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The pathogenesis of non-alcoholic steatohepatitis (NASH) involves the simultaneous interaction of multiple factors such as lipid accumulation, oxidative stress, and inflammatory response. Here, the effect of human serum albumin (HSA) fused to thioredoxin (Trx) on NASH was investigated. Trx is known to have anti-oxidative, anti-inflammatory, and anti-apoptotic effects. However, Trx is a low molecular weight protein and is rapidly eliminated from the blood. To overcome the low availability of Trx, HSA-Trx fusion protein was produced and evaluated the therapeutic effect on high-fat diet (HFD)-induced NASH model mice. HSA-Trx administered before the formation of NASH pathology showed it to have a preventive effect. Specifically, HSA-Trx was found to prevent the pathological progression to NASH by suppressing lipid accumulation, liver injury markers, and liver fibrosis. When HSA-Trx was administered during the early stage of NASH there was a marked reduction in lipid accumulation, inflammation, and fibrosis in the liver, indicating that HSA-Trx ameliorates NASH pathology. The findings indicate that HSA-Trx influences multiple pathological factors, such as oxidative stress, inflammation, and apoptosis, to elicit a therapeutic benefit. HSA-Trx also inhibited palmitic acid-induced lipotoxicity in HepG2 cells. Taken together, these results indicate that HSA-Trx has potential as a therapeutic agent for NASH pathology.

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Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-ß, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.

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Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.

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INTRODUCTION: Prophylactic treatment of hemophilia B with recombinant factor IX (rFIX) molecules with enhanced pharmacokinetics including rIX-FP (albutrepenonacog alfa; Idelvion©) and rFIXFc (eftrenonacog alfa; Alprolix©) have commonly been used in the clinic. In the absence of head-to-head comparative trials, the aim of this study was to estimate the efficacy of rIX-FP versus rFIXFc using matching-adjusted indirect comparisons (MAICs). METHODS: MAIC analyses leveraged individual patient data from the PROLONG-9FP trial and published summary-level data from the B-LONG trial for subjects who received weekly prophylaxis regimens. Individual patient data were used to assign weights and balance subjects from PROLONG-9FP with subjects from B-LONG on baseline disease severity, age, prior FIX regimen, and body mass index (BMI). Six efficacy outcomes were analyzed including annualized bleeding rate (ABR), annualized spontaneous bleeding rate (AsBR), annualized joint bleeding rate (AjBR), and the proportion of subjects without bleeding events (for total, spontaneous, and joint bleeding events). RESULTS: After adjustment for baseline disease severity, age, prior FIX regimen, and BMI, rIX-FP was associated with a statistically significant decrease in AsBR (rate ratio [RR] 0.42; 95% confidence interval [CI] 0.22, 0.82; P = 0.0107), and the proportion of patients without bleeding events (odds ratio [OR] 3.24; 95% CI 1.41, 7.45; P = 0.0057), spontaneous bleeding events (OR 3.47; 95% CI 1.56, 7.73; P = 0.0023), and joint bleeding events (OR 2.41; 95% CI 1.10, 5.26; P = 0.0274) compared with rFIXFc. Prophylactic treatment with rIX-FP was also associated with a numerically lower ABR (RR 0.75; 95% CI 0.32, 1.75; P = 0.5095) and AjBR (RR 0.82; 95% CI 0.37, 1.82; P = 0.6178). CONCLUSION: The MAICs demonstrated that weekly prophylaxis treatment of severe hemophilia B with rIX-FP resulted in favorable efficacy outcomes as compared to rFIXFc. These findings suggest rIX-FP may offer improved clinical benefits over rFIXFc.

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Human serum albumin (HSA) is a multi-domain macromolecule with diverse ligand binding capability because of its ability to allow allosteric modulation despite being a monomeric protein. Physiologically, HSA act as the primary carrier for various exogenous and endogenous compounds and fatty acids, and alter the pharmacokinetic properties of several drugs. It has antioxidant properties and is utilized therapeutically to improve the drug delivery of pharmacological agents for the treatment of several disorders. The flexibility of albumin in holding various types of drugs coupled with a variety of modifications makes this protein a versatile drug carrier with incalculable potential in therapeutics. This review provides a brief outline of the different structural properties of HSA, and its various binding sites, moreover, an overview of the genetic, biomedical, and allosteric modulation of drugs and drug delivery aspects of HSA is also included, which may be helpful in guiding advanced clinical applications and further research on the therapeutic potential of this extraordinary protein.

