Pivotal Shigella Vaccine Efficacy Trials-Study Design Considerations from a Shigella Vaccine Trial Design Working Group.

Vaccine candidates for Shigella are approaching phase 3 clinical trials in the target population of young children living in low- and middle-income countries. Key study design decisions will need to be made to maximize the success of such trials and minimize the time to licensure and implementation. We convened an ad hoc working group to identify the key aspects of trial design that would meet the regulatory requirements to achieve the desired indication of prevention of moderate or severe shigellosis due to strains included in the vaccine. The proposed primary endpoint of pivotal Shigella vaccine trials is the efficacy of the vaccine against the first episode of acute moderate or severe diarrhea caused by the Shigella strains contained within the vaccine. Moderate or severe shigellosis could be defined by a modified Vesikari score with dysentery and molecular detection of vaccine-preventable Shigella strains. This report summarizes the rationale and current data behind these considerations, which will evolve as new data become available and after further review and consultation by global regulators and policymakers.

Early effects of lasonolide a on pancreatic cancer cells.

Lasonolide A, a novel polyketide-derived macrolide, was previously identified from an extract of the marine sponge Forcepia sp. in an assay for protein kinase C (PKC) inhibitors. Cytotoxicity testing and profiling of lasonolide A in the National Cancer Institute (NCI) 60 cell panel screen revealed that it was potent toward a broad range of cell lines and also suggested a unique mechanism of action. Contrary to expected results, we found lasonolide A to be a strong activator of PKC in Panc-1 pancreatic carcinoma cells. Downstream mitogen-activated protein kinases, ERK 1/2 and p38 were also rapidly phosphorylated in response to lasonolide A, as was Akt. Microscopy studies revealed that lasonolide A induced blebbing and contraction of the cells within minutes of exposure, and the eventual loss of adherence. However, membrane integrity was maintained and the effects were reversible if lasonolide A was washed from the cells after their loss of adherence. Pretreatment of cells with a myosin II inhibitor, blebbistatin, slowed the early onset, but did not prevent the morphological effects of lasonolide A. Cells stained for actin filaments showed some reduction in stress fiber structure after lasonolide A exposure; however, it did not affect the polymerization of purified actin in vitro. Bisindolemaleimide, a PKC inhibitor, and wortmannin, a phosphoinositide 3-kinase; inhibitor, did not reduce lasonolide A-induced contraction or blebbing or the activation of mitogen-activated protein kinases, although Akt phosphorylation was prevented by wortmannin pretreatment. Our results indicate that lasonolide A activates multiple signal transduction pathways and suggest that the origin is upstream of PKC.

Tubulin polymerizing activity of dictyostatin-1, a polyketide of marine sponge origin.

Dictyostatin-1 had previously been isolated from a marine sponge of the genus Spongia sp. and described as a cytotoxic agent to murine and human cancer cells, but its mechanism of activity was unknown. In a routine screening assay used to detect cytotoxic compounds of marine origin, dictyostatin-1 was identified as a highly active component in an extract from a Lithistida sponge and exploration into its pharmacology was pursued. Initial studies demonstrated that dictyostatin-1 arrested cells in the G(2)/M phase of the cell cycle. Staining of these cells with antitubulin revealed cells having multiple aster formations and microtubule matrix bundling patterns similar to that seen in cells exposed to paclitaxel. Dictyostatin-1 was able to induce the polymerization of purified bovine brain tubulin in vitro and the polymerized tubulin remained stable at cold temperatures. Dictyostatin-1 also proved to be highly potent in two paclitaxel-resistant human cancer cell lines expressing active P-glycoprotein. Together, these results indicate that dictyostatin-1 is a potent inducer of tubulin polymerization and retains activity in cells expressing the P-glycoprotein efflux pump.

Modified binding assay for improved sensitivity and specificity in the detection of residual pertussis toxin in vaccine preparations.

A binding assay was recently published that differentiates between pertussis toxin (PTx) and the pertussis toxoid (PTd) used in acellular pertussis vaccines based on the selective binding of PTx to fetuin and detection with a polyclonal antibody. We found that the assay specificity for PTx was affected by both pH and salt. A monoclonal antibody (mAb) was identified that eliminated specificity problems and improved the sensitivity to 0.4 ngPTx/ml. This mAb was previously shown to neutralize PTx toxicity in vivo, thereby supporting the assay's potential biological relevance as an alternative to the mouse histamine sensitivity test for the safety of pertussis vaccines.

Semisynthetic analogues of the microtubule-stabilizing agent discodermolide: preparation and biological activity.

A series of 12 semisynthetic discodermolide analogues, 2-13, have been prepared using natural (+)-discodermolide (1) and evaluated for in vitro cytotoxicity against cultured murine P-388 leukemia and A-549 human adenocarcinoma cells. These semisynthetic analogues showed a significant variation of cytotoxicity and confirmed the importance of the C-7 through C-19 molecular fragment for potency. Specifically, these analogues suggested the importance of the C-11 and C-17 hydroxyl groups and the C-13 double bond for the potency of discodermolide. The preparation, structure elucidation, and biological activity of these new analogues are described.

Plakolide A, a new gamma-lactone from the marine sponge Plakortis sp.

Plakolide A (1), a new alpha-exomethylene-gamma-lactone isolated from the marine sponge Plakortis sp., was found to inhibit inducible nitric oxide synthase (iNOS) activity. The isolation, structure elucidation, and biological activity of plakolide A is described.

Five new discodermolide analogues from the marine sponge Discodermia species.

Discodermolide (1) and five new discodermolide analogues trivially named 2-epi-discodermolide (2), 2-des-methyldiscodermolide (3), 5-hydroxymethyldiscodermolate (4), 19-des-aminocarbonyldiscodermolide (5), and 9(13)-cyclodiscodermolide (6) have been isolated from marine sponges belonging to the genus Discodermia collected from the Caribbean Sea. The isolation, structure elucidation, and biological activities of 2-6 are described. The natural analogues, which were isolated in trace amounts, exhibited significant variation of cytotoxicity against the cultured murine P-388 leukemia and A-549 human adenocarcinoma cells and suggested the importance of the C(7) through C(17) moiety for potency against cultured tumor cell lines.