Improving physicochemical characteristics and cytotoxicity of baicalin esters by liposome encapsulation.

The instability of ester bonds, low water solubility, and increased cytotoxicity of flavonoid glycoside esters significantly limit their application in the food industry. Therefore, the present study attempted to resolve these issues through liposome encapsulation. The results showed that baicalin butyl ester (BEC4) and octyl ester (BEC8) have higher encapsulation and loading efficiencies and lower leakage rate from liposomes than baicalin. FTIR results revealed the location of BEC4 and BEC8 in the hydrophobic layer of liposomes, which was different from baicalin. Additionally, liposome encapsulation improved the water solubility and stability of BEC4 and BEC8 in the digestive system and PBS but significantly reduced their cytotoxicity. Furthermore, the release rate of BEC4 and BEC8 from liposomes was lower than that of baicalin during gastrointestinal digestion. These results indicate that liposome encapsulation alleviated the negative effects of fatty chain introduction into flavonoid glycosides.

Synthesis of pyrazoline fatty chain derivatives and its effects on melanoma cells.

Skin cancer is the most common type of cancer in Brazil, representing 30% of all cases. Among these, melanoma represents only 3% of malignant neoplasms; however, it is the most serious and has a high capacity for metastasis. For this reason, it is extremely important to identify more efficient compounds and treatments that stop or decrease the proliferation of melanoma, even in its more advanced stages. This work reports the synthesis and biological evaluation of two homologous series of pyrazoline fatty chain derivatives as potent antitumoral agents in the melanoma B16F10 cell line. Cells were treated with pyrazoline fatty chain compounds (3, 30, 300, and 3000 µM) for 0, 24, 48, and 72 h. Decreased cell viability was observed when using most compounds at different concentrations and times. The structure-activity relationship (SAR) between antitumoral activity and the number of carbons and lipophilicity, as well as the oxygen-sulfur bioisosteric exchange, was evaluated. Among the tested derivatives, the lipophilic compounds 5-hydroxy-5-(trifluoromethyl)-3-undecyl-4,5-dihydro-1H-pyrazole-1-carboxamide (2d) and 5-hydroxy-5-(trifluoromethyl)-3-undecyl-4,5-dihydro-1H-pyrazole-1-thiocarboxamide (3d) showed the best results in the B16F10 cell line, as they produced the best cell viability decrease effects. The presence of fatty unbranched undecyl chain in the molecular structure appears to be important for its antimelanoma properties.

Novel Site-Specific Fatty Chain-Modified GLP-1 Receptor Agonist with Potent Antidiabetic Effects.

Glucagon-like peptide-1 receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). Here, we designed a high-throughput GLP-1R extracellular domain (ECD)-based system that enabled the screening of high-potency receptor-biased GLP-1R agonists demonstrating new pharmacological virtues. Firstly, six 12-mer peptides (termed PEP01⁻06), screened from a large phage displayed peptide library were fused to the N-terminus of Exendin-4 (29⁻39) to generate PEP07⁻12. By the use of four lysine-altered PEP07 (PEP13⁻16) as the starting point, a series of fatty chain conjugates (PEP17⁻20) were synthesized and evaluated by in vitro GLP-1R-based cell assays. In addition, the acute and long-term in vivo effects on diet-induced obesity (DIO) mice were further evaluated. All four conjugates showed good receptor activation efficacy, and PEP20 was selected to undergo further assessment. Preclinical experiments in DIO mice demonstrated that PEP20 had significant insulinotropic activities and glucose-lowering abilities. Moreover, a prolonged antidiabetic effect of PEP20 was also observed by the hypoglycemic test in DIO mice. Furthermore, long-term treatment with PEP20 achieved beneficial effects on the food intake, weight gain, hemoglobin A1C (HbA1C) lowering activity, and glucose tolerance compared with the control and was similar to the Liraglutide. In conclusion, PEP20, a GLP-1R ECD-biased agonist, may provide a novel therapeutic approach to T2DM.

Preparation and Characterization of Site-Specific Fatty Chain-Modified Recombinant Human Granulocyte Colony Stimulating Factor.

The clinical use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is limited by its short serum half-life. In this study, a long-acting strategy for site-specific modification of rhG-CSF with 1-pentadecyl-1H-pyrrole-2,5-dione (C15 fatty chain-maleimide, C15-MAL) was studied in mixed DMSO-aqueous solutions. The factors influencing the conjugation reaction were investigated and optimized, and a high yield of the desired product (C15-rhG-CSF) was achieved. Subsequently, C15-rhG-CSF product was efficiently purified using preparative liquid chromatography, and further characterized. Circular dichroism spectroscopy analysis showed that the secondary structure of C15-rhG-CSF had no significant difference from unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of in vitro bioactivity of unmodified rhG-CSF. The pharmacokinetic study showed that the serum half-life of C15-rhG-CSF in mice was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study demonstrated the potential of C15-rhG-CSF being developed into a novel drug candidate as well as an efficient process for the development of long-acting protein and peptide drugs.