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The successful expression and secretion of recombinant proteins in cell factories significantly depend on the correct folding of nascent peptides, primarily achieved through disulfide bond formation. Thus, optimizing cellular protein folding is crucial, especially for proteins with complex spatial structures. In this study, protein disulfide isomerases (PDIs) from various species were introduced into Saccharomyces cerevisiae to facilitate proper disulfide bond formation and enhance recombinant protein secretion. The impacts of these PDIs on recombinant protein production and yeast growth metabolism were evaluated by substituting the endogenous PDI1. Heterologous PDIs cannot fully compensate the endogenous PDI. Furthermore, protein folding mediators, PDI and ER oxidoreductase 1 (Ero1), from different species were used to increase the production of complex human serum albumin (HSA) fusion proteins. The validated folding mediators were then introduced into unfolded protein response (UPR)-optimized strains, resulting in a 7.8-fold increase in amylase-HSA and an 18.2-fold increase in albiglutide compared with the control strain. These findings provide valuable insights for optimizing protein folding and expressing HSA-based drugs.

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Despite the fact that liver fibrosis is an intractable disease with a poor prognosis, effective therapeutic agents are not available. In this study, we focused on bone morphogenetic factor 7 (BMP7) that inhibits transforming growth factor (TGF)-ß signaling, which is involved in liver fibrosis. We prepared an albumin-fused BMP7 (HSA-BMP7) that is retained in the blood and evaluated its inhibitory effect on liver fibrosis. Bile duct ligated mice were used as an acute liver fibrosis model, and carbon tetrachloride-induced liver fibrosis mice were used as a chronic model. All mice were administered HSA-BMP7 once per week. In the mice with bile duct ligation, the administration of HSA-BMP7 significantly suppressed the infiltration of inflammatory cells, the area of fibrosis around the bile duct, and decreased in the level of hydroxyproline as compared with saline administration. The mRNA expression of TGF-ß and its downstream fibrosis-associated genes (α-SMA and Col1a2) were also suppressed by the administration of HSA-BMP7. In the carbon tetrachloride-induced liver fibrosis mice, the HSA-BMP7 administration significantly decreased the hepatic fibrosis area and the level of hydroxyproline. Based on these results, it appears that HSA-BMP7 has the potential for serving as a therapeutic agent for the treatment of liver fibrosis.

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Albumin fusion or conjugation is a well-established technique for tumor delivery and is mainly mediated by albumin-induced caveolae-dependent endocytosis. We report that caveolae-dependent endocytic signaling activated by human serum albumin (HSA) is not sufficiently strong to induce cellular uptake, mainly due to its electrostatic repulsion from the negatively charged cell surface sulfated glycosaminoglycans (GAGs), and fusion of the cell-surface-retained protein with HSA is an effective strategy to activate the HSA-induced endocytic signal, thereby improving its intracellular uptake. In this study, human lactoferrin (hLF), a protein that accumulates on the cell surface along with GAGs, was selected for delivery into human lung adenocarcinoma PC-14 cells. When added exogenously, hLF-fused HSA (hLF-HSA) was successfully endocytosed, whereas the simultaneous addition of HSA and hLF did not result in endocytosis, indicating less efficient activation of endocytic signaling by HSA alone and the importance of its fusion. Importantly, the treatment of cells with chlorate, a known inhibitor of GAG sulfation, dramatically suppressed the endocytosis of hLF-HSA owing to the loss of the hLF-GAG interaction. Therefore, the cell-surface localization of HSA imposed by fusion with the cell-surface-retained protein enhances its binding to the relevant receptor, which improves intracellular delivery as an albumin-fusion platform.

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Non-alcoholic fatty liver disease (NAFLD) currently affects about 25% of the world's population, and the numbers continue to rise as the number of obese patients increases. However, there are currently no approved treatments for NAFLD. This study reports on the evaluation of the therapeutic effect of a recombinant human serum albumin-fibroblast growth factor 21 analogue fusion protein (HSA-FGF21) on the pathology of NAFLD that was induced by using two high-fat diets (HFD), HFD-60 and STHD-01. The HFD-60-induced NAFLD model mice with obesity, insulin resistance, dyslipidemia and hepatic lipid accumulation were treated with HSA-FGF21 three times per week for 4 weeks starting at 12 weeks after the HFD-60 feeding. The administration of HSA-FGF21 suppressed the increased body weight, improved hyperglycemia, hyperinsulinemia, and showed a decreased accumulation of plasma lipid and hepatic lipid levels. The elevation of C16:0, C18:0 and C18:1 fatty acids in the liver that were observed in the HFD-60 group was recovered by the HSA-FGF21 administration. The increased expression levels of the hepatic fatty acid uptake receptor (CD36) and fatty acid synthase (SREBP-1c, FAS, SCD-1, Elovl6) were also suppressed. In adipose tissue, HSA-FGF21 caused an improved adipocyte hypertrophy, a decrease in the levels of inflammatory cytokines and induced the expression of adiponectin and thermogenic factors. The administration of HSA-FGF21 to the STHD-01-induced NAFLD model mice resulted in suppressed plasma ALT and AST levels, oxidative stress, inflammatory cell infiltration and fibrosis. Together, HSA-FGF21 has some potential for use as a therapeutic agent for the treatment of NAFLD.

