Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29.

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

Effect of intraperitoneal PERIDAN™ concentrate adhesion reduction device on clinical findings, infection, and tissue healing in an adult horse jejunojejunostomy model.

OBJECTIVE: To evaluate the effect of PERIDAN™ Concentrate on clinical findings, infection, and tissue healing in adult horses undergoing celiotomy and jejunojejunostomy. STUDY DESIGN: Block randomized blinded experimental in vivo study. ANIMALS: Adult horses (n = 12). METHODS: Horses had jejunojejunostomy at 2 sites and were administered 5 L of diluted PERIDAN™ Concentrate (6 horses) or Lactated Ringer's Injection (LRS) control intraperitoneally (6 horses) before body wall closure. Postoperative monitoring comprised physical examinations, serial hematology, coagulation and chemistry panels, and ultrasonographic examination. Horses were euthanatized 10 days postoperatively. Anastomoses and linea alba incisions were tested for mechanical strength; and tissue healing, inflammation, and infection were assessed by histological evaluation. Data were analyzed using a mixed model ANOVA. Level of significance was P < .05. RESULTS: No physical examination differences were observed between groups. Statistically significant differences were observed in leukocyte and neutrophil counts, prothrombin time, antithrombin III activity, intestinal bursting pressures, and histologic healing grade in the mid region of the linea alba. These differences were minimal, and of no observable clinical significance. Other blood variable and histologic differences between groups were not significant. CONCLUSIONS: PERIDAN™ Concentrate was safely administered intraperitoneally to healthy horses undergoing celiotomy and anastomosis.

Fucoidan film safely inhibits surgical adhesions in a rat model.

BACKGROUND: The purpose of this study was to evaluate the in vivo efficacy of 13 compounds and to further characterize the load limiting and potential toxicity of the most efficacious compound. The cascade of biochemical and molecular events that results in the formation of postsurgical adhesions provides numerous theoretical opportunities for prophylactic intervention. METHODS: Candidate agents were loaded into sodium hyaluronate (HA) films and administered to male Sprague-Dawley rats using a cecal-sidewall model of surgical adhesions. An adhesion score was obtained for each rat based on the strength and extent of the adhesions. The most efficacious agent, fucoidan, was further evaluated in a load-limiting study with a concentration range of 0.0033 to 33% w/w per film. The potential toxicity of fucoidan was evaluated in a separate study by comparison of hematology findings, blood chemistry, urinalysis, and incision thickness from rats administered control films or 33% w/w fucoidan films 1 to 4 d prior to sacrifice. RESULTS: Fucoidan loaded films reduced adhesion scores by approximately 90% compared with control films (P<0.05). A total of 50% to 100% of animals were adhesion free at fucoidan film loadings of 0.33% to 33% w/w compared with all control film animals having adhesions. No adverse effects were observed from 33% w/w fucoidan films equivalent to approximately 30 mg fucoidan/kg body weight. CONCLUSIONS: Local administration of fucoidan film during rat cecal-sidewall surgery safely reduced adhesion scores by approximately 90% and resulted in 50% to 100% of animals being adhesion free.

Controlled sulfation of mixed-linkage glucan by Response Surface Methodology for the development of biologically applicable polysaccharides.

Endogenous and exogenous sulfated polysaccharides exhibit potent biological activities, including inhibiting blood coagulation and protein interactions. Controlled chemical sulfation of alternative polysaccharides holds promise to overcome limited availability and heterogeneity of naturally sulfated polysaccharides. Here, we established reaction parameters for the controlled sulfation of the abundant cereal polysaccharide, mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG), using Box-Behnken Design of Experiments (BBD) and Response Surface Methodology (RSM). The optimization of the degree-of-substitution (DS) was externally validated through the production of sulfated MLGs (S-MLGs) with observed DS and Mw values deviating less than 20% and 30% from the targeted values, respectively. Simultaneous optimization of DS and Mw resulted in the same range of deviation from the targeted value. S-MLGs with DS > 1 demonstrated a modest anticoagulation effect versus heparin, and a greater P-selectin affinity than fucoidan. As such, this work provides a route to medically important polymers from an economical agricultural polysaccharide.

Efficacy of an intratumoral controlled release formulation of clusterin antisense oligonucleotide complexed with chitosan containing paclitaxel or docetaxel in prostate cancer xenograft models.

PURPOSE: To develop and evaluate an injectable, controlled release delivery system for a phosphorothioate antisense oligonucleotide (ASO) based on complexed ASO:chitosan dispersed in a biodegradable polymeric paste for intratumoral treatment of solid tumors. METHODS: Clusterin ASO was complexed with chitosan particles and incorporated into a paste based on a 60:40 blend of methoxy-poly(ethylene glycol) (MePEG) and triblock copolymer of poly(D: ,L: -lactic acid-co-caprolactone)-PEG-(D: ,L: -lactic acid-co-caprolactone). In vitro release profiles of clusterin ASO into phosphate-buffered saline at 37 degrees C were obtained under sink conditions and assayed by anionic exchange high-performance liquid chromatography. In vivo efficacy studies were carried out in human prostate PC-3 and LNCaP tumors grown subcutaneously in mice. Paste formulations of clusterin ASO with or without paclitaxel or docetaxel were injected intratumorally and tumor volumes and serum prostate specific antigen (PSA) levels were measured. RESULTS: Controlled release of clusterin ASO was obtained over several weeks. The rate and extent of ASO release was proportional to the ratio of ASO to chitosan in the paste. Treatment of mice bearing PC-3 tumors with clusterin ASO plus paclitaxel or docetaxel paste had reduced mean tumor volume by greater than 50% at 4 weeks. Treatment of mice bearing LNCaP tumors with clusterin ASO plus paclitaxel reduced mean tumor volume and serum PSA level by more than 50% and 70%, respectively. CONCLUSIONS: Complexation of clusterin ASO with chitosan and incorporation into polymeric paste with paclitaxel or docetaxel produced in vitro controlled release of the ASO and in vivo efficacy over 4 weeks following a single intratumoral injection in solid human prostate tumors in mice.