In silico evaluation of limited sampling strategies for individualized dosing of extended half-life factor IX concentrates in hemophilia B patients.

PURPOSE: Hemophilia B is a bleeding disorder, caused by a factor IX (FIX) deficiency. Recently, FIX concentrates with extended half-life (EHL) have become available. Prophylactic dosing of EHL-FIX concentrates can be optimized by assessment of individual pharmacokinetic (PK) parameters. To determine these parameters, limited sampling strategies (LSSs) may be applied. The study aims to establish adequate LSSs for estimating individual PK parameters of EHL-FIX concentrates using in silico evaluation. METHODS: Monte Carlo simulations were performed to obtain FIX activity versus time profiles using published population PK models for N9-GP (Refixia), rFIXFc (Alprolix), and rIX-FP (Idelvion). Fourteen LSSs, containing three or four samples taken within 8 days after administration, were formulated. Bayesian analysis was applied to obtain estimates for clearance (CL), half-life (t1/2), time to 1% (Time1%), and calculated weekly dose (Dose1%). Bias and precision of these estimates were assessed to determine which LSS was adequate. RESULTS: For all PK parameters of N9-GP, rFIXFc and rIX-FP bias was generally acceptable (range: -5% to 5%). For N9-GP, precision of all parameters for all LSSs was acceptable (< 25%). For rFIXFc, precision was acceptable for CL and Time1%, except for t1/2 (range: 27.1% to 44.7%) and Dose1% (range: 12% to 29.4%). For rIX-FP, all LSSs showed acceptable bias and precision, except for Dose1% using LSS with the last sample taken on day 3 (LSS 6 and 10). CONCLUSION: Best performing LSSs were LSS with samples taken at days 1, 5, 7, and 8 (N9-GP and rFIXFc) and at days 1, 4, 6, and 8 (rIX-FP), respectively.

Tailoring patchy nanoparticle design to modulate serum albumin adsorption and membrane interaction.

When nanoparticles (NPs) enter into the biological system, a wide range of proteins will coat on their surfaces forming protein corona, which changes the initial synthetic characteristics of NPs to the biological identity, resulting in the loss of their targets or specially designed properties. Although pre-coating with proteins would reduce the protein corona formation, they may diminish the targeting moieties in the transport process. Patchy NPs can offer unique advantages of asymmetry, heterogeneity, and multi-functions. This has inspired us to use the asymmetry to realize the versatility of NPs, to accommodate stealth and targeting functions. In this study, we performed molecular dynamics simulations to investigate the adsorption mechanism between patchy NPs and human serum albumin, and the interaction mechanism between NP-HSA and the membrane. The results show that there is a high probability for HSA to interact with the hydrophobic, or charged brushes of patchy NPs. The adsorption sites, as calculated through the contact probability between NPs and the residues, depend on the NP surface properties. Furthermore, the HSA adsorption on NPs could improve the NP-membrane interaction. The simulation results provide deep understanding of the NP interaction mechanism, which would help the NP design for their biomedical applications.

Expression and characterization of albumin fusion protein canine IFNγ-CSA in baculovirus-insect cell expression system.

In this study, canine IFNγ was fused by a flexible linker with canine serum albumin to construct the fusion protein IFNγ-CSA for the purpose to design a long-acting canine IFNγ. The fusion protein was successfully expressed in baculovirus-infected Sf9 insect cells and was purified by salting-out and ion exchange chromatography. The IFNγ-CSA fusion possessed potent anti-viral assay against vesicular stomatitis virus in cultured cells. IFNγ-CSA was also stable at 37 °C up to 72 h compared with 8 h for IFNγ alone. In vivo pharmacokinetics demonstrated a significantly longer half-life for IFNγ-CSA (15.42 h) than for canine reIFNγ (1.51 h) in KM mice. These results indicate that IFNγ-CSA expression in the baculovirus system was successful and provide a promising long-acting cytokine for veterinary clinical applications.

