Therapeutic effect of long-acting FGF21 with controlled site-specific modification on nonalcoholic steatohepatitis.

FGF21 plays an active role in the treatment of type 2 diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). However, the short half-life and poor stability of wild-type FGF21 limit its clinical application. Previous studies found that PEGylation can significantly increase the stability of FGF21. However, the uneven distribution of PEGylation sites in FGF21 makes it difficult to purify PEG-FGF21, thereby affecting its yield, purity, and activity. To obtain long-acting FGF21 with controlled site-specific modification, we mutated lysine residues in FGF21, resulting in PEGylation only at the N-terminus of FGF21 (mFGF21). In addition, we modified mFGF21 molecules with different PEG molecules and selected the PEG-mFGF21 moiety with the highest activity. The yield of PEG-mFGF21 in this study reached 1 g/L (purity >99 %), and the purification process was simple and efficient with strong quality controllability. The half-life of PEG-mFGF21 in rats reached 40.5-67.4 h. Pharmacodynamic evaluation in mice with high-fat, high-cholesterol- and methionine and choline deficiency-induced NASH illustrated that PEG-mFGF21 exhibited long-term efficacy in improving liver steatosis and reducing liver cell damage, inflammation, and fibrosis. Taken together, PEG-mFGF21 could represent a potential therapeutic drug for the treatment of NASH.

Preparation of Ribes nigrum L. polysaccharides-stabilized selenium nanoparticles for enhancement of the anti-glycation and α-glucosidase inhibitory activities.

Seven kinds of selenium nanoparticles (RP-SeNPs) were prepared by using the polysaccharides extracted from Ribes nigrum L. (RP) as the stabilizer and dispersant. Among them, RP-SeNPs-1 (94.2 nm), RP-SeNPs-2 (101.2 nm) and RP-SeNPs-3 (107.6 nm) with relatively smaller mean particle size exhibited stronger α-glucosidase inhibitory activity than other RP-SeNPs (115.3-164.2 nm) and SeNPs (288.9 nm). Ultraviolet-visible spectrophotometry, Fourier transform-infrared, X-ray diffraction, energy dispersive X-ray and X-ray photoelectron spectroscopy analyses confirmed that SeNPs were ligated with RP to form nanocomposites and displayed amorphous form. Electron microscopy images revealed that RP-SeNPs-1 - RP-SeNPs-3 were regular shape spherical nanocomposites with much better dispersion than SeNPs. Compared with SeNPs, RP-SeNPs displayed relatively high thermal, storage, pH and salt ion stability. Moreover, RP-SeNPs-1-RP-SeNPs-3 showed significantly better anti-glycation and α-glucosidase inhibitory activity than SeNPs, especially RP-SeNPs-1 with the smallest particle size. Inhibitory kinetics analysis indicated that SeNPs and RP-SeNPs inhibited α-glucosidase with competitive type and reversible mechanism. In addition, the conformation of the α-glucosidase was changed after binding with the SeNPs and RP-SeNPs-1. Fluorescence quenching and isothermal titration calorimetry assays revealed that these two nanoparticles could interact with α-glucosidase to form non-fluorescent complexes through hydrogen bonding, and the formation was spontaneously driven by enthalpy.

Sulforaphane ameliorates non-alcoholic fatty liver disease in mice by promoting FGF21/FGFR1 signaling pathway.