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The fusion of human serum albumin (HSA) with human lactoferrin (hLF) (designated as hLF-HSA) has improved the pharmacokinetic properties and anti-proliferative activities of hLF against cancer cells. In this study, we evaluated the anti-migratory activities of hLF and hLF-HSA against the human lung adenocarcinoma PC-14 cell line using wound healing and Boyden chamber assays. Despite the unexpected hLF-induced migration, hLF-HSA clearly demonstrated the complete inhibition of PC-14 cell migration. To examine the mechanism underlying the enhanced PC-14 cell migration by hLF alone but suppressed migration by hLF-HSA, we focused on the matrix metalloproteinase (MMP) family of endopeptidases because MMPs are often reported to play important roles in facilitating the migration and metastasis of cancer cells. Furthermore, hLF is a transactivator of MMP1 transcription. As expected, treatment of cells with hLF and hLF-HSA led to the upregulation and downregulation of MMP1, respectively. In contrast, MMP9 expression levels, which are often associated with cancer migration, were unchanged in the presence of either protein. An MMP inhibitor attenuated hLF-induced migration of PC-14 cells. Therefore, specific enhancement and suppression of MMP1 expression by hLF and hLF-HSA have been implicated as causes of a marked increase and decrease in PC-14 cell migration, respectively. In conclusion, the fusion of HSA with hLF (hLF-HSA) promoted its anti-migratory effects against cancer cells. Therefore, hLF-HSA is a promising anti-cancer drug candidate based on its improved anti-migratory activity towards cancer cells.

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Albutrepenonacog-alfa (Idelvion®, CSL Behring) is a recombinant fusion protein in which the recombinant FIX (rFIX) links a recombinant human albumin, extending the half-life of rFIX even beyond 100 hours. In 2016, this drug was approved worldwide for the treatment of pediatric and adult persons with hemophilia B (PWH-B). Its efficacy and safety were described in the PROLONG-9FP program and subsequently confirmed in the real-world practice, even if to date there are not many manuscripts that extensively and completely deal with the use of albutrepenonacog-alfa in daily practice, also evaluating its impact on the quality of life of patients treated with this drug; this review therefore aims to analyze all the publications currently available regarding the real-world use of this extended half-life concentrate, also noting which topics need further study and research.

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The bone morphogenetic protein-7 (BMP7) is capable of inhibiting TGF-ß/Smad3 signaling, which subsequently results in protecting the kidney from renal fibrosis, but its lower blood retention and osteogenic activity are bottlenecks for its clinical application. We report herein on the fusion of carbohydrate-deficient human BMP7 and human serum albumin (HSA-BMP7) using albumin fusion technology and site-directed mutagenesis. When using mouse myoblast cells, no osteogenesis was observed in the glycosylated BMP7 derived from Chinese hamster ovary cells in the case of unglycosylated BMP7 derived from Escherichia coli and HSA-BMP7. On the contrary, the specific activity for the Smad1/5/8 phosphorylation of HSA-BMP7 was about 25~50-times lower than that for the glycosylated BMP7, but the phosphorylation activity of the HSA-BMP7 was retained. A pharmacokinetic profile showed that the plasma half-life of HSA-BMP7 was similar to that for HSA and was nearly 10 times longer than that of BMP7. In unilateral ureteral obstruction mice, weekly dosing of HSA-BMP7 significantly attenuated renal fibrosis, but the individual components, i.e., HSA or BMP7, did not. HSA-BMP7 also attenuated a cisplatin-induced acute kidney dysfunction model. The findings reported herein indicate that HSA-BMP7 has the potential for use in clinical applications for the treatment of renal injuries.

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Although the number of patients with heart failure is increasing, a sufficient treatment agent has not been established. Oxidative stress and inflammation play important roles in the development of myocardial remodeling. When thioredoxin (Trx), an endogenous anti-oxidative and inflammatory modulator with a molecular weight of 12 kDa, is exogenously administered, it disappears rapidly from the blood circulation. In this study, we prepared a long-acting Trx, by fusing human Trx (HSA-Trx) with human serum albumin (HSA) and evaluated its efficacy in treating drug-induced heart failure. Drug-induced cardiomyopathy was created by intraperitoneally administering doxorubicin (Dox) to mice three times per week. A decrease in heart weight, increased myocardial fibrosis and markers for myocardial damage that were observed in the Dox group were suppressed by HSA-Trx administration. HSA-Trx also suppressed the expression of atrogin-1 and myostatin, myocardial atrophy factors in addition to suppressing oxidative stress and inflammation. In the Dox group, a decreased expression of endogenous Trx in cardiac tissue and an increased expression of macrophage migration inhibitory factor were observed, but these changes were restored to normal levels by HSA-Trx administration. These findings suggest that HSA-Trx improves the pathological condition associated with Dox-induced cardiomyopathy by its anti-oxidative/anti-inflammatory and myocardial atrophy inhibitory action.

