High expression of recombinant human catalase and its immunomodulatory effects on H1N1 influenza virus infection

Catalase is a key antioxidant enzyme and has been implicated in many pathophysiological processes of human diseases. In a previous study, we developed a yeast-based expression system for recombinant human catalase (rhCAT), and proved its therapeutic effects for treating H1N1 virus-induced pneumonia. However, the preparation of rhCAT was insufficient for further research applications. Here, we describe a much more convenient construction strategy for rhCAT based on the Pichia pastoris GS115 strain in a 14 L bioreactor. Quantitative-PCR, Western blotting, and activity assay were used to demonstrate the stable and efficient high-level expression (1500 U/mL; 77% recovery) achieved by this newly developed simple two-step purification procedure. The rhCAT synthesized by this new procedure was applied to an H1N1-infected mouse model (doses of 50 and 100 kU/mice/day) to assess its capabilities for inducing immunomodulatory effects. The results showed that the rhCAT could restore the impaired phagocytosis, alleviate the induced reductions in spleen and thymus organ weights, and markedly reduce the lung tissue viral load, all in a dose-dependent manner. Thus, this study provides not only a simple method for large-scale preparation of active rhCAT, but also in vivo evidence of the recombinant protein's immunomodulatory activity which may have clinical applications in treating H1N1 or other viral infections.     

Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A capillary electrophoretic method (CE) for characterizing PEGylated human parathyroid hormone 1-34 (PTH) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. CE was used to optimize the PEGylation of PTH through control of the reaction pH and the molar ratio of reactants with the advantages of minimal sample consumption and high separation capacity. The mono-PEGylated PTH (mono-PEG-PTH) was isolated and then digested with endoproteinase Lys-C. Resistance to Lys-C digestion on the PEGylation sites in the mono-PEG-PTH resulted in patterns of CE electropherograms different from that of the native PTH, and the PEGylation sites were assigned accordingly. The extent of positional isomers present in the mono-PEG-PTH was also determined by quantifying PEGylated fragments in the same CE electropherogram. In conclusion, the CE analysis of the Lys-C-digested sample allowed for simultaneous analysis of the PEGylation site and the extent of positional isomers in the mono-PEG-PTH. The results were confirmed by MALDI-TOF MS. This method will be applicable for characterizing PEGylation of other therapeutic peptides.     

Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.     

A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.     

PEGylated protein separation using different hydrophobic interaction supports: Conventional and monolithic supports

Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, β‐lactoglobulin, and lysozyme. All proteins were PEGylated in the N‐terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono‐ and di‐PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of β‐lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:702–707, 2016     

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).     

Proteolytically stabilizing fibronectin without compromising cell and gelatin binding activity

Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:277–288, 2015     

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, its application as anticancer drug is hampered by its poor stability under physiological conditions in the bloodstream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half-life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. To boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved T_m values (M31: 40°C; M36: 40°C; polyethylene glycol [PEG]-M31: 54°C; PEG-M36: 64°C) and half-life in human serum (M31: 1.9 days; M36: 2.0 days; PEG-M31: 3.2 days; PEG-M36: 4.8 days). These proved that surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine-auxotrophic tumors.     

干扰素a-2b的聚乙二醇修饰

采用分子量为20 kD的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰重组人干扰素a-2b(IFN a-2b), 建立了修饰反应及分离纯化工艺。考察了修饰反应各因素对单修饰转化率以及单修饰产物体外活性的影响, 获得了优化的修饰反应条件, 即在pH 6.5, 20 mmol/L的磷酸氢二钠-柠檬酸缓冲溶液中, 干扰素a-2b的浓度为4 mg/mL, PEG与IFN a-2b的摩尔比为8:1, 4oC时反应20 h; 在优化的反应条件下, 单修饰PEG-IFN a-2b的转化率达到55%。并且, 采用离子交换层析对修饰产物进行分离纯化, 单修饰产品纯度达到97%, 体外活性保留达到未修饰干扰素a-2b的13.4%, 其在SD大鼠体内的循环半衰期得到了较大的延长, 且具有较好的水溶液稳定性。     

Molecular Insight into the Steric Shielding Effect of PEG on the Conjugated Staphylokinase: Biochemical Characterization and Molecular Dynamics Simulation

PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.     

Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method

Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process.     

Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

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.     

Combined optimization of N‐terminal site‐specific PEGylation of recombinant hirudin using response surface methodology and kinetic analysis

In this study, a combined optimization method was developed to optimize the N‐terminal site‐specific PEGylation of recombinant hirudin variant‐2 (HV2) with different molecular weight mPEG‐propionaldehyde (mPEG‐ALD), which is a multifactor‐influencing process. The HV2‐PEGylation with 5 kDa mPEG‐ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono‐PEGylated product using combined statistical methods, including the Plackett–Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG‐ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono‐PEGylated‐HV2 was modified at the N‐terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N‐terminal site‐specific PEGylation process to obtain a homogeneous mono‐PEGylated protein with desirable yield.     

Lysine-deficient lymphotoxin-α mutant for site-specific PEGylation

The cytokine lymphotoxin-α (LTα) is a promising anticancer agent; however, its instability currently limits its therapeutic potential. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation occurs randomly at lysine residues and the N-terminus. Therefore, PEGylated proteins are generally heterogeneous and have lower bioactivity than their non-PEGylated counterparts. Previously, we created phage libraries expressing mutant LTαs in which the lysine residues of wild-type LTα (wtLTα) were substituted for other amino acids. Here, we attempted to create a lysine-deficient mutant LTα with about the same bioactivity as wtLTα by using these libraries and site-specific PEGylation of the N-terminus. We isolated a lysine-deficient mutant LTα, LT-K0, with almost identical bioactivity to that of wtLTα against mouse LM cells. The bioactivity of wtLTα decreased to 10% following random PEGylation, whereas that of LT-K0 decreased to 50% following site-specific PEGylation; PEGylated LT-K0 retained five times the bioactivity of randomly PEGylated wtLTα. These results suggest that site-specific PEGylated LT-K0 may be useful in cancer therapy.     

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.     

Mono-PEGylation of ribonuclease A: High PEGylation efficiency by thiolation with small molecular weight reagent

PEGylation can improve the therapeutic potential of ribonuclease A (RNase A), a cancer chemotherapeutic agent. However, the common PEGylation that targets at the ?-amino groups of proteins can lead to imprecise control of the stoichiometry of the protein-PEG conjugate (i.e., mono-, di- and multi-PEGylated protein). To prepare a PEGylated therapeutic protein, it is desirable that the protein is mono-PEGylated for industrial production, convenient purification and analytical characterization. Here, N-hydroxysuccinimide esters of S-acetylthioacetic acid (SATA) and 2-iminothiolane (IT) were used to introduce thiol groups on RNase A, followed by maleimide chemistry based PEGylation of the thiolated RNase A. Interestingly, the yield of mono-PEGylated RNase A was higher than 60%, and di- or multi-PEGylated RNase A were absent in the PEGylated product. Presumably, the limited number and low solvent accessibility of the introduced thiol group favored mono-PEGylation of RNase A. As compared to the unmodified RNase A, the mono-PEGylated RNase A showed slightly decreased enzymatic activity, increased anti-proliferative ability and unchanged structural properties. Our study is expected to control the PEGylation process and optimize the industrial pharmaceutical production of PEGylated proteins.     

PEGylated human plasma fibronectin is proteolytically stable,supports cell adhesion,cell migration,focal adhesion assembly,and fibronectin fibrillogenesis

Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS‐PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 493–504, 2013     

Intrapulmonary potential of polyethylene glycol-modified glucagon-like peptide-1s as a type 2 anti-diabetic agent

