Exploring a novel long-acting glucagon-like peptide-1 receptor agonist built on the albumin-binding domain and XTEN scaffolds.

In recent years, glucagon-like peptide-1 (GLP-1) has demonstrated considerable potential in the treatment of type 2 diabetes (T2D) and obesity. However, the half-life of naturally occurring GLP-1 is quite short in vivo. Two common strategies employed for half-life extension are the use of the Albumin-binding domain (ABD) and XTEN polypeptide, which operate through different mechanisms. In this study, we designed an innovative GLP-1 receptor agonist with an extended duration of action. This new construct incorporated an albumin binding domain (ABD) and an XTEN sequence (either XTEN144 or XTEN288) as carriers. We referred to these fusion proteins as GLP-ABD-XTEN144 and GLP-ABD-XTEN288. In an E. coli system, the said constructs were efficaciously produced in substantial quantity. It was observed from in vitro studies that the fusion protein GLP-ABD-XTEN144 demonstrated a five times stronger affinity towards human serum albumin (HSA), boasting a binding affinity (Kd) of 5.50 nM. This was in contrast to GLP-ABD-XTEN288, whose Kd value was registered at 27.78 nM. Moreover, GLP-ABD-XTEN144 presented a half-life of 12.9 h in mice, thus exceeding the corresponding value for GLP-ABD-XTEN288, 7.32 h in mice. Both these fusion proteins significantly mitigated non-fasting blood sugar levels and overall food consumption for 48 h subsequent to a one-time injection in mice. Notably, GLP-ABD-XTEN144 exhibited more pronounced hypoglycemic activity and food inhibitory effects than GLP-ABD-XTEN288. The designed GLP-ABD-XTEN144 fusion protein shows promising prospects for clinical application in T2D treatment. Our findings also suggest that ABD and XTEN polypeptides synergistically contribute to half-life extension, further enhancing the pharmacokinetic characteristics of a payload.

Design of a Dual Agonist of Exendin-4 and FGF21 as a Potential Treatment for Type 2 Diabetes Mellitus and Obesity.

Background: Fibroblast growth factor 21 (FGF21) is a metabolic, endocrine hormone regulating insulin sensitivity, energy expenditure, and lipid metabolism. It has significant potential as a therapeutic drug for treating type 2 diabetes and obesity. However, the clinical efficacy of FGF21 analogs is limited due to their instability and short half-life. Glucagon-like peptide 1 (GLP-1) receptor agonists have been recognized as effective medications for type 2 diabetes mellitus and obesity over the past two decades. Methods: This study designed a new long-acting dual-agonist, exendin-4/FGF21, utilizing albumin-binding-designed ankyrin repeat proteins (DARPins) as carriers. The purified fusion proteins were subcutaneously injected into mice for pharmacokinetic and biological activity studies. Results: Ex-DARP-FGF21 had a high binding affinity for human serum albumin (HSA) in vitro and a prolonged half-life of 27.6 hours in vivo. Bioactivity results reveal that Ex-DARP-FGF21 significantly reduced blood glucose levels in healthy mice. Moreover, compared to Ex-DARP alone, the Ex-DARP-FGF21 dual agonist displayed enhanced blood glucose lowering bioactivity and superior body weight management in the diet-induced obesity (DIO) mouse model. Conclusions: These results indicate that the long-acting dual agonist of exendin-4 and FGF21 holds considerable potential as a treatment for type 2 diabetes mellitus (T2DM) and obesity in the future.

Albumin-binding DARPins as scaffold improve the hypoglycemic and anti-obesity effects of exendin-4 in vivo.

