Carboxymethyl Starch Excipients for Drug Chronodelivery.

Carboxymethyl starch (CMS) is a pH-responsive excipient exhibiting also interesting properties for applications in delayed drug delivery systems. This work was aimed to investigate the release properties of monolithic and dry-coated tablets based on ionic sodium CMS and on protonated CMS, formulated with three model tracers: acetaminophen, acetylsalicylic acid (ASA), and sodium diclofenac. The sodium or protonated CMS were obtained from the same CMS synthesis by controlling the final pH of reaction media. The two forms of CMS were confirmed by the Fourier transform infrared spectroscopy. The in vitro dissolution profiles for monolithic and double core tablets were different and allowed a better understanding of characteristics of the two excipient forms. It was found that the protonated CMS exhibited a better stability in simulated gastric fluid in comparison to its sodium salt in monolithic dosage forms, whereas both excipients afforded a complete gastric protection of drugs when formulated as dry-coated dosages. Determination of water uptake and erosion rate of monolithic matrices based on the two CMS forms showed different mechanisms involved in the delivery of the three model active molecules in simulated intestinal media. When pancreatic enzymes were added in dissolution media, the drug release was accelerated showing that CMS is still a substrate for alpha-amylase. Both sodium and protonated starch excipients, formulated as dry-coated dosages, afforded a good gastro-protection and allowed a drug chronodelivery at various intervals up to 4-5 h. They could be considered as an alternative for delayed delivery and a solvent-free coating procedure.

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.

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.

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-1ß) 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 plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis.

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 degrees C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 degrees C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin.

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 high amylose starch for F4 fimbriae gastro-resistant oral formulation.

The carboxymethyl high amylose starch (CM-HAS) was proposed as excipient able to protect F4 fimbriae oral vaccine against gastric acidity and pepsin, allowing its subsequent liberation in the intestinal fluid. Thus, F4 fimbriae formulated with CM-HAS as tablets displayed a markedly higher stability after 2h of incubation in simulated gastric fluid (containing pepsin) than the free, non-protected F4 fimbriae, which, in these conditions, were almost completely digested after 120 min. In the presence of pancreatin (with alpha-amylase, lipase and proteolytic activities) in simulated intestinal conditions, the F4 fimbriae were liberated from CM-HAS tablets over a period of up to 5 h. The presence of pancreatin in intestinal medium did not affect the structural stability of the F4 fimbriae major subunits. Thus, F4 fimbriae formulated with CM-HAS would retain their receptor binding activity essential for the induction of an intestinal mucosal immune response.

Carboxymethyl high amylose starch (CM-HAS) as excipient for Escherichia coli oral formulations.

Carboxymethyl high amylose starch (CM-HAS) is proposed as a novel excipient for oral tablet formulation of bioactive agents ensuring their protection in the stomach and delivery in the intestine. Three variants of CM-HAS, with different degrees of substitution, were synthesized by starch treatment with various amounts of monochloroacetic acid. The products were dried in powder form and tablets were obtained by direct compression of mixed powders of polymeric excipient and lyophilized Escherichia coli (E. coli) bacteria. Dosage forms of CM-HAS are unswollen and compact in acidic medium, ensuring protection of active agents against acidity. Release of bacteria from CM-HAS tablets is based on the fast swelling of the tablets during the passage from gastric acidity to alkaline intestinal medium, enzymatic hydrolysis triggering their rapid, almost total dissolution. The bacteria thus formulated displayed higher survival rates in acidic gastric conditions and for longer periods than the free bacteria or than the bacteria formulated with the non-derivatized starch. The CM-HAS selected matrix also assured a good viability of bacteria after 6 months under refrigeration.