Hence, this thermo-responsive adsorbent has a potential for size-selective separation of protein especially for the recovery of large biomolecule.
Assuntos
Metacrilatos/química , Polietilenoglicóis/química , Proteínas/química , Adsorção , Vírus da Hepatite B/química , Insulina Aspart/química , Mioglobina/química , Soroalbumina Bovina/química , Soluções , Temperatura , Vírion/químicaRESUMO
PURPOSE: To study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot. METHODS: Formulations were made of 0.6 mM degludec at 3 and 5 Zn/6 insulin monomers, and 0.6 mM aspart (2 Zn/6 insulin monomers). Self-association was assessed using size-exclusion chromatography (SEC) monitored by UV and orthogonal reverse-phase chromatography. RESULTS: Simulating pharmaceutical formulation, degludec eluted as dihexamers, whereas aspart eluted as hexamers and monomers. Combining degludec at low zinc with aspart increased dihexamer content, indicating hybrid hexamer formation. At high zinc concentration, however, there was no evidence of this. Simulating the subcutaneous depot by removing preservative, degludec eluted as multihexamers and aspart as monomers. Aspart was incorporated into the multihexamer structures when combined with degludec at low zinc, but there was no such interaction with high-zinc degludec. SEC using progressively diluted concentrations of phenol and m-cresol showed that dissociation of aspart into monomers occurs before the formation of degludec multihexamers. CONCLUSION: Insulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart.
Assuntos
Hipoglicemiantes/química , Insulina Aspart/química , Insulina de Ação Prolongada/química , Química Farmacêutica , Cromatografia em Gel , Cromatografia de Fase Reversa , Combinação de Medicamentos , Difusão Dinâmica da Luz , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Injeções Subcutâneas , Insulina Aspart/metabolismo , Insulina de Ação Prolongada/administração & dosagem , Insulina de Ação Prolongada/metabolismo , Modelos Biológicos , Conservantes Farmacêuticos/químicaRESUMO
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.
Assuntos
Cresóis/química , Excipientes/química , Hipoglicemiantes/química , Insulina Aspart/química , Insulina Lispro/química , Insulina/análogos & derivados , Fenol/química , Estabilidade de Medicamentos , Insulina/química , Agregados Proteicos , TemperaturaRESUMO
The interfacial behavior of regular insulin (Reg-insulin) and aspart insulin (Asp-insulin) was critically affected by the presence of Zn(2+) in the subphase. This cation induced a condensed-like behavior in the compression isotherms, especially apparent for Reg-insulin films when observed by Brewster angle microscopy. Immediately after spreading, Reg-insulin, but not Asp-insulin, showed bright patches that moved in a gaseous-like state. Moreover, Zn(2+) caused marked variations of the surface electrostatics of both insulin monolayers and considerable hysteresis of their molecular organization. By oscillatory compression-expansion cycles, we observed in all cases the development of a dilatational response to the surface perturbation, and both monolayers exhibited well-defined shear moduli in the presence of Zn(2+), which was higher for Reg-insulin. Development of a shear modulus indicates behavior resembling a nominal solid, more apparent for Reg-insulin than for Asp-insulin, suggesting the presence of viscoelastic networks at the surface.
Assuntos
Ar , Insulina Aspart/química , Insulina/química , Reologia , Água/química , Zinco/química , Força Compressiva , Módulo de Elasticidade , Humanos , Pressão , Propriedades de Superfície , Termodinâmica , ViscosidadeRESUMO
Insulin shows a complex equilibrium between monomers and hexamers, involving varying conformers and association states. We sought to perform a structural characterization of the fast-acting human insulin variant B28Asp ("aspart"). Small-angle X-ray scattering measurements reveal similar globular behavior in both the aspart and regular human insulin, with a Rg of 19Å and a Dmax of approximately 50Å, indicating similar mean quaternary assembly distribution. Crystallographic assays revealed a T3R3 assembly of the aspart insulin formed by the TR dimer in the asymmetric unit, with all the first 8 residues of the B chain in the R-state monomer in helical conformation and the participation of its B3Asn in the stabilization of the hexamer. Our data provide access to novel structural information on aspart insulin such as an aspart insulin dimer in solution, the aspart insulin in T conformation and a pure R-state conformer establishing a T3R3 assembly, providing further insight on the stepwise conformational transition and assembly of this fast-insulin.
