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31.
Omran Z Kay G Di Salvo A Knott RM Cairns D 《Bioorganic & medicinal chemistry letters》2011,21(1):45-47
The genetic disease, nephropathic cystinosis is characterized by lysosomal accumulation of the amino acid cystine. Crystallization of cystine in affected organs, if untreated, results in mortality of the affected individuals by their middle to late teens. The only approved treatment for cystinosis is administration of cysteamine. However, cysteamine is associated with an offending odor and taste and this, coupled to a rapid first pass metabolism and a 6 h dosing regimen, suggest a clear need to improve the therapy. A number of PEGylated derivatives of cystamine, the disulfide counterpart of cysteamine, have been synthesised and evaluated in cultured cystinotic fibroblasts for toxicity and efficacy. All of the tested compounds were non-cytotoxic and displayed a remarkable depletion of intralysosomal cystine. 相似文献
32.
Summary. Due to the obvious advantages of long-acting peptide and protein drugs, strategies to prolong plasma half life time of such
compounds are highly on demand. Short plasma half life times are commonly due to fast renal clearance as well as to enzymatic
degradation occurring during systemic circulation. Modifications of the peptide/protein can lead to prolonged plasma half
life times. By shortening the overall amino acid amount of somatostatin and replacing l-analogue amino acids with d-amino acids, plasma half life time of the derivate octreotide was 1.5 hours in comparison to only few minutes of somatostatin.
A PEG2,40 K conjugate of INF-α-2b exhibited a 330-fold prolonged plasma half life time compared to the native protein. It was the aim
of this review to provide an overview of possible strategies to prolong plasma half life time such as modification of N- and
C-terminus or PEGylation as well as methods to evaluate the effectiveness of drug modifications. Furthermore, fundamental
data about most important proteolytic enzymes of human blood, liver and kidney as well as their cleavage specificity and inhibitors
for them are provided in order to predict enzymatic cleavage of peptide and protein drugs during systemic circulation. 相似文献
33.
Due to potential problems that can occur during blood transfusion and increasing blood shortages, our group engineered methoxypolyethylene glycol conjugated bovine red blood cells (mPEG-bRBCs) as a potential universal oxygen therapeutic. This current work investigates the immunological properties of mPEG-bRBCs incubated with human plasma (hP) and correlates these properties to exposed Galalpha(1,3)Gal xenoantigens. After mPEG-bRBCs were incubated with hP, the amount of bound IgG and IgM was assessed via flow cytometry. Flow cytometry also assessed the amount of GS-IB4 bound to exposed Galalpha(1,3)Gal xenoantigens. The results of this study demonstrate that most hP samples strongly promote agglutination of mPEG-bRBCs regardless of the extent of mPEG surface coverage or donor blood type. IgG and IgM from hP bound strongly to mPEG-bRBCs. In general, the Galalpha(1,3)Gal xenoantigen remains exposed at all levels of PEG surface coverage. PEGylation did block some of the xenoantigens as the amount of exposed Galalpha (1,3)Gal decreased with increased mPEG surface coverage. However, this was not sufficient to prevent a strong agglutination reaction. Taken together, the results of this study indicate that the current strategy for PEGylating bRBCs is unsatisfactory for the development of immunologically silent oxygen therapeutics. 相似文献
34.