Most studies regarding the beneficial effect of sulforaphane (SFN) on non-alcoholic fatty liver disease (NAFLD) have focused on nuclear factor E2-related factor 2 (Nrf2). But the molecular mechanisms underlying the beneficial effect of SFN in the treatment of NAFLD remain controversial. Fibroblast growth factor (FGF) 21 is a member of the FGF family expressed mainly in liver but also in adipose tissue, muscle and pancreas, which functions as an endocrine factor and has been considered as a promising therapeutic candidate for the treatment of NAFLD. In the present study we investigated whether FGF21 was involved in the therapeutic effect of SFN against NAFLD. C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to generate NAFLD and continued on the HFD for additional 6 weeks with or without SFN treatment. We showed that administration of SFN (0.56 g/kg) significantly ameliorated hepatic steatosis and inflammation in NAFLD mice, along with the improved glucose tolerance and insulin sensitivity, through suppressing the expression of proteins responsible for hepatic lipogenesis, while enhancing proteins for hepatic lipolysis and fatty acids oxidation. SFN administration significantly increased hepatic expression of FGFR1 and fibroblast growth factor 21 (FGF21) in NAFLD mice, along with decreased phosphorylation of p38 MAPK (the downstream of FGF21). HepG2 cells were treated in vitro with FFAs (palmitic acid and oleic acid) followed by different concentrations of SFN. We showed that the effects of SFN on FGF21 and FGFR1 protein expression were replicated in FFAs-treated HepG2 cells. Moreover, the increased FGFR1 protein occurred earlier than increased FGF21 protein. Interestingly, the rapid effect of SFN on FGFR1 protein was not regulated by the FGFR1 gene transcription. Knockdown of FGFR1 and p38 genes weakened SFN-reduced lipid deposition in FFAs-treated HepG2 cells. SFN administration in combination with rmFGF21 (1.5 mg/kg, i.p., every other day) for 3 weeks further suppressed hepatic steatosis in NAFLD mice. In conclusion, SFN ameliorates lipid metabolism disorders in NAFLD mice by upregulating FGF21/FGFR1 pathway. Our results verify that SFN may become a promising intervention to treat or relieve NAFLD.

The recombinant Newcastle disease virus Anhinga strain expressing human TRAIL exhibit antitumor effects on a glioma nude mice model.

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. However, the potential oncolytic ability of the recombinant newcastle disease virus (NDV) Anhinga strain carried with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has not been fully explored at present. In the present study, the recombinant NDV/Anh-TRAIL that secretes soluble TRAIL was constructed and the experiment results suggested NDV/Anh-TRAIL as a promising candidate for glioma therapy. Growth kinetic and TRAIL secreted quantity of recombinant NDV/Anh-TRAIL virus were measured. Cytotoxic and cell apoptosis were analyzed for its anti-glioma therapy in vitro. Nude mice were used for the in vivo evaluation. Both tumor volume and mice behavior after injection were observed. The recombinant virus replicated with the same kinetics as the parental virus and the highest expression of TRAIL (77.8 ng/L) was found at 48 hours. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole and flow cytometry data revealed that the recombinant NDV/Anh-TRAIL (56.1 ± 8.2%) virus could induce a more severe apoptosis rate, when compared with the NDV wild type (37.2 ± 7.0%) and mock (7.0 ± 1.8%) groups (P < .01), in U251 cells. Furthermore, in the present animal study, the average tumor volume was smaller in the NDV/Anh-TRAIL group (97.21 mm3 ), when compared with the NDV wild type (205.03 mm3 , P < .05) and PBS (310.30 mm3 , P < .01) groups.

Pulmonary toxicity and RNA sequencing analyses of mouse in response to exposure to cellulose nanofibrils.

OBJECTIVE: The growing applications of nanocelluloses in the fields of advanced nanocomposites, electronics, and medical devices necessitate investigation of their potential adverse effects on human health. The lungs are the primary and the most important route for the entry of nanocelluloses into the human body in occupational settings. However, data on the pulmonary toxicity of cellulose nanofibrils (CNFs) and its molecular mechanism are limited. This study investigated the pulmonary toxicity of CNFs and its genomic expression using the RNA sequencing approach. MATERIALS AND METHODS: Female C57BL/6 mice were administered CNFs at 50 µg/mouse by oropharyngeal aspiration. Samples were collected at 3 and 14 days after exposure to CNFs (DAEC). RESULTS: At three DAEC, the microscopic sections of lungs revealed a significant inflammatory response. In terms of gene expression alterations, 94 genes were up-regulated, and 107 genes were down-regulated. Most of these differentially expressed genes were involved in the inflammatory and immune responses, including chemokines, NK cells, killer cell lectin-like receptors, CD antigens, T cell-specific GTPases, immunity-related GTPase family M members, and interferon-induced proteins encoding genes. However, only 9 and 26 genes at 14 DAEC were significantly up- and down-regulated, respectively. CONCLUSIONS: The pathological analysis of lung sections and the analysis of sequencing data suggested that the homeostasis of mice lungs was restored at 14 DAEC. The findings of this study provide insights into the pulmonary toxicity, and underlying toxicological mechanisms, caused by exposure to CNFs, and are useful for the assessment of the potential toxicity of nanocelluloses.

