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1.
PEGylation has been suggested to improve the stability of insulin, but evidence for that is scarce. Here, we compared the forced aggregation behavior of insulin and mono-PEGylated insulin. Therefore, recombinant human insulin was conjugated on lysine B29 with 5-kDa PEG. PEG-insulin was purified by size-exclusion chromatography (SEC) and characterized by mass spectrometry (MS). Next, insulin and PEG-insulin were subjected to heating at 75 °C, metal-catalyzed oxidation, and glutaraldehyde cross-linking. The products were characterized physicochemically by complementary analytical methods. Mono-PEGylation of insulin was confirmed by SEC and MS. Under each of the applied stress conditions, insulin and PEG-insulin showed comparable degradation profiles. All the stressed samples showed submicron aggregates in the size range between 50 and 500 nm. Covalent aggregates and conformational changes were found for both oxidized products. Insulin and its PEGylated counterpart also exhibited similar characteristics when exposed to heat stress, that is, slightly changed secondary and tertiary structures, covalent aggregates with partially intact epitopes, and separation of chain A from chain B. Both glutaraldehyde-treated insulin and PEG-insulin contained covalent and noncovalent aggregates with intact epitopes, showed partially perturbed secondary structure, and substantial loss of tertiary structure. From these results, we conclude that PEGylation does not protect insulin against forced aggregation.  相似文献   

2.
Purpose. In the pH range 2–5, human insulin degrades via deamidation at the A-21 asn and covalent dimerization. Both products form via a common cyclic anhydride intermediate, a product of intramolecular nucleophilic attack by the A-21 carboxyl terminus. This study examines the influence of [insulin] and self-association on the partitioning of the intermediate to products. Methods. Insulin self-association was characterized (pH 2–4) by concentration difference spectroscopy. Deamidation rates (pH 2–4) and concurrent rates of covalent dimer formation (pH 4) were determined versus [insulin] at 35°C by initial rates. A mathematical model was developed to account for the overall rate and product composition profile versus pH and [insulin]. Results. Between pH 2–4, insulin self-associates to form non-covalent dimers with a pH independent association constant of 1.8 × 104 M –1. The overall rate of degradation is governed by intermediate formation, while product distribution is determined by competition between water and the phe B-l amino group of insulin for the anhydride. In dilute solutions, deamidation is first-order in [insulin] while covalent dimerization is second-order. Thus, deamidation predominates in dilute solutions but the fraction of covalent dimer formed increases with [insulin]. At high [insulin], self-association inhibits covalent dimer formation, preventing exclusive degradation via this pathway. The model accurately predicts a maximum in covalent dimer formation near pH 4. Conclusions. A mechanism is described which accounts for the complex dependence of insulin's degradation rate and product distribution profile on pH (between 2–5) and [insulin]. If these results can be generalized, they suggest that covalent aggregation in proteins may be inhibited by self-association.  相似文献   

3.
Conventional slow‐acting insulin preparations for subcutaneous injection, e.g., suspensions of the complex with protamine and/or zinc, were reformulated as dry powders for inhalation and the insoluble aerosol tested for providing sustained insulin plasma levels. Large porous particles made of lactose, albumin, and dipalmitoylphosphatidylcholine, and incorporating insulin, protamine, and/or zinc chloride were prepared using spray‐drying. Integrity of insulin after spray‐drying and insulin insolubilization in spray‐dried particles was verified in vitro. The pharmacokinetic profile of the formulation delivered by inhalation and subcutaneous injection was assessed in vivo in the rat. The formulation process of insulin as dry powders did not alter insulin integrity and did not impede, in most cases, insulin insolubilization by protamine and/or zinc. Large porous insulin particles presented 7 μm mass mean geometric particle diameters, 0.1 g/cm3 bulk powder tap densities and theoretical aerodynamic diameters suitable for deep lung deposition (in the range of 2.2–2.5 μm). The dry powders exhibited 40% respirable fractions in the Andersen cascade impactor and 58–75% in the Aero‐Breather™. Insoluble inhaled insulin provided sustained insulin plasma levels for half a day, similar to injected insulin, and exhibited a bioavailability of 80.5% relative to subcutaneous injection of the same formulation. Drug Dev. Res. 48:178–185, 1999. ©1999 Wiley‐Liss, Inc.  相似文献   