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Hemophilia B is a bleeding disorder caused by a deficiency of coagulation factor IX (FIX). Treatment with FIX replacement products can increase FIX activity levels to minimize or prevent bleeding events. However, frequent dosing with standard-acting FIX products can create a high treatment burden. Long-acting products have been developed to maintain bleed protection with extended dosing intervals. Recombinant factor IX-albumin fusion protein (rIX-FP) is a long-acting product indicated for the treatment and prophylaxis of bleeding events and perioperative management in adult and pediatric patients. This review outlines data from all previously treated patients in the Prophylaxis and On-Demand Treatment using Longer Half-Life rIX-FP (PROLONG-9FP) clinical trial program and summarizes real-world data evaluating the use of rIX-FP in routine clinical practice. In the PROLONG-9FP program, rIX-FP demonstrated effective hemostasis in all patients at dose regimens of up to 21 days in patients aged ≥ 18 years and up to 14 days in patients aged < 12 years. rIX-FP has a favorable pharmacokinetic profile and an excellent safety and tolerability profile. Extended dosing intervals with rIX-FP led to high levels of adherence and reduced consumption compared with other FIX therapies. Data from real-world practice are encouraging and reflect the results of the clinical trials.

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An effective strategy is highly desirable for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Thioredoxin-1 (Trx), a redox-active protein that has anti-oxidative and anti-inflammatory properties, would be a candidate for this but its short half-life limits its clinical application. In this study, we examined the renoprotective effect of long-acting Trx that is comprised of human albumin and Trx (HSA-Trx) against AKI to CKD transition. AKI to CKD mice were created by renal ischemia-reperfusion (IR). From day 1 to day 14 after renal IR, the recovery of renal function was accelerated by HSA-Trx administration. On day 14, HSA-Trx reduced renal fibrosis compared with PBS treatment. At the early phase of fibrogenesis (day 7), HSA-Trx treatment suppressed renal oxidative stress, pro-inflammatory cytokine production and macrophage infiltration, thus ameliorating tubular injury and fibrosis. In addition, HSA-Trx treatment inhibited G2/M cell cycle arrest and apoptosis in renal tubular cells. While renal Trx protein levels were decreased after renal IR, the levels were recovered by HSA-Trx treatment. Together, HSA-Trx has potential for use in the treatment of AKI to CKD transition via its effects of modulating oxidative stress and inflammation.

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The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (KD ) 0·5 nM] over wild-type FIX-HSA (KD 164·4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2·7 days, P < 0·0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.

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Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

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Synthetic ligand affinity adsorbents offer an efficient means for purification of biopharmaceuticals. Single-isomer textile dye C.I. Reactive Blue and newer ligands developed by rational design and screening of chemical combinatorial libraries based on a triazine scaffold are routinely used for the capture and purification of these proteins from engineered recombinant expression systems. Here, we describe methods for the purification of recombinant human serum albumin and related fusion proteins using synthetic ligand affinity adsorbents.

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Fibroblast growth factor 21 (FGF21) is a hormone-like protein that improves blood glucose and lipid metabolism. However, its short half-life and instability are bottlenecks to its clinical applications. In this study, to extend its pharmacological action, we created a stabilized mutant FGF21 (mFGF21:ΔHPIP, P171G, A180E, L118C-A134C, S167A) and then genetically fused it with human albumin (HSA-mFGF21) via a polypeptide linker. Physicochemical analyses suggested that HSA-mFGF21 was formed from both intact HSA and mFGF21. Pharmacokinetic findings indicated the half-life of HSA-mFGF21 was 20 times longer than that of FGF21. In addition, HSA-mFGF21 was persistently distributed in adipose tissue as a target tissue. The in vivo hypoglycemic activity of HSA-mFGF21 using streptozotocin (STZ)-induced type I diabetes model mice, in which insulin secretion was suppressed, showed that a single intravenous administration of HSA-mFGF21 rapidly alleviated hyperglycemia. At that time, HSA-mFGF21 increased GLUT1 mRNA expression in adipose tissue without having any effect on insulin secretion. A twice weekly administration of HSA-mFGF21 continuously suppressed blood glucose levels and ameliorated the abnormalities of adipose tissue induced by STZ treatment. Interestingly, HSA-mFGF21 showed no hypoglycemic effects in healthy mice. Together, HSA-mFGF21 could be a novel biotherapeutic for the treatment of metabolic disorders including diabetes mellitus.

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