Resolution of epoetin-induced pure red cell aplasia, successful re-challenge with roxadustat.

The application of erythropoietin (EPO) can bring about a rare but serious complication called anti-EPO antibody-mediated pure red cell aplasia (PRCA). Once the disease is diagnosed, EPO administration should be stopped immediately. However, after the removal of the anti-EPO antibody, treating anaemia in these patients with chronic renal disease with EPO therapy is difficult, as restarting EPO therapy risks the recurrence of anti-EPO antibody-mediated PRCA. A 26-year-old man with anaemia related to renal failure, who was administered recombinant human EPO subcutaneously, developed anti-EPO antibody-mediated PRCA. After removal of antibodies by treatment with corticosteroids and cyclosporine, therapy for anaemia of chronic renal disease with roxadustat achieved good results. Roxadustat is a new type of drug for the treatment of anaemia, and it can stimulate endogenous EPO within or near the physiologic range and increase haemoglobin levels.

An efficient large-scale refolding technique for recovering biologically active recombinant human FGF-21 from inclusion bodies.

Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.

Experimental Evaluation of the Transport Mechanisms of PoIFN-α in Caco-2 Cells.

For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α), the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC)-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER) value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

Recombinant Newcastle disease virus expressing human TRAIL as a potential candidate for hepatoma therapy.

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently proved for clinical trials. We have previously reported, for the first time, NDV Anhinga strain has an efficient cancer therapeutic efficacy in hepatoma. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. In this study, we have showed administration of a recombinant NDV Anhinga strain expressing soluble TRAIL (NDV/Anh-TRAIL) results in an efficient suppression of hepatocellular carcinoma without significant toxicity. The results show that recombinant NDV Anhinga strain expressing soluble TRAIL is a promising candidate for hepatoma therapy.

Pharmacological efficacy of FGF21 analogue, liraglutide and insulin glargine in treatment of type 2 diabetes.

Fibroblast growth factor 21 (FGF21) is a promising regulator of glucose and lipid metabolism with multiple beneficial effects including hypoglycemic and lipid-lowering. Previous studies have reported that FGF21 is expected to become a new drug for treatment of diabetes. Liraglutide and insulin glargine are the two representative anti-diabetic biological drugs. In the current study, we aim to compare the long-term pharmacological efficacy of mFGF21 (an FGF21 analogue), liraglutide and insulin glargine in type 2 diabetic db/db mice. Db/db mice were initially treated with three kinds of proteins (25nmol/kg/day) by subcutaneous injection once a day for 4weeks, then subsequently be treated with once every two days for next 4weeks. After 8weeks of treatments, the blood glucose levels, body weights, glycosylated hemoglobin levels, fasting insulin levels, serum lipid profiles, hepatic biochemical parameters, oral glucose tolerance tests and hepatic mRNA expression levels of several proteins (GK, G6P, GLUT-1 and GLUT-4) associated with glucose metabolism of the experimental mice were detected. Results demonstrated that three proteins could significantly decrease the fed blood glucose levels of db/db mice. After treatment for 1week, the fed blood glucose levels of db/db mice in liraglutide group were significantly lower than those in mFGF21 and insulin glargine groups. However, after 2weeks of administration, the long-lasting hypoglycemic effect of mFGF21 was superior to liraglutide and insulin glargine up to the end of the experiments. Compared with liraglutide and insulin glargine, mFGF21 significantly reduced the glycosylated hemoglobin levels and improved the ability on glycemic control, insulin resistance, serum lipid and liver function states in db/db mice after 8weeks treatments. In addition, mFGF21 regulated glucose metabolism through increasing the mRNA expression levels of GK and GLUT-1, and decreasing the mRNA expression level of G6P. But liraglutide and insulin glargine could only up-regulate the mRNA expression of GLUT-4. In summary, as a hypoglycemic drug for long-term treatment, mFGF21 has the potential to be an ideal drug candidate for the therapy of type 2 diabetes.

Study on antitumor, antioxidant and immunoregulatory activities of the purified polyphenols from pinecone of Pinus koraiensis on tumor-bearing S180 mice in vivo.

