聚乙二醇化尿酸酶体内外稳定性研究

目的研究聚乙二醇化尿酸酶体内外稳定性。方法以酶活为指标,考察聚乙二醇修饰尿酸酶和尿酸酶的温度稳定性(4~80℃)、酸碱稳定性、抗胰蛋白酶水解能力和小鼠体内半衰期。结果4~60℃条件下,修饰的尿酸酶的稳定性大于尿酸酶,在70℃时,两者活性均迅速降低。pH 5.2~6.0及pH 9.2~10.0之间,尿酸酶活性迅速降低,而修饰的尿酸酶却保留了较高的活性。抗胰蛋白酶水解中,尿酸酶在作用200 min后,活性降至最高值的20%;而修饰的尿酸酶仍保留70%的活性。体内稳定性试验表明,修饰的尿酸酶和尿酸酶的半衰期分别为1 530和45 min。结论聚乙二醇修饰可以增加尿酸酶的稳定性和抗胰蛋白酶水解能力,延长体内半衰期。     

Production and Optimization of Site-Specific monoPEGylated Uricase Conjugates Using mPEG-Maleimide Through RP–HPLC Methodology

Purpose

Uricase (Uc), a therapeutic enzyme, is widely used in its PEGylated form to treat hyperuricemia and is largely manufactured by means of random/first generation PEGylation approach. Currently available randomly PEGylated uricase conjugates exhibit inadequacies like reduced uricolytic activity, risk of inducing immunogenic reactions, lack of selectivity, and molecular heterogeneity. In the present study, site-specific/second generation PEGylation strategy involving modification of specific and rare amino acids by means of terminally functionalized PEG polymers was applied.

Methods

Uricase was conjugated with methoxypolyethyelenglycol-maleimide (mPEG-mal) by means of thiol PEGylation to synthesize monoPEGylated uricase conjugates. For enhancing the yield of monoPEGylated uricase conjugates, response surface methodology was employed to determine the yield of monoPEGylated conjugates using reverse phase high performance liquid chromatography. Using the optimized conditions, the developed method was validated for the production of monoPEGylated uricase conjugates which were further purified by size exclusion fast protein liquid chromatography (SE-FPLC). The molecular weights of the purified conjugates were determined by sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE).

Results

The optimum values of reaction conditions were determined as 1:12 concentration ratio of Uc to mPEG-mal, 2.76 kDa as mPEG-mal molecular weight and 3.55 mM EDTA concentration which resulted in a very high conjugate yield of 95.16 %. The conjugate synthesized using the optimized method retained a residual uricolytic activity of 84 % and a thiol group modification extent of 68.3 %.

Conclusion

The PEGylation reaction was optimized using OVAT and statistical methods. Using the optimized conditions very high yield of conjugates were obtained and RP–HPLC method was used to quantify the PEGylated uricase.

     

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50°C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.     

Uricases as therapeutic agents to treat refractory gout: Current states and future directions

Treatment of refractory gout remains a challenge on drug development. While pegloticase, a recombinant mammalian uricase modified with monomethoxyl-poly(ethylene glycol) (mPEG) is effective in treating refractory gout, after continued treatment for three months biweekly at a therapeutic dose of 0.14 mg/kg body weight, it elicits an immune response against mPEG in nearly 20% of patients. For continued treatment of refractory gout PEGylated uricases at monthly therapeutic doses below 4 μg/kg body weight have promise. To formulate uricases to achieve monthly therapeutic regimens requires pharmacodynamics simulation and experimentation including: (a) molecular engineering of uricases based on rational design and evolution biotechnology in combination to improve their inherent catalytic efficiency, thermostability and selectivity for urate over xanthine and; (b) optimization of the number and distribution of accessible reactive amino acid residues in native uricases for site-specific PEGylation with PEG derivatives with lower of immunogenicity than mPEG to retain activity, minimize immunogenicity and enhance the pharmacokinetics of the PEGylated uricase. These issues are briefly reviewed as a means to stimulate the development of safer uricase formulations for continued treatment of refractory gout.     