Lactosylated Albumin Nanoparticles: Potential Drug Nanovehicles with Selective Targeting Toward an In Vitro Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) ranks fifth in occurrence and second in mortality of all cancers. The development of effective therapies for HCC is urgently needed. Anticancer drugs targeted to the liver-specific asialoglycoprotein receptors (ASGPRs) are viewed as a promising potential treatment for HCC. ASGPRs facilitate the recognition and endocytosis of molecules, and possibly vehicles with galactose end groups, by the liver. In this study, bovine serum albumin (BSA) was conjugated with lactose using a thermal treatment. The formation of lactosylated BSA (BSA-Lac) was confirmed by a change of the chemical structure, increased molecular mass, and Ricinus communis lectin recognition. Subsequently, the low-crosslinking BSA-Lac nanoparticles (LC BSA-Lac NPs) and high-crosslinking BSA-Lac nanoparticles (HC BSA-Lac NPs) were synthesized. These nanoparticles presented spherical shapes with a size distribution of 560 ± 18.0 nm and 539 ± 9.0 nm, as well as an estimated surface charge of -26 ± 0.15 mV and -24 ± 0.45 mV, respectively. Both BSA-Lac NPs were selectively recognized by ASGPRs as shown by biorecognition, competition, and inhibition assays using an in vitro model of HCC. This justifies pursuing the strategy of using BSA-Lac NPs as potential drug nanovehicles with selective direction toward hepatocellular carcinoma.

Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell.

PURPOSE: Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. METHODS: FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. RESULTS: FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. CONCLUSION: AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

RSM optimization of HSA/IL1Ra in Pichia pastoris overexpression strain and study of its in vivo activity in reducing hyperglycemia of GK rats.

Human serum albumin (HSA) and interleukin-1 receptor antagonist (IL1Ra) fusion protein is a potential long-acting drug in the treatment of type 2 diabetes. Previously, the expression level of HSA/IL1Ra in Pichia pastoris was successfully improved by increasing the gene copy number and coexpression with chaperone (protein disulfide isomerase) in our laboratory. However, the overexpression strain resulted in low production of high- cell-density fermentation. In this study, the culture medium was optimized in both flask and fermenter, and the optimum culture medium notably increased the productivity and stability of HSA/IL1Ra. To further improve the expression, response surface methodology was used to further optimize the culture condition through modeling three selected parameters (induction pH, induction temperature [T], and maximum methanol feed rate [Vm ]). The maximum yield of HSA/IL1Ra reached 1.1 g/L (10-fold higher than original fermentation condition) under the optimized culture condition (pH 7.0, T = 29 â„ƒ and Vm = 4.82 mL/L/H) in a 5-L fermenter. In addition, the degradation position of HSA/IL1Ra during fermentation was determined to be K571, serving as a potential target for genetic modification strategies to reduce the degradation. Finally, the in vivo activity study showed that HSA/IL1Ra maintained the therapeutic effect of IL1Ra in type 2 diabetes model rats meanwhile reducing the frequency of administration.

In†Vivo Gold Complex Catalysis within Live Mice.

Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-AuIII complex was designed and developed that enables organ-specific, localized propargyl ester amidation with nearby proteins within live mice. The targeted reactivity can be imaged through the use of Cy7.5- and TAMRA-linked propargyl ester based fluorescent probes. This targeting system could enable the exploitation of other metal catalysis strategies for biomedical and clinical applications.

A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys.

Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

Changes in intestinal permeability and nutritional status after cytotoxic therapy in patients with cancer.