4.
目的 建立精蛋白胰岛素注射液的无菌检查方法。方法 根据中国药典2015年版四部通则1101无菌检查法进行方法适用性试验,对药典收载的精蛋白胰岛素注射液无菌检查方法进行优化,使用无微生物毒性的肝素钠溶液中和胰岛素注射液中的精蛋白,使精蛋白胰岛素注射液由白色混悬液变为澄清透明液体,便于薄膜过滤法处理。结果 使用薄膜过滤法,无菌检查方法适用性试验中6株阳性对照菌均可正常生长。结论 建立了可行的精蛋白胰岛素注射液的无菌检查方法,为下一版中国药典修订已收载的精蛋白胰岛素注射液无菌检查方法提供了实验依据。  相似文献   

5.
目的 对酸性胰岛素注射液、中性胰岛素注射液、精蛋白锌胰岛素注射液以及低精蛋白锌胰岛素注射液中的脱酰胺胰岛素进行检查,并对不同储存时间的这几种制剂中的脱酰胺胰岛素进行比较。方法 利用高效液相色谱分析仪,对胰岛素制剂中的脱酰胺胰岛素进行检测。结果 酸性胰岛素注射液中的脱酰胺胰岛素的含量随储存时间的延长而增大;中性胰岛素注射液、精蛋白锌胰岛素注射液以及低精蛋白锌胰岛素注射液中的脱酰胺胰岛素含量很少,而且储存时间的长短对其影响很小。结论 本试验对酸性胰岛素注射液的不宜应用提供依据,也为其他剂型提高质量标准提供简易方法。  相似文献   

6.
目的对酸性胰岛素注射液、中性胰岛素注射液、精蛋白锌胰岛素注射液以及低精蛋白锌胰岛素注射液中的脱酰胺胰岛素进行检查,并对不同储存时间的这几种制剂中的脱酰胺胰岛素进行比较。方法利用高效液相色谱分析仪,对胰岛素制剂中的脱酰胺胰岛素进行检测。结果酸性胰岛素注射液中的脱酰胺胰岛素的含量随储存时间的延长而增大;中性胰岛素注射液、精蛋白锌胰岛素注射液以及低精蛋白锌胰岛素注射液中的脱酰胺胰岛素含量很少,而且储存时间的长短对其影响很小。结论本试验对酸性胰岛素注射液的不宜应用提供依据,也为其他剂型提高质量标准提供简易方法。  相似文献   

7.
Lipid peroxidation produces many reactive byproducts including 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) derived from the peroxidation of n-3 and n-6 polyunsaturated fatty acids, respectively. HNE and HHE can modify circulating biomolecules through the formation of covalent adducts. It remains, however, unknown whether HHE and HNE could induce functional and structural changes in the insulin molecule, which may in turn be pivotal in the development of insulin resistance and diabetes. Recombinant human insulin was incubated in the presence of HHE or HNE, and the formation of covalent adducts on insulin was analyzed by mass spectrometry analysis. Insulin tolerance test in mice and stimulation of glucose uptake by 3T3 adipocytes and L6 muscle cells were used to evaluate the biological efficiency of adducted insulin compared with the native one. One to 5 adducts were formed on insulin through Michael adduction, involving histidine residues. Glucose uptake in 3T3-L1 and L6C5 cells as well as the hypoglycemic effect in mice was significantly reduced after treatment with adducted insulin compared to native insulin. The formation of HNE- and HHE-Michael adducts significantly disrupts the biological activity of insulin. These structural and functional abnormalities of the insulin molecule might contribute to the pathogenesis of insulin resistance.  相似文献   

