Carmen Calinescu

Carboxymethyl high amylose starch: Chitosan self-stabilized matrix for probiotic colon delivery.

A new hydrophilic tablet dosage system based on an ionic self-stabilization of a carboxylated (carboxymethyl high amylose starch, CM-HAS) and an amino (Chitosan) excipient was proposed for probiotic colon delivery. CM-HAS (protonated and compacted in acidic medium) ensures gastro-protection and Chitosan (low soluble in intestinal media) prevents early release of Lactobacillus rhamnosus bacteria. Thus, in CM-HAS:Chitosan monolithic tablets, increasing percentage and molecular weight (MW) of Chitosan generated a decrease of bacteria release rate, bacteria being the most effectively retarded by the highest MW of Chitosan (2.2 x 10(6)g/mol). The monolithic formulations containing high percentages of CM-HAS (80%) delivered bacteria after 2h of incubation in gastrointestinal conditions for all the Chitosan MWs used. A combined mechanism of bacteria release is proposed for CM-HAS:Chitosan monolithic tablets, involving the swelling of the tablets (due to the Chitosan), followed by the erosion and dissolution of CM-HAS. In addition, a gel-forming barrier of Chitosan in acidic conditions also contributed to the delay of the bacteria delivery. The CM-HAS dry-coated monolithic tablets changed the effect of Chitosan molecular weight on bacteria liberation and improved the percentage of delivered bacteria in simulated intestinal conditions.

Carboxymethyl starch: Chitosan monolithic matrices containing diamine oxidase and catalase for intestinal delivery

The capacity of carboxymethyl starch (CMS):Chitosan monolithic tablets to protect diamine oxidase and/or catalase therapeutic enzymes against simulated gastric fluid (SGF) and to control their delivery in simulated intestinal fluid (SIF) was investigated. Enzyme formulations loaded with grass pea seedlings diamine oxidase (PSDAO) vegetal extract, catalase, or PSDAO associated to catalase, were obtained by direct compression. The CMS:Chitosan (1:1) matrix afforded a good gastric protection to PSDAO and to catalase, when each enzyme was formulated separately. Variable amounts of DAO were delivered in the SIF containing pancreatin, with maximal release reached at about 8h, a time convenient for tablets to attain the colon. Up to 50% of the initial enzymatic activity of catalase formulated with CMS:Chitosan was found after 8 h in SIF. For the CMS:Chitosan tablets of bi-enzymatic formulations containing PSDAO:Catalase, the releases of DAO and of catalase were synchronized. The hydrogen peroxide (product of DAO activity) was decomposed by the catalase liberated in the same SIF environment. The proposed formulations could allow novel therapeutic approaches for the treatment of inflammatory bowel diseases, intestinal cancers or pseudo-allergic reactions.

Oral Immunization with F4 Fimbriae and CpG Formulated with Carboxymethyl Starch Enhances F4-Specific Mucosal Immune Response and Modulates Th1 and Th2 Cytokines in Weaned Pigs

PURPOSE F4 fimbriae are a potential candidate for an oral subunit vaccine for prevention of post-weaning diarrhea in swine due to infection with F4-positive enterotoxigenic Escherichia coli. However, large quantities of F4 fimbriae are required to induce a specific antibody response. The aim of the present study was to evaluate the effect of supplementation of F4 fimbriae with Cytosine-phosphate-Guanosine-oligodeoxynucleotide (CpG-A D19) or with complete cholera toxin (CT) as adjuvants on the F4-specific antibody response and cytokine production in weaned pigs following oral administration of F4 fimbrial antigen formulated with Carboxymethyl Starch (CMS). METHODS Oral dosage forms of F4 fimbriae alone or supplemented with CpG-A D19 or with CT were formulated with CMS as monolithic tablets, obtained by direct compression, and administered to weaned pigs. Blood and faecal samples were collected to determine the systemic and mucosal immune status of animals at various times until necropsy. During necropsy, contents of the jejunum and ileum were collected for determination of mucosal F4 specific antibodies. Segments of jejunum and ileum were also used to measure mRNA cytokine production. RESULTS The presence of CpG in the formulation of the fimbriae significantly increased F4-specific immunoglobulin (Ig) IgM and IgG levels in intestinal secretions, and enhanced Th1 (Interferon-gamma / IFN-γ, Tumour Necrosis Factor-alpha / TNF-α, Interleukin-12p40 / IL-12p40, IL-1β) and Th2 (IL-4, IL-6) cytokine production in intestinal tissues. Supplementation with CT did not result in induction of F4-specific antibodies in secretions, although a significant Th1 response (IFN-α, IFN-γ, IL-18) was detected in tissues. Neither F4-specific systemic antibodies, nor intestinally secreted IgA were detected throughout the immunization trial for all groups. CONCLUSIONS CpG-A D19 appeared to be a promising adjuvant for an oral F4 subunit vaccine formulated with CMS excipient as monolithic tablets. This matrix afforded gastro-protection and delivered the F4 fimbriae at their intestinal sites.

Zymographic assay of oxidases using peroxidase or hemin entrapped in polyacrylamide gel.

This chapter describes a zymographic assay of oxidases which is based on a coupled peroxidase or hemin reaction. The enzymatic activity of oxidases (i.e., diamine oxidase/DAO, glucose oxidase, galactose oxidase) can be directly monitored on polyacrylamide gels containing horseradish peroxidase or hemin, in the presence of their specific substrates and ortho-phenylenediamine (OPDA), an oxidizable chromogen. In the presence of hydrogen peroxide, OPDA is oxidized to azo-aniline, which led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of oxidases.

Effects of amine oxidases in allergic and histamine-mediated conditions.

This review provides an update on histamine, on diamine oxidase (DAO) and on their implications in allergy and various conditions or affections, such as food histaminosis, ischemia and inflammatory bowel diseases (IBD). The review also presents, in brief, patent coverage on therapies for allergy and IBD with the focus on histamine-related treatments.