Damage to intestinal mucosa may impair nutritional status and increase the demand for nutrients involved in intestinal cell proliferation (retinol and folate). It is still unclear if cytotoxic therapy affects serum concentrations of these nutrients in patients with cancer and if this would be associated with disturbances of intestinal mucosa. Intestinal permeability, serum folate, and retinol and nutritional status of 22 patients with hematologic malignancies and 17 healthy volunteers [control group (CG)] were assessed before (T0) and after cytotoxic therapy (T1). Ingestion of lactulose and mannitol was used to assess intestinal permeability. Anthropometric, body composition, phase angle (PA), and biochemical analysis (albumin, retinol, and folate) were also performed. Lactulose/mannitol ratio (0.026 ± 0.014 vs. 0.052 ± 0.037) and lactulose excretion (0.27 ± 0.18% vs. 0.53 ± 0.6%) increased at T1. PA decreased (7.2 ± 1.9° vs. 6.2 ± 0.9°). Serum folate and albumin (20.7 ± 9.5 nmol/L, 37.7 ± 5.5 g/L) were lower than CG (39.2 ± 16.4 nmol/L, 42.9 ± 5.2 g/L) but did not change at T1 (17.5 ± 7.0 nmol/L, 35.9 ± 4.5 g/L). Serum retinol did not differ from CG and did not change at T1 (1.83 ± 0.30 µmol/L vs. 1.69 ± 0.3 µmol/L; CG: 1.86 ± 0.20 µmol/L). Abnormal intestinal permeability, low serum folate levels, and its possible relationship with intestinal alterations, and reduced PA, may be associated with poor nutritional status in cancer patients.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Permeability and contractile responses of collecting lymphatic vessels elicited by atrial and brain natriuretic peptides.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are cardiac hormones released into the bloodstream in response to hypervolaemia or fluid shifts to the central circulation. The actions of both peptides include natriuresis and diuresis, a decrease in systemic blood pressure, and inhibition of the renin-angiotensin-aldosterone system. Further, ANP and BNP elicit increases in blood microvessel permeability sufficient to cause protein and fluid extravasation into the interstitium to reduce the vascular volume. Given the importance of the lymphatic vasculature in maintaining fluid balance, we tested the hypothesis that ANP or BNP (100 nM) would likewise elevate lymphatic permeability (Ps) to serum albumin. Using a microfluorometric technique adapted to in vivo lymphatic vessels, we determined that rat mesenteric collecting lymphatic Ps to rat serum albumin increased by 2.0 ± 0.4-fold (P = 0.01, n = 7) and 2.7 ± 0.8-fold (P = 0.07, n = 7) with ANP and BNP, respectively. In addition to measuring Ps responses, we observed changes in spontaneous contraction amplitude and frequency from the albumin flux tracings in vivo. Notably, ANP abolished spontaneous contraction amplitude (P = 0.005) and frequency (P = 0.006), while BNP augmented both parameters by ∼2-fold (P < 0.01 each). These effects of ANP and BNP on contractile function were examined further by using an in vitro assay. In aggregate, these data support the theory that an increase in collecting lymphatic permeability opposes the absorptive function of the lymphatic capillaries, and aids in the retention of protein and fluid in the interstitial space to counteract volume expansion.

Synthesis and bioactive evaluation of novel hybrids of metronidazole and berberine as new type of antimicrobial agents and their transportation behavior by human serum albumin.

A series of novel hybrids of metronidazole and berberine as new type of antimicrobial agents were synthesized and characterized by (1)H NMR, (13)C NMR, IR, MS and HRMS spectra. Bioactive assay manifested that most of the prepared compounds exhibited effective antibacterial and antifungal activities and some showed comparable or superior potency against Methicillin-resistant Staphylococcus aureus to reference drugs Norfloxacin, Chloromycin and Berberine. The transportation behavior of human serum albumin (HSA) to the highly active compound 5g was evaluated and revealed that the association of imidazole derivative 5g with HSA was spontaneous and the electrostatic interactions played important roles in the transportation of HSA to 5g. The calculated parameters indicated that compound 5g could be effectively stored and carried by HSA.

Wettability versus electrostatic forces in fibronectin and albumin adsorption to titanium surfaces.

OBJECTIVES: Although the enhancement of plasma protein adsorption to titanium ( Ti ) following wetting has been recognized, the relationship between wettability and electrostatic forces has remained unclear. Thus, we have carried out a series of studies to determine the role of wettability and electrostatic forces on protein adsorption. METHODS: Titanium disks with different surfaces were wetted with a range of solutions, two of which contained divalent positive ions ( Ca and Mg ). Unwetted disks served as a control. Subsequently, the wetted disks were subjected to three treatment regimes: (1) incubation in human serum albumin (HSA) or human serum fibronectin (HSF); (2) drying the wetted disks, followed by incubation in HSA or HSF; and (3) following protein adsorption, the Ca originating in the wetting solutions was removed by divalent positive ions chelator treatment (EGTA), and the remaining quantities were assessed. The quantity of the adsorbed proteins was determined by ELISA. RESULTS: It was found that in the case of HSA, adsorption was enhanced by the wettability, the presence of Ca and Mg in the wetting solution, and the existence of rough surfaces. For HSF, the wettability and rough surfaces enhanced adsorption. CONCLUSION: The results demonstrate that in addition to wettability, the composition of the wetting solution affects the protein adsorption. While wetting reduces the time for the HSA and HSF adsorption to reach saturation, the electrostatic forces enhance the amount of HSA adsorption. Thus, the protein adsorption capacity of titanium rough surfaces can be selectively manipulated by changing of the wetting solution.

Fiberoptic microneedle device facilitates volumetric infusate dispersion during convection-enhanced delivery in the brain.