Characterization, efficacy, pharmacokinetics, and biodistribution of 5kDa mPEG modified tetrameric canine uricase variant

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ? amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated. Approximately 9.4 5 kDa mPEG chains were coupled to each monomeric uricase and the main conjugates contained 7-11 mPEG chains per subunit. mPEG-UHC showed significantly therapeutic or preventive effect on uric acid nephropathy and acute urate arthritis based on three different animal models. The clearance rate from an intravenous injection of mPEG-UHC varied significantly between species, at 2.61 mL/h/kg for rats and 0.21 mL/h/kg for monkeys. The long elimination half-life of mPEG-UHC in non-human primate (191.48 h, intravenous injection) indicated the long-term effects in humans. Moreover, the acceptable bioavailability of mPEG-UHC after subcutaneous administration in monkeys (94.21%) suggested that subcutaneous injection may be regarded as a candidate administration route in clinical trails. Non-specific tissue distribution was observed after administration of (125)I-labeled mPEG-UHC in rats, and elimination by the kidneys into the urine is the primary excretion route.     

PEGylation of Octreotide: I. Separation of Positional Isomers and Stability Against Acylation by Poly(D,L-lactide-<Emphasis Type="Italic">co</Emphasis>-glycolide)

Purpose. To investigate the mechanism by which polyethylene glycol (PEG) conjugation (PEGylation) prevents the acylation of octreotide by poly(d,l-lactide-co-glycolide) (PLGA).Methods. Octreotide was chemically modified by reaction with succinimidyl propionate-monomethoxy PEG. Each PEGylated octreotide species with different PEG number and modified position was separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with endoproteinase Lys-C digestion. Acylation of octreotide and PEGylated octreotides was observed with hydrophobic and hydrophilic PLGA.Results. Two mono- and one di-PEGylated octreotides were separated by RP-HPLC. MALDI-TOF MS of the PEGylated products after Lys-C digestion at different pH revealed that the two mono-PEGylated octreotides were modified at the N-terminus and Lys⁵ residue, respectively. The interaction of octreotide with PLGA involved an initial adsorption followed by acylation and the subsequent release of octreotide and acylated octreotide. The initial adsorption of octreotide was dependent on the acidity of PLGA. PEGylation of octreotide significantly inhibited the initial adsorption and acylation by PLGA. In particular, the acylation could be completely prevented by mono-PEGylation at the N-terminus of octreotide.Conclusions. This study shows that the N-terminus of octreotide is the preferred PEGylation site to prevent acylation in degrading PLGA microspheres. The mono-N-terminally PEGylated octreotide may possibly serve as a new source for somatostatin microsphere formulation.     

Stability of PEGylated salmon calcitonin in nasal mucosa

The purpose of this study was to evaluate the stabilization of salmon calcitonin (sCT) by PEGylation in nasal mucosa. Degradation of native sCT in the homogenates of rat nasal mucosa was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The initial cleavage of sCT was due to tryptic-like endopeptidase activity, and the subsequent degradation followed the sequential pattern of aminopeptidase activity. To prepare PEGylated sCT resistant to the proteolytic degradation, the lysine residues susceptible to tryptic activity were selectively PEGylated by controlling reaction pH. The PEGylated sCT showed strong resistance against enzymatic degradation in rat nasal mucosa, with 56-fold prolonged half-life compared with that of native sCT. In the MALDI-TOF MS spectrum, the PEGylated sCT did not show any degradation peak for incubation of 120 min in the homogenates of rat nasal mucosa. The improved stability may be responsible for enhancing nasal absorption of PEGylated sCT.     

N-terminal specificity of PEGylation of human bone morphogenetic protein-2 at acidic pH

Site-specific PEGylation offers the possibility to modify a therapeutic protein without interfering with its biological activity. Previously, a preferential N-terminal PEGylation has been reported for several proteins when the reaction was performed at acidic pH. In the present study it was explored if acidic pH favors N-terminal PEGylation of bone morphogenetic protein-2 (BMP-2). PEGylation by poly(ethylene glycol) aldehyde (PEG-AL) or poly(ethylene glycol) carboxymethyl succinimidyl ester (PEG-NHS) was performed at moderate acidic pH of 4. Comparing with PEG-NHS, PEG-AL converted more BMP-2 mainly to mono- or di-PEGylated derivatives at much less molar excess and shorter duration. Analysis of Tryptic fragments of the PEGylated derivatives indicated a partial N-terminal PEGylation specificity. PEG-AL exhibited higher specificity than PEG-NHS. UV spectrometry proved that PEGylation improved the solubility of BMP-2 in PBS. Surface plasmon resonance showed that PEGylation decreased the binding of BMP-2 proteins to a type II receptor. Remarkably, mono-PEGylated BMP-2 with PEG-AL showed higher cellular bioactivity than unmodified protein. Higher N-terminal PEGylation specificity correlates with higher receptor binding affinity and cellular activity. In summary, PEGylation of BMP-2 by PEG-AL and PEG-NHS at acidic pH exhibits a partial N-terminal specificity which however might be sufficient for an efficient site-specific PEGylation process.     