Polysaccharide from flammuliana velutipes improves colitis via regulation of colonic microbial dysbiosis and inflammatory responses.

The aim of this study is to investigate whether Flammuliana Velutipes Polysaccharide (FVP) could aid in the prevention of colitis. Effect of FVP on colitis was evaluated using dextran sulfate sodium (DSS)-induced colitis in rats. Influence of FVP on the expression of inflammation related biomarkers and signal pathway element of TLR4\NF-κB were assessed. The composition and taxonomy of colonic microbiota were analyzed by 16S rDNA high throughput sequencing, and the concentrations of caecal short fatty chain acids were assessed by chromatography-mass spectrometry. Our results showed that FVP treatment could regulate the colonic microbial dysbiosis and promote the levels of caecal short fatty chain acids, leading to down-regulation of TLR4\NF-κB signal pathway, which finally ameliorate the colitis. Thus, the present study is the first attempt to elucidate the effect of FVP on colitis and support the potential application of FVP as a functional food ingredient or preventive drugs for colitis.

Rapid selection of a novel GLP-1/GIP dual receptor agonist with prolonged glycemic control and weight loss in rodent animals.

BACKGROUND: Glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) co-agonists have emerged as treatment options for reversing diabetes and obesity. Here, we screened the high potency receptor-biased GLP-1R agonists via a newly designed high-throughput GLP-1R extracellular domain (ECD)-based system and demonstrated its in vitro and in vivo therapeutic characters. METHODS: Twelve 9-mer peptides (named XEL1-XEL12) which were screened from a large phage-displayed peptide library were fused to the N-terminus of GIP (3-30) to generate another twelve fusion peptides, termed XEL13-24. Using the six lysine-altered XEL17 as leading sequences, eighteen fatty chain modified fusion peptides were further assessed via in vitro GLP-1R/GIPR-based cell assay. Moreover, the acute and long-acting in vivo effects of selected candidate on diabetic db/db mice and diet-induced obesity (DIO) rats were both carefully evaluated. RESULTS: XEL17 exhibited balanced activation potency on GLP-1R/GIPR in stable cell lines, and further assessment was performed to evaluate the XEL32, a fatty chain modified XEL17 derivative. Preclinical pharmacodynamic results in diabetic db/db mice demonstrated that XEL32 held outstanding insulinotropic and glucose-lowering activities. In addition, protracted antidiabetic effects of XEL32 were also proved by the hypoglycemic test and multiple oral glucose tolerance test. Furthermore, chronic treatment of XEL32 in DIO rats exhibited outstanding beneficial effects on body weight control, fat loss, food intake control, hemoglobin A1C (HbA1C) reduction as well as the glucose tolerance. CONCLUSIONS: XEL32, as a novel GLP-1/GIP dual receptor agonist, may supply efficient glycemic control and weight loss.

Site-specific fatty chain-modified exenatide analogs with balanced glucoregulatory activity and prolonged in vivo activity.

The therapeutic utility of exenatide (Ex-4) is limited due to short plasma half-life of 2.4h and thus numerous approaches have been used to obtain a longer action time. However, such strategies often attend to one thing and lose another. The study aimed to identify a candidate with balanced glucoregulatory activity and prolonged in vivo activity. A series of fatty chain conjugates of Ex-4 were designed and synthesized. First, thirteen cysteine modified peptides (1-13) were prepared. Peptides 1, 10, and 13 showed improved glucagon-like peptide-1 (GLP-1) receptor activate potency and were thus selected for second step modifications to yield conjugates I-1-I-9. All conjugates retained significant GLP-1 receptor activate potency and more importantly exerted enhanced albumin-binding properties and in vitro plasma stability. The protracted antidiabetic effects of the most stable I-3 were further confirmed by both multiple intraperitoneal glucose tolerance test and hypoglycemic efficacies test in vivo. Furthermore, once daily injection of I-3 to streptozotocin (STZ) induced diabetic mice achieved long-term beneficial effects on hemoglobin A1C (HbA1C) lowering and glucose tolerance. Once daily injection of I-3 to diet induced obesity (DIO) mice also achieved favorable effects on food intake, body weight, and blood chemistry. Our results suggested that I-3 was a promising agent deserving further investigation to treat obesity patients with diabetes.