Human serum albumin–thioredoxin fusion protein with long blood retention property is effective in suppressing lung injury

Abstract

Thioredoxin (Trx) is a redox-active protein with anti-inflammatory effects but with a short half life of 1 h. Genetic fusion of Trx to human serum albumin (HSA) extended its half life without causing significant loss of its biological activities. HSA–Trx caused a decrease in the number of cells in brochoalveolar lavage fluid, the wet/dry ratio and the inflammation at the respiratory tract of the ovalbumin (OVA) induced lung injury model mouse. Three intraperitoneal doses of Trx alone produced the same extent of suppression of those three detrimental effects of OVA as one intravenous dose of HSA–Trx. Inhibition experiments confirmed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) involved in the progression of the injury. HSA–Trx inhibited the production of ROS as confirmed in the EPR experiment, but lung tissue staining suggested that induced nitrogen oxide synthase (iNOS) was not suppressed by the fusion protein. Instead, the production of nitrotyrosine, 8-nitro-cGMP, and 8-hydroxy-2′-deoxyguanosine downstream to the iNOS has been inhibited. This suggested that HSA–Trx produced lung protection effect via different mechanisms from Trx alone. HSA–Trx retains the biological properties of Trx thus has great potential in treating oxidative stress related diseases.

Introduction

Thioredoxin (Trx) is a small ubiquitous protein in human body that is redox active and is induced in response to various oxidative stress conditions. Trx contains 5 cysteine residues, with those located at 32 and 35, -Cys-Gly-Pro-Cys-, responsible for its main redox activity via dithiol–disulfide exchange to exert its anti-oxidative effect. It regulates the redox conditions of cells intracellularly and extracellularly. In addition, Trx also possesses oxygen radical removal ability in collaboration with other peroxidase [1]. Recently, a number of studies on exploiting the biological activities of Trx in treating diseases caused by oxidative stress have been attempted. For example, rat or rabbit hemorrhagic shock reperfusion model showed that Trx has good organ protection effect. It was also effective in bleomycin induced interstitial pneumonia or chronic obstructive pulmonary disease [2], [3], [4]. In transgenic mouse that expresses high level of Trx, a lot of beneficial effects such as reduction of diabetes mellitus and cerebral infarction/stroke, suppression of hemorrhagic renal failure, improvement of interstitial pneumonia and pulmonary fibrosis have been observed [5], [6].

Although Trx has great potential as a new therapeutic agent, being a peptide with small molecular weight it will be eliminated extensively via glomerular filtration. In fact, the half life of Trx is about 1 h, which is extremely short. As a result, constant rate infusion or repeated administration is required [6], [7]. This will add on to the burden of the patients as well as the health care professionals, not to mention increased medical cost. Therefore, it is crucial to improve the poor blood retention property of Trx before it will have practical use clinically.

A number of methods have been developed to improve the blood retention property of a biological active peptide such as nanoparticulation with polylactic acid, macromolecularization via micelle formation, peglytization with polyethylene glycol, and other different types of drug delivery systems [8], [9], [10]. The advancement of recombinant DNA technology has made genetic fusion of two protein molecules possible. The biologically active peptides or low molecular weight protein can be fused genetically to human serum albumin (HSA) which has a longer plasma half life so that the half life of the short peptide can be extended [11], [12]. In comparison to chemical modification method such as peglytization, albumin fusion method has not been reported to produce an accelerated blood clearance phenomenon, a phenomenon of increased clearance with increasing dosing [13].

With this background, in a previous study we have successfully produced the fusion protein of HSA and Trx, and performed structural and functional properties evaluation of the fusion protein, HSA–Trx. The HSA–Trx fusion protein exhibited similar pharmacokinetic property as HSA, thus a much improved blood retention property than that of Trx alone. Although fusion to HSA caused about 40% reduction in Trx's in vitro biological activity, HSA–Trx has proven to be effective therapeutically in the septic shock mouse model. It is noteworthy that HSA–Trx showed higher distribution to the lungs than to other organs, as well as 10 times longer plasma half life than Trx in normal mice [14].

Parenteral administration of Trx has been shown in a number of studies to be effective in treating lung disorder, due either or both of Trx antioxidative and anti-inflammatory properties. Nakamura et al. reported the usefulness of Trx in treating different types of diseases including severe acute lung diseases where Trx is likely to contribute with its anti-inflammatory properties. Trx has been shown to inhibit the asthmatic response after OVA sensitization by Ichiki et al. [15], [16]. On the other hand, Callister et al. reported that extracellular Trx levels are raised in patients with acute lung injury, particularly of pulmonary origin [17]. Furthermore, priming of donor lungs with Trx before transplant attenuates acute allograft injury in a rat model of lung transplantation [18].

Using the results of our previous study of HSA–Trx in normal mice and septic shock mouse model as a starting point, we further investigated the usefulness of HSA–Trx as a therapeutic agent for treating oxidative stress related lung disorders, taking advantage of the HSA–Trx high lung distribution property [14]. The effects of HSA–Trx were assessed using an ovalbumin (OVA) induced lung injury model mouse [15].