Pinecone polyphenols are bioactive dietary constituents that enhance health and help prevent and treat cancer through improving antioxidant and immunoregulatory activities. This study was designed to investigate the antitumor, antioxidant and immunoregulatory activities of the 40% ethanol eluent of polyphenols from pinecone of pinus koraiensis (PPP-40) in Sarcoma 180 (S180)-bearing mice models in vivo. The results of antitumor activity indicated that PPP-40 significantly inhibited S180 tumor growth and the dose of 150mg/kg exhibited the highest antitumor activity. Moreover, TdT-mediated dUTP nick end labeling (TUNEL) assay results further confirmed the apoptosis of S180 tumor cells. In addition, PPP-40 could obviously promote the expressions of Bax protein and inhibit the Bcl-2 protein, accordingly improve the expressions of activated Caspase-3 as well, which resulted in the activation of mitochondrial apoptotic pathway of tumor cells in S180 mice eventually. The results of antioxidant activity showed that the S180 mice treated with PPP-40 had the higher superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, the more glutathione (GSH) content, and the lower malondialdehyde (MDA) level in plasma comparing with non-treated control group. Moreover, the administration with PPP-40 (150mg/kg) significantly accelerated the proliferation of splenocytes (p<0.01) and increased the monocyte phagocytosis activity in vivo simultaneously. These results revealed that PPP-40 exerts an effective antitumor activity by activating the mitochondrial apoptotic pathway and improving the antioxidant and immunoregulatory activities.

EGF and curcumin co-encapsulated nanoparticle/hydrogel system as potent skin regeneration agent.

Wound healing is a complex multifactorial process that relies on coordinated signaling molecules to succeed. Epidermal growth factor (EGF) is a mitogenic polypeptide that stimulates wound repair; however, precise control over its application is necessary to reduce the side effects and achieve desired therapeutic benefits. Moreover, the extensive oxidative stress during the wound healing process generally inhibits repair of the injured tissues. Topical applications of antioxidants like curcumin (Cur) could protect tissues from oxidative damage and significantly improve tissue remodeling. To achieve much accelerated wound healing effects, we designed a novel dual drug co-loaded in situ gel-forming nanoparticle/hydrogel system (EGF-Cur-NP/H) which acted not only as a supportive matrix for the regenerative tissue, but also as a sustained drug depot for EGF and Cur. In the established excisional full-thickness wound model, EGF-Cur-NP/H treatment significantly enhanced wound closure through increasing granulation tissue formation, collagen deposition, and angiogenesis, relative to normal saline, nanoparticle/hydrogel (NP/H), Cur-NP/H, and EGF-NP/H treated groups. In conclusion, this study provides a biocompatible in situ gel-forming system for efficient topical application of EGF and Cur in the landscape of tissue repair.

FGF21 ameliorates nonalcoholic fatty liver disease by inducing autophagy.

The aim of this study is to evaluate the role of fibroblast growth factor 21 (FGF21) in nonalcoholic fatty liver disease (NAFLD) and seek to determine if its therapeutic effect is through induction of autophagy. In this research, Monosodium L-glutamate (MSG)-induced obese mice or normal lean mice were treated with vehicle, Fenofibrate, and recombinant murine FGF21, respectively. After 5 weeks of treatment, metabolic parameters including body weight, blood glucose and lipid levels, hepatic and fat gene expression levels were monitored and analyzed. Also, fat-loaded HepG2 cells were treated with vehicle or recombinant murine FGF21. The expression levels of proteins associated with autophagy were detected by western blot, real-time PCR, and transmission electron microscopy (TEM). Autophagic flux was monitored by laser confocal microscopy and western blot. Results showed that FGF21 significantly reduced body weight (P < 0.01) and serum triglyceride, improved insulin sensitivity, and reversed hepatic steatosis in the MSG model mice. In addition, FGF21 significantly increased the expression of several proteins related to autophagy both in MSG mice and fat-loaded HepG2 cells, such as microtubule associated protein 1 light chain 3, Bcl-2-interacting myosin-like coiled-coil protein-1 (Beclin-1), and autophagy-related gene 5. Furthermore, the evidence of TEM revealed an increased number of autophagosomes and lysosomes in the model cells treated with FGF21. In vitro experimental results also showed that FGF21 remarkably increased autophagic flux. Taken together, FGF21 corrects multiple metabolic parameters on NAFLD in vitro and in vivo by inducing autophagy.