BACKGROUND AND OBJECTIVES: A fiberoptic microneedle device (FMD) was designed and fabricated for the purpose of enhancing the volumetric dispersal of macromolecules delivered to the brain through convection-enhanced delivery (CED) by concurrent delivery of sub-lethal photothermal hyperthermia. This study's objective was to demonstrate enhanced dispersal of fluid tracer molecules through co-delivery of 1,064 nm laser energy in an in vivo rodent model. MATERIALS AND METHODS: FMDs capable of co-delivering fluids and laser energy through a single light-guiding capillary tube were fabricated. FMDs were stereotactically inserted symmetrically into both cerebral hemispheres of 16 anesthetized rats to a depth of 1.5 mm. Laser irradiation (1,064 nm) at 0 (control), 100, and 200 mW was administered concurrently with CED infusions of liposomal rhodamine (LR) or gadolinium-Evans blue-serum albumin conjugated complex (Gd-EBA) at a flow rate of 0.1 µl/min for 1 hour. Line pressures were monitored during the infusions. Rodents were sacrificed immediately following infusion and their brains were harvested, frozen, and serially cryosectioned for histopathologic and volumetric analyses. RESULTS: Analysis by ANOVA methods demonstrated that co-delivery enhanced volumetric dispersal significantly, with measured volumes of 15.8 ± 0.6 mm(3) for 100 mW compared to 10.0 ± 0.4 mm(3) for its fluid only control and 18.0 ± 0.3 mm(3) for 200 mW compared to 10.3 ± 0.7 mm(3) for its fluid only control. Brains treated with 200 mW co-delivery exhibited thermal lesions, while 100 mW co-deliveries were associated with preservation of brain cytoarchitecture. CONCLUSION: Both lethal and sub-lethal photothermal hyperthermia substantially increase the rate of volumetric dispersal in a 1 hour CED infusion. This suggests that the FMD co-delivery method could reduce infusion times and the number of catheter insertions into the brain during CED procedures.

S-Nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects.

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.

177Lu-DO3A-HSA-Z EGFR:1907: characterization as a potential radiopharmaceutical for radionuclide therapy of EGFR-expressing head and neck carcinomas.

Epidermal growth factor receptor 1 (EGFR) is an attractive target for radionuclide therapy of head and neck carcinomas. Affibody molecules against EGFR (Z(EGFR)) show excellent tumor localizations in imaging studies. However, one major drawback is that radiometal-labeled Affibody molecules display extremely high uptakes in the radiosensitive kidneys which may impact their use as radiotherapeutic agents. The purpose of this study is to further explore whether radiometal-labeled human serum albumin (HSA)-Z(EFGR) bioconjugates display desirable profiles for the use in radionuclide therapy of EGFR-positive head and neck carcinomas. The Z(EFGR) analog, Ac-Cys-Z(EGFR:1907), was site-specifically conjugated with HSA. The resulting bioconjugate 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A)-HSA-Z(EGFR:1907) was then radiolabeled with either (64)Cu or (177)Lu and subjected to in vitro cell uptake and internalization studies using the human oral squamous carcinoma cell line SAS. Positron emission tomography (PET), single photon emission computed tomography (SPECT), and biodistribution studies were conducted using SAS-tumor-bearing mice. Cell studies revealed a high (8.43 ± 0.55 % at 4 h) and specific (0.95 ± 0.09 % at 4 h) uptake of (177)Lu-DO3A-HSA-Z(EGFR:1907) as determined by blocking with nonradioactive Z(EGFR:1907). The internalization of (177)Lu-DO3A-HSA-Z(EGFR:1907) was verified in vitro and found to be significantly higher than that of (177)Lu-labeled Z(EFGR) at 2-24 h of incubation. PET and SPECT studies showed good tumor imaging contrasts. The biodistribution of (177)Lu-DO3A-HSA-Z(EGFR:1907) in SAS-tumor-bearing mice displayed high tumor uptake (5.1 ± 0.44 % ID/g) and liver uptake (31.5 ± 7.66 % ID/g) and moderate kidney uptake (8.5 ± 1.08 % ID/g) at 72 h after injection. (177)Lu-DO3A-HSA-Z(EGFR:1907) shows promising in vivo profiles and may be a potential radiopharmaceutical for radionuclide therapy of EGFR-expressing head and neck carcinomas.

Influence of molecular shape, conformability, net surface charge, and tissue interaction on transscleral macromolecular diffusion.