Type 2 diabetes mellitus (T2DM) and obesity have been considered epidemics and threats to public health worldwide. Exendin-4 (Ex), a GLP-1R agonist, has potential for treating T2DM and obesity. However, Ex has a half-life of only 2.4 h in humans and needs to be administered twice daily, which hampers its clinical application. In this study, we synthesized four new GLP-1R agonists by genetically fusing Ex to the N-terminus of HSA-binding ankyrin repeat proteins (DARPins) via linkers of different lengths, denoted as Ex-DARPin-GSx fusion proteins (x = 0, 1, 2, and 3). The Ex-DARPin fusion proteins were substantially stable, resulting in incomplete denaturation even at 80 °C. The in vitro bioactivity results demonstrated that Ex-DARPin fusion proteins could bind to HSA and activate GLP-1R. The Ex-DARPin fusion proteins had a comparable half-life (29-32 h), which is much longer than that of native Ex (0.5 h in rats). Subcutaneous injection of 25 nmol/kg Ex-DARPin fusion protein normalized blood glucose (BG) levels for at least 72 h in mice. The Ex-DARPin fusion proteins, injected at 25 nmol/kg every three days, significantly lowered BG, inhibited food consumption, and reduced body weight (BW) for 30 days in STZ-induced diabetic mice. Histological analysis of pancreatic tissues using H&E staining revealed that Ex-DARPin fusion proteins significantly improved the survival of pancreatic islets in diabetic mice. The differences in in vivo bioactivity of fusion proteins with different linker lengths were not significant. According to the findings in this study, long-acting Ex-DARPin fusion proteins designed by us hold promise for further development as antidiabetic and antiobesity therapeutic agents. Our findings also indicate that DARPins are a universal platform for generating long-acting therapeutic proteins via genetic fusion, thus broadening the application scope of DARPins.

Monitoring the Methyl Eugenol Response and Non-Responsiveness Mechanisms in Oriental Fruit Fly Bactrocera dorsalis in China.

Bactrocera dorsalis is a notorious polyphagous pest in China, and its management strategies largely depend on methyl eugenol (ME), which has been widely used as an attractant to monitor and eradicate B. dorsalis populations for seven decades. However, the non-responsiveness levels in field B. dorsalis populations to ME is unknown. In this study, we monitored the response to ME in field populations from the four most heavily infested provinces in China, and the results showed that the populations had lower sensitivity to ME relative to GZS susceptible strain. The percent responsiveness of the lowest sensitivity population was 5.88-, 3.47-, and 1.47-fold lower relative to the susceptible strain at doses of 1, 10, and 100 µL of ME, respectively. Gene expression analysis and inhibitor assays further revealed that odorant binding protein (BdorOBP2, BdorOBP83b) and the P450 enzyme system may be associated with the lower response to ME. To our knowledge, this work is the first to report that the P450 enzyme system confers a lower responsiveness to lure insects. These findings provided valuable insights for exploiting ME non-responsiveness to protect sterile males from ME-based control strategies and the use of lures combined with insecticides.

LC-MS/MS assay for the quantification of foretinib in rat plasma and its application to preclinical pharmacokinetic study.

A simple and sensitive ultra-high-performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of foretinib in rat plasma. The analyte and internal standard were extracted from the bio-samples with acetonitrile and then separated on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm) using 0.1% formic acid aqueous and acetonitrile as mobile phase, at a flow rate of 0.4 ml/min. The mass detection was performed in positive selected reaction monitoring mode with precursor-to-product transitions at m/z 317.1 > 128.1 for foretinib and m/z 502.2 > 323.1 for internal standard. The assay was demonstrated to be linear in the concentration range of 0.5-1000 ng/ml, with correlation coefficient >0.999. The mean extraction recovery of foretinib from rat plasma was within the range of 84.55-88.09%, while the matrix effect was in the range of 88.56-99.21%. The intra- and inter-day precisions were <12.95% and the accuracy ranged from -7.55 to 8.57%. Foretinib was stable in rat plasma under the tested storage conditions. The validated assay was successfully applied to the pharmacokinetic study of foretinib in the rats. The results revealed that foretinib showed moderate elimination half-life, low clearance and dose-independent pharmacokinetic profiles inrats.

New-Onset Diabetes After Renal Transplantation (NODAT): Is It a Risk Factor for Renal Cell Carcinoma or Renal Failure?