Recombinant Newcastle disease virus expressing P53 demonstrates promising antitumor efficiency in hepatoma model.

BACKGROUND: Numerous studies have demonstrated that the NDV-mediated gene therapy is a promising new approach for treatment of cancers. P53 plays a vital role in tumor suppression and surveillance. Therefore, we hypothesize that a recombinant NDV expressing P53 would be an ideal agent for the hepatoma therapy. RESULTS: In the essay, the human P53 gene was incorporated into the genome of a lentogenic strain (named rNDV-P53), which did not affect viral replication kinetics and magnitude in HepG2 cells. Compared to the vehicle virus, rNDV-P53 increased cell growth suppressor ratio and early apoptosis by 2 folds, and decreased the mitochondrial membrane potential in HepG2 cells. In vivo studies, treatment with rNDV-P53 reduced tumor volume of tumor-bearing mice by more than 4 folds, tumor weight by more than 5 folds comparing with rNDV. The 120-day survival rate of rNDV-P53-treated mice was 75 %, survival rate of rNDV-treated mice was 12.5 %. TUNEL analysis showed a significant increase in the apoptosis rate in the tumor tissues of rNDV-P53-treated mice than that of rNDV-treated mice. Moreover, serum chemistries revealed an insignificant change of blood urea nitrogen (BUN), creatinine levels, alanine aminotransferase (ALT) and aspartate transaminase (AST) in rNDV-P53-treated group compared to normal mice, suggesting treatment with the recombinant virus was not toxic. CONCLUSION: rNDV-P53 is a potent candidate for carcinoma therapy especially for hepatocarcinoma.

Recombinant Newcastle disease virus (NDV/Anh-IL-2) expressing human IL-2 as a potential candidate for suppresses growth of hepatoma therapy.

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical study and are currently approved for clinical trials. NDV Anhinga strain which is a mesogenic strain should be classified as lytic strain and has a therapeutic efficacy in hepatocellular cancer. In this study, we evaluated the capacity of NDV Anhinga strain to elicit immune reaction in vivo and the possibility for using as a vaccine vector for expressing tumor therapeutic factors. Interleukin-2 (IL-2) could boost the immune response against the tumor cells. Therefore, we use NDV Anhinga strain as backbone to construct a recombinant virus (NDV/Anh-IL-2) expressing IL-2. The virus growth curve showed that the production of recombinant NDV/Anh-IL-2 was slightly delayed compared to the wild type. The NDV/Anh-IL-2 strain could express soluble IL-2 and effectively inhibit the growth of hepatocellular carcinoma in vivo. 60 days post-treatment, mice which were completely cured by previous treatment were well protected when rechallenged with the same tumor cell. From the H&E-stained sections, intense infiltration of lymphocyte was observed in the NDV Anhinga strain treated group, especially in NDV/Anh-IL-2 group. The NDV Anhinga strain could not only kill the tumor directly, but could also elicit immune reaction and a potent immunological memory when killing tumor in vivo. In conclusion, the Anhinga strain could be an effective vector for tumor therapy; the recombinant NDV/Anh-IL-2 strain expressing soluble IL-2 is a promising candidate for hepatoma therapy.

Pilot-scale production and characterization of PEGylated human FGF-21 analog.

