Human serum albumin–thioredoxin fusion protein with long blood retention property is effective in suppressing lung injury
Graphical abstract
HSA–Trx prevent the OVA induced lung injury via suppression of ROS and RNS in the lungs.
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].
Section snippets
Materials
OVA and allopurinol were purchased from Sigma (Osaka, Japan). Aluminum hydroxide was from SERVA. Nω-nitro-l-arginine methyl ester (L-NAME) was purchased from Dojindo Laboratories (Kumamoto, Japan). Anti-8-nitro-cGMP antibody (1G6) was obtained from Department of Microbiology, Faculty of Life Sciences, Kumamoto University. The Pichia Expression Kit was purchased from Invitrogen Corp. (Carlsbad, CA, USA). A DNA sequence kit (Dye Terminator Cycle Sequencing FS Ready Reaction Kit) was obtained from
Evaluation of the OVA induced lung injury mouse model
After sensitizing the mice with OVA, the mice were nebulised with the OVA to produce Lung disorder mice. The validity of the mouse model was evaluated from 3 aspects: 1. inflammatory cell numbers in BALF that evaluates the lung infiltration; 2. wet/dry ratio that evaluates the extent of lung edema; and 3. HE staining that reveals the site of inflammation in the tissue.
Firstly, the number of cells in BALF of the OVA(+) group increased significantly than the OVA(−) group (Fig. 2A). Based on the
Discussion
Trx has been reported to not only have antioxidation and anti-inflammatory effects but also able to suppress excessive immune response. We have shown in a previous study that HSA–Trx sustained 60% insulin reducing assay activity of Trx as well as producing beneficial effect in endotoxic shock mouse model. In addition, HSA–Trx was found to accumulated significantly in lungs. Trx was found to be effective in asthmatic treatment where Ichiki et al. reported the usefulness of exogenous Trx in
Acknowledgments
This research was supported [in part] by grant-in-aid for scientific research from the Japan Society for the Promotion of Science (JSPS) (KAKENHI 21390177).
References (30)
- et al.
Amelioration of ischemia-reperfusion injury by human thioredoxin in rabbit lung
J. Thorac. Cardiovasc. Surg.
(1997) PEGylated antibodies and antibody fragments for improved therapy: a review
Adv Drug Deliv Rev
(2002)- et al.
Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins
J. Pharm. Sci.
(2005) - et al.
Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes
J. Control. Release
(2007) - et al.
Albumin fusion of thioredoxin—the production and evaluation of its biological activity for potential therapeutic applications
J. Control. Release
(2010) - et al.
Thioredoxin suppresses airway hyperresponsiveness and airway inflammation in asthma
Biochem. Biophys. Res. Commun.
(2005) - et al.
Thioredoxin 1 delivery as new therapeutics
Adv Drug Deliv Rev
(2009) - et al.
Priming donor lungs with thioredoxin-1 attenuates acute allograft injury in a rat model of lung transplantation
J Heart Lung Transplant
(2008) - et al.
EPR spin-trapping study of nitric oxide formation during bilateral carotid occlusion in the rat
Biochim Biophys Acta
(1993) - et al.
Anti-asthmatic effect of schizandrin on OVA-induced airway inflammation in a murine asthma model
Int. Immunopharmacol.
(2010)
Effects of thioredoxin on established airway remodeling in a chronic antigen exposure asthma model
Biochem. Biophys. Res. Commun.
Human thioredoxin-1 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production
Gastroenterology
Physiological functions of thioredoxin and thioredoxin reductase
Eur. J. Biochem.
Protective effect of adult T-cell leukemia-derived factor on retinal ischemia-reperfusion injury in the rat
Invest Ophthalmol Vis Sci
Recombinant human thioredoxin-1 becomes oxidized in circulation and suppresses bleomycin-induced neutrophil recruitment in the rat airway
Free. Radic. Res.
Cited by (40)
Applications of serum albumins in delivery systems: Differences in interfacial behaviour and interacting abilities with polysaccharides
2021, Advances in Colloid and Interface ScienceThioredoxin-albumin fusion protein prevents urban aerosol-induced lung injury via suppressing oxidative stress-related neutrophil extracellular trap formation
2021, Environmental PollutionCitation Excerpt :To improve the pharmacokinetics of Trx, we previously synthesized human serum albumin-fused thioredoxin (HSA-Trx) and found that the plasma half-life of HSA-Trx is over 10-fold higher than that of Trx (Ikuta et al., 2010). Over several subsequent studies, we found that a single intravenous administration of HSA-Trx is effective against a variety of diseases, including ovalbumin-induced lung injury, contrast nephropathy, rhabdomyolysis-associated acute kidney injury, and bleomycin-induced pulmonary fibrosis, via its long-lasting antioxidant action (Furukawa et al., 2011; Kodama et al., 2013; Nishida et al., 2015; Tanaka et al., 2013). Here, we examined the effect of HSA-Trx on urban aerosol-induced lung injury in mice.
Development of a long acting FGF21 analogue-albumin fusion protein and its anti-diabetic effects
2020, Journal of Controlled ReleaseDrug delivery systems based on nanoparticles and related nanostructures
2020, European Journal of Pharmaceutical SciencesThioredoxin-albumin fusion protein prevents copper enhanced zinc-induced neurotoxicity via its antioxidative activity
2018, International Journal of PharmaceuticsSingle chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo
2016, Journal of Controlled ReleaseCitation Excerpt :Then the early endosomes containing complex of FcRn and IgG/SA are sorted to recycle back to cell surface and IgG/SA released by exocytic processes close to the physiological pH [24–28]. Many studies had used the FcRn binding domains from SA or IgG and made fusion proteins containing these domains to achieve recycling and circulation extension [29–32], there had been also some efforts made to improve drugs' circulation properties by engineering or developing new peptide or protein binding domains of FcRn [33–43]. But most of these attempts including Fc-fusion proteins could not match the endogenous IgGs' long circulation half-life.
- 1
First three authors contributed equally to this work.