Fee CJ 《Biotechnology and bioengineering》2007,98(4):725-731
Therapeutic proteins conjugated with branched poly(ethylene glycol) (PEG) have extended in vivo circulation half-lives compared to linear PEG-proteins, thought to be due partly to a greater hydrodynamic volume of branched PEG-proteins, which reduces the glomerular sieving coefficient. In this paper, viscosity radii of PEGylated alpha-lactalbumin (M(r) = 14.2 kDa) and bovine serum albumin (M(r) = 67 kDa) prepared with linear and branched PEGs (with nominal molecular weights 5, 10, 20 and 40 kDa) were compared experimentally using size exclusion chromatography (SEC). PEG adduct:protein molecular weight ratios of the PEGylated proteins covered the range 1:12 to 6:1. Direct comparisons of experimentally measured viscosity radii were found to be misleading due to differences between actual and nominal molecular weights of the PEG reagents used. Comparison with predicted viscosity radii shows that there is no significant difference between the viscosity radii of branched and linear PEG-proteins having the same total molecular weight of PEG adducts. Therefore, longer in vivo circulation half-lives of branched PEG-proteins compared to linear PEG-proteins are not explained by size difference. It is also calculated that the molecular size cut-off for glomerular filtration, 60 A for a 30 kDa PEG, matches the 30-50 A size range for the pores of the glomerular basement membrane. Finally, it is confirmed that prediction of PEG-protein viscosity radii should be based upon conservation of the total PEG adduct surface area to volume ratio for both linear and branched PEG-proteins regardless of PEGylation extent. 相似文献
35.
Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications. 相似文献
36.
分别用氰脲酰氯法及N-羟基丁二酰亚胺活性酯法合成了蛋白质的氨基PEG化试剂mPEGcc和mPEG-GS,并研究了它们对蛋白质的修饰作用。在合成过程中,通过分析反应体系中微量水分的存在对mPEGcc合成效率的影响,及溶剂中小分子可活化杂质成分对mPEG-GS合成产物质量的影响,发现去水剂的存在,可使mPEGce产率提高7倍;二氧六环优于DMF,且二氧六环的预处理也很重要。同时,为了测定活化PEG修饰蛋白质的效能,首次以BSA为模型蛋白,建立起一种测定活化PEG修饰能力的方法,应用此方法能直观而又准确地比较各种方法活化的PEG对蛋白质的修饰能力,具有普遍的意义。 相似文献
37.
天花粉蛋白的定点聚乙二醇修饰 总被引:3,自引:0,他引:3
用一种定点修饰天花粉蛋白(trichosanthin,TCS)的方法,将聚乙二醇(PEG)偶联到预先选定的位点.利用nTCS无半胱氨酸(Cys)残基这一特点,通过定点突变将一个Cys残基引入TCS以取代第7位的丝氨酸(Ser)残基.然后,与巯基反应的PEG-m aleim ide 即可偶联到新引入的Cys 残基上.经纯化得到均一的PEG-TCS复合物,在SDS-PAGE上显示一条区带,表观分子量为38 kD.复合物的体外致核糖体失活活性降低了6倍,但其体内引产活性与nTCS相同.定点PEG修饰方法为改造TCS提供了新途径. 相似文献
38.
Qingqing Wang Lijing Sun Shaoyang Ji Dawei Zhao Jiaxin Liu Zhiguo Su Tao Hu 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(7):1201-1207
As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic. 相似文献
39.
40.
Ilhem F. Hakem Anna M. Leech Justin Bohn Jeremy P. Walker Michael R. Bockstaller 《Biopolymers》2013,99(7):427-435
The compositional heterogeneity associated with polymer conjugation reactions of biomolecules is analyzed for the particular case of nonspecific PEGylation reactions. It is shown that the distribution of the number of PEG moieties grafted to biomolecules such as proteins is a binomial‐type function of two parameters—the reaction efficiency as well as the number of binding sites per biomolecule. The nature of this distribution implies that uniform compositions are favored for increasing number of coupling sites per biomolecule as well as for increasing efficiency of the modification process. Therefore, the binomial distribution provides a rationale for the pronounced heterogeneity that is observed for PEGylated small enzyme systems even at high coupling efficiencies. For the particular case of PEGylated trypsin it is shown that the heterogeneity results in a broad distribution of deactivation times that is captured by a stretched exponential decay model. The presented analysis is expected to apply to general modification processes of compounds in which partial functionalization of a fixed number of reactive sites is achieved by means of a nonspecific coupling reaction. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 427–435, 2013. 相似文献