8.
Purpose To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands.Materials and Methods The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry.Results An excellent correlation between the thermal stabilization by ligand binding and the deamidation of AsnB3 was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37°C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated.Conclusions Thermal stability can be used to predict the rate of AsnB3 deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.  相似文献   

9.
Formation of in vitro adducts between different classes of xenobiotics and the lysine-containing peptide Lys-Tyr was monitored by high-performance liquid chromatography and electrospray ionization mass spectrometry. The molecular structures of the main resulting products could be sensitively analyzed by mass spectrometry (flow injection analysis), enabling the detection of characteristic binding formations. Aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde were shown to form stable linkages to lysine amino groups via Schiff bases. Other electrophilic substances (e.g., toluene-2,4-diisocyanate, 2,4-dinitro-1-fluorobenzene, 2,4,6-trinitrobenzene sulfonic acid, dansyl chloride, and phthalic acid anhydride) also formed covalent adducts with lysine residues. The reactivity of the compounds was quantified by measuring the amount of peptide that remained unchanged after incubation for a certain period with the xenobiotic. Although reactivity levels within this group of aldehydes varied only to a small extent, as would be expected, extreme differences were seen among the structurally heterogeneous group of nonaldehyde xenobiotics. These results support the hypothesis that simple chemical reactions may lead to the adduction of nucleophilic macromolecules such as peptides or proteins. Such reactions, in particular, Schiff base formation of aldehydes, have previously been shown to be capable of specifically interfering with costimulatory signaling on T cells. Our results suggest that electrophilic xenobiotics of other classes may also inherit the capacity to exert similar effects. Forming covalent linkage to peptides may represent a possible molecular mechanism of electrophilic xenobiotics in vivo, yielding immunotoxic effects. The model utilized in this study is appropriate for monitoring the adduction of xenobiotics to basic peptides and for analyzing the resulting molecular structures.  相似文献   

10.
Insulin NPH (neutral protamine hagedorn) has for long been one of the most important therapeutic formulations for the treatment of diabetes. The protracted action profile of NPH formulations is gained from crystallizing insulin with zinc in the presence of the basic poly-arginine peptide protamine. In spite of its long history and successful use, the binding mode of the insulin–protamine complex is not known. In this study, three different systems were used to study protamine binding to insulin. In the first system, crystals of an insulin–protamine complex grown in the presence of urea and diffracting to 1.5 Å resolution were analyzed. In the second system, a shorter peptide consisting of 12 arginine residues was co-crystallized with insulin in order to reduce the flexibility and thereby improve the electron density of the peptide. Both systems yielded data to a significantly higher resolution than obtained previously. In addition, a third system was analyzed where crystals of insulin and protamine were grown in the absence of urea, with conditions closely resembling the pharmaceutical formulation. Data from these NPH microcrystals could for the first time be collected to 2.2 Å resolution at a micro focused X-ray beamline. Analysis of all three crystal forms reveal potential protamine density located close to the solvent channel leading to the centrally located zinc atoms in the insulin hexamer and support that protamine binds to insulin in a not well defined conformation.  相似文献   

11.
This paper describes the establishment and validation of a reversed phase HPLC (RP-HPLC) method for determination of protamine peptides in protamine sulphate raw material and insulin drug products, discusses the analytical results obtained for a number of protracted insulin formulations and the potential of the method in connection with protamine sulphate raw material and final drug product quality control.  相似文献   

12.
Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4–45°C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures 25°C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.To whom correspondence should be addressed at Nove Research Institute, Novo Alle, DK-2880Bagsvaerd, Denmark  相似文献   