The purpose of this study was to investigate the influence of molecular shape, conformability, net surface charge and tissue interaction on transscleral diffusion. Unfixed, porcine sclera was clamped in an Ussing chamber. Fluorophore-labelled neutral albumin, neutral dextran, or neutral ficoll were placed in one hemi-chamber and the rate of transscleral diffusion was measured over 24 h using a spectrophotometer. Experiments were repeated using dextrans and ficoll with positive or negative net surface charges. Fluorescence recovery after photobleaching (FRAP) was undertaken to compare transscleral diffusion with diffusion through a solution. All molecules were 70 kDa. With FRAP, the diffusion coefficient (D) of neutral molecules was highest for albumin, followed by ficoll, then dextran (p < 0.0001). Positive dextrans diffused fastest, followed by negative, then neutral dextrans (p = 0.0004). Neutral ficoll diffused the fastest, followed by positive then negative ficoll (p = 0.5865). For the neutral molecules, transscleral D was highest for albumin, followed by dextran, then ficoll (p < 0.0001). D was highest for negative ficoll, followed by neutral, then positive ficoll (p < 0.0001). By contrast, D was highest for positive dextran, followed by neutral, then negative dextran (p = 0.0021). In conclusion, diffusion in free solution does not predict transscleral diffusion and the molecular-tissue interaction is important. Molecular size, shape, and charge may all markedly influence transscleral diffusion, as may conformability to a lesser degree, but their effects may be diametrically opposed in different molecules, and their influence on diffusion is more complex than previously thought. Each variable cannot be considered in isolation, and the interplay of all these variables needs to be tested, when selecting or designing drugs for transscleral delivery.

Expression, purification and characterization of recombinant human interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in Pichia pastoris.

Interleukin-2 (IL-2) plays important roles in variety of immune functions. Recombinant IL-2 has become an important therapeutic protein for therapy of melanoma and renal cell carcinoma. Previously, it was proved that the therapeutic efficacy of rIL-2 expressed in Saccharomyces cerevisiae was improved by prolonging its in vivo half-life through genetic fusion with albumin. In this study, a fusion protein composed of hIL-2 genetically fused to HSA was expressed as a secretory protein under AOX1 (alcohol oxidase 1) promoter in Pichia pastoris. An effective strategy was established to express rhIL-2-HSA fusion protein in 5L scale and the optimal purification procedure was investigated. The purity of rhIL-2-HSA in final product was about 95%. The purified rhIL-2-HSA fusion protein could be recognized by both anti-hIL-2 and anti-human serum albumin monoclonal antibody. Bioactivity analysis showed that the purified rhIL-2-HSA fusion protein displayed high level activity on proliferation in IL-2 dependent manner in CTLL2-cells. rhIL-2-HSA fusion protein also showed a extended half-life in plasma compared with IL-2 when tested in a BALB/c mouse model. This study provides an alternative strategy for large-scale production of bioactive rhIL-2-HSA fusion protein using P. pastoris as an expression host.

Site-specific chemical modification of human serum albumin with polyethylene glycol prolongs half-life and improves intravascular retention in mice.

Human serum albumin (HSA) is used as an important plasma volume expander in clinical practice. However, the infused HSA may extravasate into the interstitial space and induce peripheral edema in treating the critical illness related to marked increase in capillary permeability. Such poor intravascular retention also demands a frequent administration of HSA. We hypothesize that increasing the molecular weight of HSA by PEGylation may be a potential approach to decrease capillary permeability of HSA. In the present study, HSA was PEGylated in a site-specific manner and the PEGylated HSA carrying one chain of polyethylene glycol (PEG) (20 kDa) per HSA molecule was obtained. The purity, PEGylated site and secondary structure of the modified protein were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), thiol group blockage method and circular dichroism (CD) measurement, respectively. In addition, the pharmacokinetics in normal mice was investigated, vascular permeability of the PEGylated HSA was evaluated in lipopolysaccharide (LPS)-induced lung injury mouse model and the pharmacodynamics was investigated in LPS-induced sepsis model with systemic capillary leakage. The results showed that the biological half-life of the modified HSA was approximately 2.3 times of that of the native HSA, PEG-HSA had a lower vascular permeability and better recovery in blood pressure and haemodilution was observed in rats treated with PEG-HSA. From the results it can be inferred that the chemically well-defined and molecularly homogeneous PEGylated HSA is superior to HSA in treating capillary permeability increase related illness because of its longer biological half-life and lower vascular permeability.