The effect of PEGylation on the stability of small therapeutic proteins

The influence of PEGylation on the thermal stability of small therapeutic proteins was evaluated using two model proteins. Changes in the midpoint of thermal unfolding and the ability to properly refold after thermal denaturation were monitored by differential scanning calorimetry (DSC) as a function of PEGylation and pH. The results showed that PEGylation increased the thermal stability of both model proteins as well as their ability to refold properly after thermal denaturation. The DSC results were compared to traditional accelerated stability data that were collected using size exclusion high performance liquid chromatography (SE-HPLC). The DSC data agreed reasonably well with those from SE-HPLC indicating that microcalorimetry can be an efficient screening tool for PEGylated proteins.     

Glucose-Binding Property of Pegylated Concanavalin a

Purpose. Concanavalin A (Con A) has been used in the development of sol-gel phase-reversible hydrogels for modulated insulin delivery. The usefulness of Con A has suffered from its poor aqueous solubility and stability. The goal of this study was to modify Con A with poly(ethylene glycol) (PEG) and examine the water solubility and stability of the PEGylated Con A. Methods. Con A was PEGylated using monomethoxy poly(ethylene glycol) p-nitrophenol carbonates, and the extent of PEGylation was determined by the fluorescamine method. The stability of the PEGylated Con A was examined by measuring the time-dependent absorbance at 630 nm. The binding affinities of glucose and allyl glucose to native- and PEGylated-Con A were measured by the equilibrium dialysis method. Results. The total number of PEG molecules that can be grafted to Con A was 10. As the number of grafted PEG chains per each Con A was increased up to 5, the binding affinity of glucose was gradually increased and reached the maximum. The solubility and stability of PEGylated Con A were improved significantly over those of native Con A. The binding affinity of allyl glucose to Con A was not changed much by PEGylation. When the extent of PEGylation was excessive (i.e., the number of grafted PEG chains per each Con A was larger than 5), however, the binding affinities of both glucose and allyl glucose were decreased significantly. Conclusions. PEGylation of Con A resulted in improved aqueous solubility and stability of Con A. The binding affinity of glucose increased and reached the maximum when the extent of PEGylation was 50%. Advantages of PEGylated Con A over native Con A are improved aqueous solubility, enhanced long-term stability, and higher glucose sensitivity.     

Conformational Stability of Lyophilized PEGylated Proteins in a Phase-Separating System

PEGylation of proteins is of great interest to the pharmaceutical industry as covalent attachment of poly(ethylene glycol) (PEG) molecules can increase protein sera half‐lives and reduce antigenicity. Not surprisingly, PEGylation significantly alters the surface characteristics of a protein, and consequently, its conformational stability during freezing and drying. Freeze concentration‐induced phase separation between excipients has been previously shown to cause degradation of the secondary structure in lyophilized hemoglobin. In this report we show how PEGylation of two proteins, hemoglobin‐and brain‐derived neurotrophic factor (BDNF), influences partitioning and protein secondary structure as determined by FTIR spectroscopy in a system prone to freezing‐induced phase separation. PEGylation of hemoglobin reduces the loss of structure induced by lyophilization in a PEG/dextran system that phase separates during freezing, perhaps due to altered partitioning. The partition coefficient for native hemoglobin favors the dextran‐rich phase (PEG/dextran partition coefficient = 0.3), while PEGylated hemoglobin favors the PEG phase (partition coefficient = 3.1). In addition, we demonstrate that PEGylation alters hemoglobin's stability during lyophilization in the absence of other excipients. In contrast, because native BDNF already partitions into the PEG‐rich phase, PEGylation of BDNF has a less dramatic effect on both partition coefficients and conformational stability during lyophilization. This is the first report on the effects of PEGylation on protein structural stability during lyophilization and points out the need to consider modification of formulations in response to changing protein surface characteristics.     