13.
目的 了解孝感市中心医院胰岛素类药物的使用情况和变化趋势,为促使临床合理用药提供参考。方法 通过医院信息系统(HIS)提取2014-2018年孝感市中心医院胰岛素类药物销售金额、使用频度(DDDs)、日均费用(DDC)及排序比(B/A)等进行统计和分析。结果 2014-2018年,共有12种胰岛素类药物供临床使用,胰岛素类药物的销售金额及占降糖药物总销售金额的构成比均呈增长趋势;甘精胰岛素、地特胰岛素、门冬胰岛素、门冬胰岛素(30)、门冬胰岛素(50)、精蛋白生物合成人胰岛素、精蛋白锌重组赖脯胰岛素(25R)和精蛋白锌重组赖脯胰岛素(50R)的销售金额均呈增长趋势,其中甘精胰岛素、地特胰岛素的增长更显著;精蛋白生物合成人胰岛素(30R)、精蛋白生物合成人胰岛素(50R)、精蛋白锌胰岛素的DDDs值较大,甘精胰岛素、地特胰岛素和门冬胰岛素(30)的DDDs值较小,所有胰岛素类药物的DDDs均呈增长趋势,其中甘精胰岛素、地特胰岛素的DDDs值增加最显著;甘精胰岛素、地特胰岛素的DDC值较大,普通胰岛素和精蛋白锌胰岛素的DDC值较小;大部分胰岛素类药物的B/A值在0.50~1.50,但甘精胰岛素和地特胰岛素的B/A值<0.50。结论 孝感市中心医院胰岛素类药物的使用基本合理,但个别患者经济负担较重的胰岛素类药物的增长速度过快,需密切关注,确保临床用药更为规范、合理。  相似文献   

14.
Heparin employed in extracorporeal blood circulation (ECBC) procedures (e.g. open heart operations) often leads to a high incidence of bleeding complications. Protamine employed in heparin neutralization, on the other hand, can cause severe adverse reactions. We previously developed an approach that could prevent both heparin- and protamine-induced toxic side effects concomitantly. This approach consisted of placing a hollow fiber-based bioreactor device containing immobilized protamine (termed a "protamine bioreactor") at the distal end of the ECBC procedure. This protamine bioreactor would remove heparin after heparin served its anticoagulant purpose in the ECBC device, thereby eliminating heparin-induced bleeding risks. In addition, this protamine bioreactor would prevent protamine from entering the patients, thereby aborting any protamine-induced toxic effects. Both in vitro and in vivo studies have successfully demonstrated the feasibility of this approach. Despite promises, early findings also revealed two shortcomings that must be overcome for the protamine bioreactor to be applied clinically. The first drawback was that the cyanate ester linkages, involved in conjugating protamine to the bioreactor device, were unstable and prone to hydrolysis, resulting in the leakage of a significant amount of protamine into circulation during application of the protamine bioreactor. The second deficiency was that the capacity of the protamine bioreactor in heparin removal was rather low, owing to the limited surface area of the hollow fibers for protamine immobilization and subsequently heparin adsorption. In this paper, we present novel strategies to overcome these two limitations. A new conjugation method based on the use of 4-(oxyacetyl)phenoxyacetic acid (OAPA) as the activating reagent was employed to yield stable linkages, via the abundant arginine residues of protamine, onto the hollow fibers. Results showed that while the amount of protamine immobilized on each gram of fibers was relatively comparable between the OAPA and the previous CNBr activation methods (7.45 mg/g versus 7.69 mg/g fibers), there was virtually no detectable leaching of immobilized protamine from the bioreactor by the OAPA method, comparing to 35% leaching of protamine by the previous CNBr method following 72 h of storage of the bioreactor in PBS buffer at 37 degrees C. To improve the capacity and functionality of the protamine bioreactor, two novel approaches were adopted. Long chain and high molecular weight poly-lysine was linked to the hollow fibers, prior to protamine coupling, to create multiple layers of immobilized protamine for subsequent heparin adsorption. In addition, a poly(ethylene glycol) (PEG) chain was inserted between protamine and the hollow fibers to yield a three-dimensional, free dynamic motion for immobilized protamine. Preliminary observations indicated that a four- to five-fold enhancement in heparin adsorption was attained by utilizing each of these new approaches. Aside from their current use, these new strategies can also be employed generically to improve the functionality of any affinity-type bioreactor. Indeed, efforts have been made recently in utilizing these approaches to develop a clinically usable GPIIb/IIIa bioreactor for the treatment of immune thrombocytopenic purpura (ITP)-an autoimmune disease.  相似文献   