FGF-21 has become a potential drug candidate for the treatment of type 2 diabetes. Previous studies have demonstrated that PEGylation of FGF-21 could significantly increase its in vivo half-life and provide its long-lasting blood glucose-lowering effect. To accelerate the development of PEGylated FGF-21 for clinical application as a long-acting antidiabetes drug, we prepared ahmFGF-21 (FGF-21 mutant) and PEGylated ahmFGF-21 in Escherichia coli Rosetta (DE3) by high cell density fermentation at a 50-L scale and pilot-scale purification. The physical and chemical properties of the purified proteins were analyzed in this study, including purity, molecular weight, isoelectric point, bacterial endotoxin, PEGylated site and second structure. As well as the in vitro glucose uptake activity and in vivo anti-diabetic effect were evaluated. Under the optimal fermentation and purification conditions, the average bacterial yield and expression level of target protein of three batches attained 52.2±4.6g/L and 223.92±5.41mg/L, respectively. The purity of pilot product was above 98% by SDS-PAGE (non-reducing or reducing) and HPLC (SEC or RPC) analysis and the final yield of PEGylated ahmFGF-21 was 87.91±1.49mg/L, which indicated that the pilot-scale production process was relatively stable. N-terminal sequencing and circular dichroism (CD) spectroscopy results showed that modification site of PEGylated ahmFGF-21 was alanine at N-terminal and the second structure of ahmFGF-21 had no obvious changes after PEGylation. Compared with ahmFGF-21, the long-acting hypoglycemic effect of PEGylated ahmFGF-21 prepared in the pilot-scale production was significantly improved in type 2 diabetic db/db mice. Our results demonstrated that the pilot-scale production process of PEGylated ahmFGF-21 was successfully established, which was very important for the clinical application.

Long-lasting hypoglycemic effect of modified FGF-21 analog with polyethylene glycol in type 1 diabetic mice and its systematic toxicity.

Fibroblast growth factor-21 (FGF-21) is a novel metabolic regulator and has the potential to become a powerful therapy to treat diabetes mellitus. However, we found that the clinical application of wild type FGF-21 was influenced by its low intrinsic bio-stability and poor hypoglycemic potency. In this study, The N-terminus of FGF-21 analog (mFGF-21) was PEGylated in a site-specific manner by 20kD methoxy poly-ethylene glycol-propionaldehyde (mPEG-ALD). PEGylated mFGF-21 was isolated by Capto Q anion exchange chromatography. The properties of PEGylated mFGF-21 including the in vitro bio-stability and biological activity were evaluated. As well as the anti-diabetic effect of PEGylated mFGF-21 were studied in streptozotocin (STZ)-induced type 1 diabetic mice. Results demonstrated that PEGylated mFGF-21 had a similar capacity of stimulating glucose uptake in HepG2 cells with mFGF-21 and PEGylation of mFGF-21 significantly enhanced the anti-protease ability and the long acting anti-diabetic effect in type 1 diabetic mice. Furthermore, the preliminary safety of PEGylated mFGF-21 following subcutaneously injection was assessed using healthy mice by measuring the body weight, histopathology and clinical biochemical parameters, and the results showed no subacute toxicity to major organs or tissues and no significant changes in physiological and biochemical parameters in healthy mice. Taken together, under the premise of remaining the in vitro biological activity of mFGF-21, PEGylation significantly improves the long lasting hypoglycemic effect of mFGF-21 in type 1 diabetic mice. Our valuation shows that PEGylated mFGF-21 is a potential drug for the effective treatment of type 1 diabetes.

Long-lasting anti-diabetic efficacy of PEGylated FGF-21 and liraglutide in treatment of type 2 diabetic mice.

Fibroblast growth factor-21 (FGF-21) is a new member of the FGF family and potential drug candidate for the treatment of type 2 diabetes mellitus. However, FGF-21 protein has short half-life in vivo, which severely affects its clinical application. In the present study, PEGylated FGF-21 was prepared by modifying the N-terminus of hFGF-21 with 20 kDa mPEG-ALD. The long-acting hypoglycemic effect of PEGylated FGF-21 and liraglutide was compared on type 2 diabetic db/db mice. The pharmacological efficacy of the compounds was evaluated by blood glucose levels, body weight, glycosylated hemoglobin levels, insulin levels, oral glucose tolerance test, lipid levels, and liver function parameters. We noticed that both PEGylated FGF-21 and liraglutide could significantly decrease plasma glucose in db/db mice. However, comparing to liraglutide treatments, PEGylated FGF-21 therapy resulted in more significant effect in lowering blood glucose levels and glycosylated hemoglobin levels, alleviating insulin resistance, improving lipid profile, liver function, and glucose control of the experimental mice. Our results suggest that PEGylated FGF-21 appears more beneficial anti-diabetic effect in type 2 diabetic mice than liraglutide, which holds significant promise as an ideal candidate for the treatment of type 2 diabetic patients.

Recombinant Newcastle Disease virus Expressing IL15 Demonstrates Promising Antitumor Efficiency in Melanoma Model.

Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma.