No abstract

The purpose of this investigation was to explore the structure-function relationship among naturally occurring Quillaja saponins and derivatives for their ability to stimulate insulin delivery from nosedrops and eyedrops and to test the hypothesis that stimulation of peptide drug delivery was correlated with surfactant strength. Native saponins, including QS-21, were purified from an aqueous extract of Quillaja saponaria bark by adsorption chromatography and HPLC. Native saponins were then deacylated by mild alkaline hydrolysis to form DS-1 and DS-2, derivatives that are smaller and more hydrophilic than their parent compounds. DS-1 was further treated either to reduce an aldehyde residue to form DS-1(R) or to remove the fucose-containing oligosaccharide to form QH-957. Rats receiving eyedrops or nosedrops formulated with insulin, but without any Quillaja saponins, showed no hypoglycemic response. Rats receiving eyedrops or nosedrops formulated with insulin plus saponins showed a dose-dependent hypoglycemic response, with the following rank order: QS-21 > DS-1 > DS-1(R) > DS-2 > QH-957. Surfactant strength was determined by measurement of the critical micellar concentration (cmc) and hemolysis of sheep erythrocytes. The cmc was lowest for the parent saponins QS-21 and QS-18, and increased for the deacylated saponin derivatives DS-1, DS-2, and QH-957; hemolysis of sheep erythrocytes was observed at low concentrations (approximately 0.006 mM) of the parent saponins, QS-21 and QS-18, at intermediate concentrations (0.06-0.08 mM) of DS-1 and DS-2, and at higher concentrations of DS-1(R) (0.45 mM) and QH-957 (1.5 mM). Hence, efficacy as an absorption-enhancing agent was greatest in those saponins with the lowest hemolytic titers and cmc values. However, this relationship was not a strict one, because DS-1, which differs from DS-2 only in the absence of one glucose residue, was significantly more potent than DS-2 in stimulating the absorption of insulin. DS-1 and DS-2 share a similar cmc and hemolytic titer, so this difference in efficacy must be due to some specificity beyond simple surfactant strength. Furthermore, DS-1 does not trigger an immune response when administered to animals, whereas QS-21 is a strong immune system activator. Therefore, DS-1 has emerged as an interesting candidate for inclusion in an eyedrop or nosedrop formulation.

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed inEscherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.

No abstract

DS-1, a modified Quillaja saponin, has recently been shown to promote the absorption of insulin and aminoglycoside antibiotics via the ocular and nasal route. The purpose of this study is to investigate the effect of DS-1 on intestinal permeability, the mechanism of its action, and reversibility of the effect. The permeation-enhancing activity of DS-1 was evaluated in cultured monolayers of the Caco-2 intestinal epithelial cells by examining its effect on the transepithelial electric resistance (TEER) and on transport of mannitol and a model D-decapeptide. Mucosal addition of DS-1 promptly reduced the TEER of the Caco-2 monolayers, and a propensity of recovery of the TEER was observed upon its removal. DS-1 added at 0.01-0.1% (w/v) increased the transports of both mannitol and D-decapeptide in a dose-dependent manner; a relatively "flat" concentration-dependence was seen at 0.1-0.2%. Visualization studies conducted by confocal laser scanning microscopy (CLSM) seem to suggest that DS-1 enhances the Caco-2 permeability mainly via a transcellular route. Histological examination failed to reveal noticeable morphological alterations in the cell monolayers pretreated with DS-1. The integrity of the Caco-2 monolayers, as assessed by their permeability to mannitol, was found to be recoverable following the mucosal pretreatment of DS-1. These results suggest that DS-1 is an efficacious intestinal permeation-enhancing agent with low adverse effect on the epithelial viability and barrier function.