15.
Insulin is one of the most important drugs in the treatment of diabetes. There is an increasing interest in the oral administration of insulin as it mimics the physiological pathway and potentially reduces the side effects associated with subcutaneous injection. Therefore, insulin-loaded polyelectrolyte complex (PEC) nanoparticles were prepared by the ionic cross-linking method using protamine sulfate as the polycationic and sodium alginate as the anionic polymer. Taguchi experimental design was used for the optimization of nanoparticles by varying the concentration of sodium alginate, the mass ratio of sodium alginate to protamine, and the amount of insulin. The optimized nanoparticle formulation was used for further in vitro characterization. Then, insulin-loaded PEC nanoparticles were placed in hard gelatin capsules and the capsules were enteric-coated by Eudragit L100-55 (PEC-eCAPs). Hypoglycemic effects PEC-eCAPs were determined in vivo by oral administration to diabetic rats. Furthermore, in vivo distribution of PEC nanoparticles was evaluated by fluorescein isothiocyanate (FITC) labelled nanoparticles. The experimental design led to nanoparticles with a size of 194.4 nm and a polydispersity index (PDI) of 0.31. The encapsulation efficiency (EE) was calculated as 95.96%. In vivo studies showed that PEC-eCAPs significantly reduced the blood glucose level of rats at the 8th hour compared to oral insulin solution. It was concluded that PEC nanoparticles loaded into enteric-coated hard gelatin capsules provide a promising delivery system for the oral administration of insulin.  相似文献   

16.
目的:建立高效液相色谱(HPLC)法测定精蛋白锌胰岛素注射液中硫酸鱼精蛋白的含量。方法:采用Grace Vydac C18色谱柱(250 mm×4.6 mm,5μm),以A:乙腈-0.2 mol.L-1硫酸钠溶液(4∶96);B:乙腈-水(50∶50)为流动相进行梯度洗脱(0~8 min时B0%→70%,8~15 min时B70%,15~16 min时B70%→0%,16~30 min时B0%);流速1.0 mL.min-1;柱温40℃;检测波长:214 nm。结果:硫酸鱼精蛋白线性范围为0.05~2.5 mg.mL-1(r=0.9994),方法平均回收率(n=9)为99.7%。结论:本方法准确、简便、快速,重复性好,可用于精蛋白锌胰岛素注射液中硫酸鱼精蛋白的含量测定。  相似文献   

17.
A DNA duplex containing the primary acrolein adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (2), of deoxyguanosine in a 5'-CpG sequence context spontaneously but reversibly formed an interchain cross-link with the exocyclic amino group of deoxyguanosine in the opposing chain. The linkage was sufficiently stable that the cross-linked duplex could be isolated by HPLC and characterized by MALDI-TOF mass spectrometry. Enzymatic degradation gave bis-nucleoside 6, which was independently prepared by direct reaction of 2 with dGuo.  相似文献   

