Preparation Of Long-acting Human Serum Albumin And Investigation On Its Biological Function And Pharmacokinetics

Human serum albumin (HSA) is the major protein component of human plasma with a plasma content of42±3.5g/L. HSA is responsible for80%of the colloid osmotic pressure of plasma (25～33mmHg). Because of this, its main clinical use is in increasing circulating plasma volume, maintaining colloid osmotic pressure and hemodilution. HSA is widely used to treat hypovolemia, traumatic shock, burns, hypoalbuminemia, acute liver failure, ascites, surgery, acute respiratory distress syndrome and hemodialysis.HSA is most commonly isolated by fractionation of plasma obtained from blood donors. However, since the supply for material plasma is limited in the global range, the shortage of donor blood has become a limiting factor for the production of HSA preparation. Aiming at this issue, many efforts have been made to develop a substitute for the plasma-derived HSA by means of genetic engineering. However, due to the low production yields and the high clinical dosage of HSA, there is still an unsettled problem concerning the establishment of techniques for high purity, low cost, industrial large-scale production of rHSA. Therefore, at present, plasma is still the most important source of HSA as a plasma volume expander.In addition, there are some limitations to the clincal usage of HSA. Increased vascular permeability is an important pathphysiological cause of the hypoalbuminemia commonly seen in burn injury, sepsis and trauma. Under this condition, correction of low plasma volume therefore becomes essential. However, the plasma expanding effect of HSA is transient due to a continuous leakage of macromolecules into the interstitial space, such poor intravascular retention does not only lead to interstitial edema but also demands a frequent administration of HSA infusion to maintain the desired blood concentration. These results suggest that the modification of HSA with PEG could significantly increase its hydrodynamic radius. A two-step method consisting of blockage and PEGylation was applied to determine the modification site. SDS-PAGE analysis subsequently showed that NEM-treated HSA could no longer be PEGylated by PEG-MAL, thus confirming that PEG-MAL was specifically attached to the unique thiol group of HSA, Cys34. The relative molecular mass (Mr) of N-terminal PEG-HSA was determined by gel permeation chromatography-multi angle laser scattering technique, Mr of N-terminal PEG-HSA was83.71kDa, which indicated that only one PEG chain was conjugated onto one HSA molecule.3Drug binding properties of native and PEGylated HSAHSA is the most important carrier protein in human plasma, the affinity of a drug toward HSA or PEGylated HSA is an important issue when determining its overall pharmacokinetic profile including transport, distribution and metabolism. In the present study, surface plasmon resonance (SPR) technique was used to evaluate the interaction of two model drugs (warfarin and naproxen) and proteins.SPR technique could correctly reflect the binding level of drugs to HSA, the dose-response curves showed that warfarin and naproxen binding appeared monophasic and approached saturation levels at low concentrations. The dissociation equilibrium constant (KD) was used to measure the affinity of drugs toward native and PEGylated HSA, the smaller the KD, the greater the affinity. The KD values of warfarin towards HSA, PEG-cys34HSA, N-terminal PEG-HSA and random PEG-HSA were12.9,24,14.2and67.6umol/L, respectively. The KD values of naproxen towards HSA, PEG-cys34HSA, N-terminal PEG-HSA and random PEG-HSA were24.6,36,27.7and44.7μmol/L. Site-specific PEGylation of HSA has little influence on the drug binding properties, compared with PEGylation targeting cys34, PEGylation targeting N-terminus has less influence on the drug affinity of HSA. While, random PEGylation may reduce the affinities of protein and drugs. The affinities between PEGylated HSA and small molecular drugs are different according to different modification sites. 4Investigation of pharmacokinetics and tissue distribution of native and PEGylated HSA in miceRadioiodine labelling and TCA precipitation method was used to investigate the half-life and tissue distribution of native and PEGylated HSA in mice.The results showed that plasma concentration-time profile after intravenous administration of native and PEGylated HSA fitted well using the two-compartment model. The elimination half-life (t1/2β) of HSA, PEG-cys34HSA, N-terminal PEG-HSA and random PEG-HSA was9.51±2.22,21.91±2.00,22.50±1.70and22.93±1.51h, respectively. The t1/2β of the modified HSA was approximately2.3-2.4times of that of the native HSA. In addition, decreased clearance (CL) and increased mean retention time (MRT) were observed as well. These all suggested improvements in the retention of PEGylated HSA in the circulation. No significant difference was observed in tissue distribution between native and PEGylated HSA, the modification did not alter the tissue distribution characteristics of HSA.5Pulmonary microvascular permeability of native and PEGylated HSA in acute lung injury (ALI) model and pharmacodynamics of native and PEGylated HSA in lipopolysaccharide-induced sepsis modelLPS-induced ALI model was used to compare the pulmonary microvascular permeability properties of HSA and PEG-HSA. The pharmacodynamics of native and PEGylated HSA was investigated in LPS-induced sepsis rat model with systemic capillary leakage. Haematocrit (Hct) and mean arterial pressure (MAP) was used to evaluate the therapeutic effect of native and PEGylated HSA on sepsis.A lower vascular permeability of PEGylated HSA was observed compared with that of HSA in LPS-induced ALI model, this result suggested that the PEGylation of HSA could reduce the extravasation into lung parenchyma. In the pharmacodynamics study, a significant decrease in MAP and increase in Hct was observed2h post LPS injection, which indicated the sepsis model was successfully established. At2h after fluid treatment, MAP in HSA group increased to98±15mmHg from92±6mmHg, MAP in PEG-cys34HSA group increased to117±7mmHg from92±12mmHg, the MAP recovery in PEG-cys34HSA group was better than in HSA group (P<0.05). Meanwhile, Hct was much lower in PEG-cys34HSA group than HSA group, suggesting a relatively greater increment in intravascular plasma volume. The above results suggest that the resuscitation effect of PEG-HSA is better than that of HSA in LPS-induced sepsis with systemic capillary leakage.The main conclusions were as follows:1. The conjugation of HSA with mPEG-MAL and mPEG-ALD could generate molecularly homogeneous site-specific PEGylated HSA, the reactions are characterized by simple operation, good reproducibility and high yield of monoPEGylated products. PEGylated HSA with high purity could be obtained using two-step separation process of DEAE Sepharose FF column chromatography and ultrafiltration.2. The modification of HSA with PEG did not induce significant alteration of the secondary protein structure and could significantly increase its hydrodynamic radius.3. The affinities between PEGylated HSA and small molecular drugs are different according to modification sites. Site-specific PEGylation targeting Cys34and N-terminus of HSA has little influence on the drug binding properties, while, random PEGylation may reduce the affinities of protein and drugs.4. PEGylation of HSA prolongs elimination half-life and improves retention in the circulation. PEGylated HSA exhibited no significant difference in tissue tropism with respect to HSA.5. The PEGylation of HSA could reduce transcapillary loss of albumin molecules in LPS-induced acute lung injury model. In the therapy of lipopolysaccharide-induced sepsis, the effect of MAP recovery and plasma volume expansion of PEGylated HSA is superior to HSA.