聚乙二醇化蛋白质多肽类药物定点修饰策略

聚乙二醇修饰具有抵抗蛋白酶降解、提高稳定性、延长体内半衰期、降低免疫原性等优点,能够有效地改善蛋白质多肽类药物的临床药效。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产物的产率,已经引起了广泛关注。本文概述近年来聚乙二醇定点修饰蛋白质多肽类药物方面的研究进展,并对聚乙二醇定点修饰技术的发展趋势进行了展望。     

Synthesis,Characterization and In Vivo Efficacy of PEGylated Insulin for Oral Delivery with Complexation Hydrogels

Purpose  This work evaluated the feasibility of combining insulin PEGylation with pH responsive hydrogels for oral insulin delivery. Methods  A mono-substituted PEG–insulin conjugate was synthesized and purified. The site of conjugation was determined by MALDI-TOF MS. Uptake and release of PEGylated insulin was performed in complexation hydrogels to simulate oral dosing. The bioactivity of the conjugate and PK/PD profile was measured in vivo in rats. Results  PEGylation was confirmed to be specifically located at the amino terminus of the B-chain of insulin. Higher loading efficiency was achieved with PEGylated insulin than regular human insulin in pH responsive hydrogels. The release of PEGylated insulin was lower than that of human insulin at all pH levels considered. Full retention of bioactivity of the PEG–insulin conjugate was confirmed by intravenous dosing while subcutaneous dosing exhibited a relative hypoglycemic effect 127.8% that of human insulin. Conclusions  Polyethylene glycol conjugated specifically to the amino terminus of the B-chain of insulin maintained the bioactivity of the protein and significantly extended the duration of the hypoglycemic effect. Used in combination with pH responsive hydrogels, PEGylated insulin has significant potential for oral delivery.     

Reproductible production of a PEGylated dual-acting peptide for diabetes

A PEGylated glucagon-like peptide-1 (GLP-1) agonist and glucagon antagonist hybrid peptide was engineered as a potential treatment for type 2 diabetes. To support preclinical development of this PEGylated dual-acting peptide for diabetes (DAPD), we developed a reproducible method for PEGylation, purification, and analysis. Optimal conditions for site-specific PEGylation with 22 and 43 kDa maleimide-polyethylene glycol (maleimide-PEG) polymers were identified by evaluating pH, reaction time, and reactant molar ratio parameters. A 3-step purification process was developed and successfully implemented to purify PEGylated DAPD and remove excess uncoupled PEG and free peptide. Five lots of 43 kDa PEGylated DAPD with starting peptide amounts of 100 mg were produced with overall yields of 53% to 71%. Analytical characterization by N-terminal sequencing, amino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and GLP-1 receptor activation assay confirmed site-specific attachment of PEG at the engineered cysteine residue, expected molecular weight, correct amino acid sequence and composition, and consistent functional activity. Purity and safety analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ion-exchange chromatography, reversed-phase high-performance liquid chromatography, and limulus amebocyte lysate test showed that the final products contained <1% free peptide, <5% uncoupled PEG, and <0.2 endotoxin units per milligram of peptide. These results demonstrate that the PEGylation and purification process we developed was consistent and effective in producing PEGylated DAPD preclinical materials at the 100 mg (peptide weight basis) or 1.2 g (drug substance weight basis) scale.     

PEGylation of proteins and liposomes: a powerful and flexible strategy to improve the drug delivery

PEGylation is one of the most successful strategies to improve the delivery of therapeutic molecules such as proteins, macromolecular carriers, small drugs, oligonucleotides, and other biomolecules. PEGylation increase the size and molecular weight of conjugated biomolecules and improves their pharmacokinetics and pharmacodinamics by increasing water solubility, protecting from enzymatic degradation, reducing renal clearance and limiting immunogenic and antigenic reactions. PEGylated molecules show increased half-life, decreased plasma clearance, and different biodistribution, in comparison with non-PEGylated counterparts. These features appear to be very useful for therapeutic proteins, since the high stability and very low immunogenicity of PEGylated proteins result in sustained clinical response with minimal dose and less frequent administration. PEGylation of liposomes improves not only the stability and circulation time, but also the 'passive' targeting ability on tumoral tissues, through a process known as the enhanced permeation retention effect, able to improve the therapeutic effects and reduce the toxicity of encapsulated drug. The molecular weight, shape, reactivity, specificity, and type of bond of PEG moiety are crucial in determining the effect on PEGylated molecules and, at present, researchers have the chance to select among tens of PEG derivatives and PEG conjugation technologies, in order to design the best PEGylation strategy for each particular application. The aim of the present review will be to elucidate the principles of PEGylation chemistry and to describe the already marketed PEGylated proteins and liposomes by focusing our attention to some enlightening examples of how this technology could dramatically influence the clinical application of therapeutic biomolecules.     

Site-directed PEGylation as successful approach to improve the enzyme replacement in the case of prolidase

The first aim of this work was to perform site-directed PEGylation of the enzyme prolidase at sulphydril groups by methoxy-polyethylene glycol-maleimide (Mal-PEG, Mw 5000 Da) in order to obtain a safe conjugation product more stable than the native enzyme. Prolidase is a cytosolic aminoacyl-l-proline hydrolase whose deficiency causes the onset of rare autosomal recessive disorder called prolidase deficiency (PD). The second purpose of this work was to investigate whether biodegradable chitosan nanoparticles loaded with PEGylated prolidase could be effective in releasing active enzyme inside fibroblasts as a possible therapeutic approach for PD. The SDS-PAGE analysis and the ESI-MS spectra confirmed the presence of the PEGylated prolidase: in particular the main conjugation product (m/z=about 65,000 Da) corresponded to the enzyme with two residues of Mal-PEG. In this study it was demonstrated the lack of toxicity (MTT assay) and the prolonged activity (40.6+/-2.6% after 48h of incubation at 37 degrees C) of the PEGylated enzyme. The PEGylated prolidase loaded chitosan nanoparticles had spherical shape, narrow size distribution (271.6+/-45.5 nm), a positive zeta-potential (15.93+/-0.26 mV) with a good preparation yield (54.6+/-3.6%) and protein encapsulation efficiency (44.8+/-4.6%). The ex vivo evaluation of prolidase activity on PD fibroblasts individuated a good level of prolidase activity replaced (about 72% after only 2 days of incubation) up to 10 days with improved morphological cell features.     

Preparation and characterization of lipid vesicles containing uricase

The study described the development of lipid vesicles as colloidal carriers for uricase, an enzyme with low activity at physicological conditions and low stability in vitro and in vivo. The lipid vesicles containing uricase (UOXLVs) were prepared and the process parameters were optimized with the indexes of entrapment efficiency, polydispersion, particle size, and zeta potential. The storage stability of uricase in lipid vesicles was significantly increased compared to that of free uricase at 4°C. The stability to proteolytic digestion was also increased obviously by entrapping the uricase in the lipid vesicles. In vitro and in vivo pharmacodynamic studies on the hyperuricemia rat model explicitly suggested that the uricase entrapped by UOXLVs possessed high uricolytic activity and distinctively decreased the uric acid level.     

The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg‐white lysozyme (Lyz) and its diPEGylated (5 kDa) analog as a result of typical handling stress conditions. Both proteins were subjected to mechanical stress in the absence and presence of silicone oil (SO), elevated temperatures, and freeze–thaw cycles. Flow imaging microscopy showed that PEGylated Lyz formed approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein–protein interactions, which may partly explain the reduced particle formation. Surprisingly, the PEGylated Lyz adsorbed an order of magnitude faster onto SO, despite being much larger in size, as determined by small‐angle X‐ray scattering and dynamic light scattering measurements. Thus, PEGylation may significantly reduce, but not prevent, micron aggregate formation of a protein during typical handling stresses. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3043–3054, 2014     

Preparation and characterization of lipid vesicles containing uricase

The study described the development of lipid vesicles as colloidal carriers for uricase, an enzyme with low activity at physicological conditions and low stability in vitro and in vivo. The lipid vesicles containing uricase (UOXLVs) were prepared and the process parameters were optimized with the indexes of entrapment efficiency, polydispersion, particle size, and zeta potential. The storage stability of uricase in lipid vesicles was significantly increased compared to that of free uricase at 4°C. The stability to proteolytic digestion was also increased obviously by entrapping the uricase in the lipid vesicles. In vitro and in vivo pharmacodynamic studies on the hyperuricemia rat model explicitly suggested that the uricase entrapped by UOXLVs possessed high uricolytic activity and distinctively decreased the uric acid level.