Assuntos
Insulina Aspart/química , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Insulina Aspart/genética , Insulina Aspart/metabolismo , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A 44 years-old diabetic male patient was admitted several times to the emergency department of Albi Hospital (France) for nocturnal hypoglycemias with losses of consciousness. The initial blood analysis, performed on a Cobas(®) analyzer, retrieved low levels of insulinemia. This patient was treated by analogues of insulin and did not present any comorbidities. Moreover, an extensive check-up did not retrieve any evident cause for these hypoglycemias. After a severe hypoglycemic coma that occurred during the last hospitalization when insulinotherapy was interrupted, the staff suggested the possibility of a factice hypoglycemia by surreptitious administration of insulin. Hormonal assays were then performed on a Centaur(®) analyzer, which is able to recognize insulin aspart and glargine. They revealed elevated concentrations of insulin along with low levels of C-peptide. Such a blood profile is consistent with an exogenous administration of insulin or its analogues. On the basis of this biological clue, the patient was questioned again and he finally admitted self-injection of insulin aspart. This case gives us the opportunity to review the diabetic hypoglycemia, to point out the particularities of the blood assays of insulin analogues and to confirm the need of a close collaboration between clinic and laboratory staffs in the difficult cases of factice hypoglycemias.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Hipoglicemia/induzido quimicamente , Insulina/administração & dosagem , Adulto , Sequência de Aminoácidos , Transtornos Autoinduzidos/induzido quimicamente , Transtornos Autoinduzidos/diagnóstico , Humanos , Hipoglicemia/diagnóstico , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Insulina/efeitos adversos , Insulina/análogos & derivados , Insulina/química , Insulina Aspart/administração & dosagem , Insulina Aspart/efeitos adversos , Insulina Aspart/química , Masculino , Autoadministração , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Aggregation of insulin into insoluble fibrils (fibrillation) may lead to complications for diabetes patients such as reduced insulin potency, occlusion of insulin delivery devices, or potentially increased immunological potential. Even after extensive investigation of fibril formation in regular human insulin, there are little published data about the intrinsic fibrillation of fast-acting analogs. This article investigates and compares the intrinsic fibrillation of three fast-acting insulin analogs--lispro, aspart, and glulisine--as a function of their primary protein structure and exclusive of the stabilizing excipients that are added to their respective commercial formulations. METHODS: The insulin analogs underwent a buffer exchange into phosphate-buffered saline to remove formulation excipients and then were heated and agitated to characterize intrinsic fibrillation potentials devoid of excipient stabilizing effects. Different analytical methods were used to determine the amount of intrinsic fibrillation for the analogs. After initial lag times, intrinsic fibrillation was detected by an amyloid-specific stain. Precipitation of insulin was confirmed by ultraviolet analysis of soluble insulin and gravimetric measurement of insoluble insulin. Electron microscopy showed dense fibrous material, with individual fibrils that are shorter than typical insulin fibrils. Higher resolution kinetic analyses were carried out in 96-well plates to provide more accurate measures of lag times and fibril growth rates. RESULTS: All three analogs exhibited longer lag times and slower intrinsic fibrillation rates than human insulin, with glulisine and lispro rates slower than aspart. This is the first study comparing the intrinsic fibrillation of fast-acting insulin analogs without the stabilizing excipients found in their commercial formulations. CONCLUSIONS: Data show different intrinsic fibrillation potentials based on primary molecular structures when the formulation excipients that are critical for stability are absent. Understanding intrinsic fibrillation potential is critical for evaluating insulin analog stability and device compatibility.
Assuntos
Hipoglicemiantes/química , Insulina Aspart/química , Insulina Lispro/química , Insulina/análogos & derivados , Precipitação Química , Química Farmacêutica , Dicroísmo Circular , Formas de Dosagem , Estabilidade de Medicamentos , Temperatura Alta , Humanos , Insulina/química , Cinética , Microscopia Eletrônica de Transmissão , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estabilidade Proteica , Solubilidade , Espectrofotometria UltravioletaRESUMO
RATIONALE: The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values. METHODS: Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1. RESULTS: Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS: Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2).