The pulmonary delivery of anti-diabetic peptide drugs can improve diabetic patient compliance. In this study, we tried to improve the pulmonary pharmacokinetic properties of glucagon-like peptide-1(7-36) (GLP-1) using a PEGylation approach. Initially, three types of site-specific (Lys(34)) PEGylated GLP-1 analogs were synthesized using PEGs of 2, 5, and 10 kDa, respectively. Their pharmacokinetic profiles were then examined in endotracheally cannulated rats. The results obtained show that all pharmacokinetic parameters (AUC(inf), C(max), t(1/2), V/F, and Cl/F etc.) of PEGylated GLP-1s were greatly improved by increasing PEG Mw. Specifically, the t(1/2) values of PEGylated GLP-1s (PEG Mw: 2, 5, 10 kDa) increased to 23.1+/-6.2, 41.6+/-12.3, and 81.7+/-11.7 min, respectively, vs. 8.9+/-2.9 min for intratracheally administered GLP-1. Also, PEGylated GLP-1s were found to have substantially greater C(max) values (7.4-7.8 ng/ml) than GLP-1 (4.0+/-2.4 ng/ml). Moreover, these PEGylated GLP-1s were found to have 10-20 fold more resistance to rat lung enzyme and plasma dipeptidyl peptidase IV (DPP IV). These findings indicate the dual-pharmacokinetic enhancements that PEGylated GLP-1s better survives proteolytic breakdown in the lungs than GLP-1s and better enters the systemic circulation, and that these analogs are more resistant to DPP IV-induced proteolysis and are much less rapidly removed from the systemic circulation. In conclusion, this study demonstrates the pharmaceutical usefulness of PEGylation in the context of the pulmonary delivery of GLP-1. These results show that PEGylated GLP-1s should be considered potential components of anti-diabetic inhalant preparations.     

A better anti-diabetic recombinant human fibroblast growth factor 21 (rhFGF21) modified with polyethylene glycol

As one of fibroblast growth factor (FGF) family members, FGF21 has been extensively investigated for its potential as a drug candidate to combat metabolic diseases. In the present study, recombinant human FGF21 (rhFGF21) was modified with polyethylene glycol (PEGylation) in order to increase its in vivo biostabilities and therapeutic potency. At N-terminal residue rhFGF21 was site-selectively PEGylated with mPEG20 kDa-butyraldehyde. The PEGylated rhFGF21 was purified to near homogeneity by Q Sepharose anion-exchange chromatography. The general structural and biochemical features as well as anti-diabetic effects of PEGylated rhFGF21 in a type 2 diabetic rat model were evaluated. By N-terminal sequencing and MALDI-TOF mass spectrometry, we confirmed that PEG molecule was conjugated only to the N-terminus of rhFGF21. The mono-PEGylated rhFGF21 retained the secondary structure, consistent with the native rhFGF21, but its biostabilities, including the resistance to physiological temperature and trypsinization, were significantly enhanced. The in vivo immunogenicity of PEGylated rhFGF21 was significantly decreased, and in vivo half-life time was significantly elongated. Compared to the native form, the PEGylated rhFGF21 had a similar capacity of stimulating glucose uptake in 3T3-L1 cells in vitro, but afforded a significantly long effect on reducing blood glucose and triglyceride levels in the type 2 diabetic animals. These results suggest that the PEGylated rhFGF21 is a better and more effective anti-diabetic drug candidate than the native rhFGF21 currently available. Therefore, the PEGylated rhFGF21 may be potentially applied in clinics to improve the metabolic syndrome for type 2 diabetic patients.     

Pulmonary fibrosis induced by H5N1 viral infection in mice

Background

Inflammatory process results in lung injury that may lead to pulmonary fibrosis (PF). Here, we described PF in mice infected with H5N1 virus.

Methods

Eight-week-old BALB/c mice were inoculated intranasally with 1 × 10¹ MID₅₀ of A/Chicken/Hebei/108/2002(H5N1) viruses. Lung injury/fibrosis was evaluated by observation of hydroxyproline concentrations, lung indexes, and histopathology on days 7, 14, and 30 postinoculation.

Results

H5N1-inoculated mice presented two stages of pulmonary disease over a 30-d period after infection. At acute stage, infected-mice showed typical diffuse pneumonia with inflammatory cellular infiltration, alveolar and interstitial edema and hemorrhage on day 7 postinoculation. At restoration stage, most infected-mice developed PF of different severities on day 30 postinoculation, and 18% of the survived mice underwent severe interstitial and intra-alveolar fibrosis with thickened alveolar walls, collapsed alveoli and large fibrotic areas. The dramatically elevated hydroxyproline levels in H5N1-infected mice showed deposition of collagen in lungs, and confirmed fibrosis of lungs. The dry lung-to-body weight ratio was significantly increased in infected group, which might be associated with the formation of PF in H5N1-infected mice.

Conclusion

Our findings show that H5N1-infected mice develop the typical PF during restoration period, which will contribute to the investigation of fibrogenesis and potential therapeutic intervention in human H5N1 disease.