BACKGROUND Diabetes mellitus (DM) is a risk factor for renal failure and possibly for renal cell carcinoma (RCC). Post-transplantation DM occurs frequently after solid organ transplantation. We investigated whether new-onset diabetes after renal transplantation (NODAT) is a risk factor for RCC or renal failure. MATERIAL AND METHODS Data of 96,699 discharged patients with and without NODAT were extracted from the 2005-2014 Nationwide Inpatient Sample (NIS) database, after excluding patients with DM diagnosed at least 1 year prior to renal transplantation. Main outcomes were RCC diagnosis less than 1-year post-transplantation, RCC stage, and renal failure. Univariate and multivariate regression analyses were performed to identify demographic and clinical factors associated with post-transplantation RCC or renal failure. RESULTS Significant differences were found in age and race between patients with and without NODAT (both P<0.001). The renal failure rate was 0.8% (n=1) in NODAT patients and 0.3% (n=314) in those without NODAT. Older age (OR, 1.030; 95% CI: 1.023 to 1.036), male (OR, 1.872; 95% CI: 1.409 to 2.486), Black (OR, 2.199; 95% CI: 1.574 to 3.071) and hospitalization in urban teaching hospitals were associated with increased risk of RCC. CONCLUSIONS Analysis of over 90,000 NIS hospitalizations with diagnosis-coded kidney transplantation suggested that NODAT may not be an independent risk factor for RCC and renal failure.

Research on Vegetable Pest Warning System Based on Multidimensional Big Data.

Pest early warning technology is part of the prerequisite for the timely and effective control of pest outbreaks. Traditional pest warning system with artificial mathematical statistics, radar, and remote sensing has some deficiency in many aspects, such as higher cost, weakness of accuracy, low efficiency, and so on. In this study, Pest image data was collected and information about four major vegetable pests (Bemisia tabaci (Gennadius), Phyllotreta striolata (Fabricius), Plutella xylostella (Linnaeus), and Frankliniella occidentalis (Pergande) (Thysanoptera, Thripidae)) in southern China was extracted. A multi-sensor network system was constructed to collect small-scale environmental data on vegetable production sites. The key factors affecting the distribution of pests were discovered by multi-dimensional information, such as soil, environment, eco-climate, and meteorology of vegetable fields, and finally, the vegetable pest warning system that is based on multidimensional big data (VPWS-MBD) was implemented. Pest and environmental data from Guangzhou Dongsheng Bio-Park were collected from June 2017 to February 2018. The number of pests is classified as level I (0⁻56), level II (57⁻131), level III (132⁻299), and level IV (above 300) by K-Means algorithm. The Pearson correlation coefficient and the grey relational analysis algorithm were used to calculate the five key influence factors of rainfall, soil temperature, air temperature, leaf surface humidity, and soil moisture. Finally, Back Propagation (BP) Neural Network was used for classification prediction. The result shows: I-level warning accuracy was 96.14%, recall rate was 97.56%; II-level pest warning accuracy was 95.34%, the recall rate was 96.45%; III-level pest warning accuracy of 100%, the recall rate was 96.28%; IV-level pest warning accuracy of 100%, recall rate was 100%. It proves that the early warning system can effectively predict vegetable pests and achieve the early warning of vegetable pest’s requirements, with high availability.

Influence of two common polymorphisms in the EPHX1 gene on warfarin maintenance dosage: a meta-analysis.

We conducted a meta-analysis to investigate the influence of two common single nucleotide polymorphisms (SNPs) (rs2292566 G>A and rs4653436 A>G) in the EPHX1 gene on warfarin maintenance dosages. Relevant literatures were searched using the PubMed, Embase, Web of Science, Cochrane Library, CISCOM, CINAHL, Google Scholar, CBM, and CNKI databases without any language restrictions. STATA Version 12.0 software (Stata Corporation, College Station, TX, USA) was used for this meta-analysis. Standard mean difference and its corresponding 95% confidence interval (95% CI) were calculated. Seven studies met the inclusion criteria, including 2,063 warfarin-treated patients. Meta-analysis results illustrated that EPHX1 rs2292566 G>A polymorphism might be strongly correlated with a higher maintenance dose of warfarin. However, no interaction of EPHX1 rs4653436 A>G polymorphism with warfarin maintenance dosage was detected. A further subgroup analysis based on stratification by ethnicity indicated that EPHX1 rs2292566 G>A polymorphism was positively correlated with warfarin maintenance dosage among Caucasians, but not Asians. No associations were observed between EPHX1 rs4653436 A>G polymorphism warfarin maintenance dosage among both Caucasians and Asians. Our meta-analysis provides robust and unambiguous evidence that EPHX1 rs2292566 polymorphism may affect the maintenance dose of warfarin in Caucasians.