18.
Mixing pharmaceutical preparations of soluble neutral regular insulin solution (NRI) and neutral protamine Hagedorn (NPH) crystalline insulin suspension leads to a reduction in the measurable amount of soluble insulin in the formulation supernatant. However in spite of the loss in soluble insulin, the time-actions of these components have been shown, in clinical trials, to be unaffected. The interaction between these different physical forms of insulin has been studied using reversed-phase HPLC, isothermal titrating calorimetry, and Doppler electrophoretic light scattering analysis. Sorbent surface and solution perturbation studies revealed that the NRI adsorbs to the surface of the NPH crystal with an equilibrium constant ranging from 104 M–1 to 107 M–!, depending on the protamine concentration, pH, ionic strength, and temperature. This adsorption behavior suggests that the binding is mediated by electrostatic interactions arising between the positively-charged NPH crystal and the negatively-charged NRI hexamer. Doppler electrophoretic light scattering results, used to probe the pH-dependent surface charge of NPH and soluble insulin hexamer, support the conclusion that electrostatic interactions mediate the adsorption process. Adsorption studies under physiological conditions indicate that the elevated temperature and ionic strength, in a subcutaneous depot, are sufficient to lead to the dissociation of the NRI/NPH complex that exists in these NPH mixture formulations.  相似文献   

19.
The strategy of oral administration of bioactive macromolecules using cell-penetrating peptides (CPPs) is restricted to covalent linkage or electrostatic interaction between the cargo and CPPs. In the present study, we devised an approach utilizing CPP-functionalized poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a carrier for oral delivery of insulin. Pegylated PLGA nanoparticles were modified with poly(arginine)8 enantiomers (l-R8 and d-R8) via a maleimide-mediated covalent conjugating procedure. The physical and chemical features of the nanoparticles were characterized, which confirmed the successful immobilization of R8 to the nanoparticles. Using a Caco-2 cell monolayer model, R8-modified nanoparticles were found to exhibit significantly increased cellular uptake and transportation. Pharmacokinetics and pharmacodynamics of the insulin-loaded nanoparticles were evaluated with rats by intestinal administration. Compared to the unmodified nanoparticles, l-R8 and d-R8 modified-nanoparticles increased the relative bioavailabilities of insulin by 3.2- and 4.4-times, meanwhile, improved the hypoglycemic effects by 2.5- and 3.7-times, respectively. Neither of the R8-modified nanoparticles caused perceptible histological toxicities. The results implied that surface modification of biodegradable nanoparticles with poly(arginine)8, especially with the d-form enantiomer, showed remarkable advancement in promoting the intestinal absorption of insulin. This delivery system is also promising for the delivery of a wide variety of bioactive macromolecules by oral administration.  相似文献   

20.
Acyl glucuronides are ubiquitous metabolites formed from acidic xenobiotics and endogenous compounds, such as bilirubin. Previous studies indicated that the covalent binding of acyl glucuronides to proteins occurs via an imine intermediate in a manner analogous to the glycation of proteins via reducing sugars. When glucuronic acid was incubated in solution with albumin, it formed 10 and 4 times more fluorescent, Maillard reaction products with albumin after 25 days than did glucose or fructose, respectively. However, radiolabeled glucuronic acid exhibited less covalent binding to albumin than either glucose or fructose. Circular dichroism measurements indicate that glucuronic acid is about 0.02% open chain form with exposure of the reactive aldehyde, whereas fructose and glucose have 2 and 0.0026% present in solution as the open chain; thus, differences in reactivity of the reducing sugars were not correlated with exposure of the free aldehyde. Methyl glucuronate formed little fluorescent product with albumin, suggesting that the C-6 carboxylate of glucuronic acid may facilitate the reactions after covalent binding that lead to the formation of fluorescent products. When acyl glucuronide metabolites of two previously marketed acidic drugs, zomepirac and suprofen, were incubated with albumin at a concentration of 2.5 mM, more fluorescent product was formed than by 500 mM glucose. Reversible binding of the acyl glucuronides to albumin was 60-90%, but almost zero for the free reducing sugars, which indicates that reversible binding may explain the enhanced reactivity of the acyl glucuronides in forming fluorescent products with albumin. These results indicate that acyl glucuronides are reactive metabolites that may cause significant glycation of proteins with glucuronic acid in vivo.  相似文献   

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