An R848 adjuvanted influenza vaccine promotes early activation of B cells in the draining lymph nodes of non-human primate neonates.

Impaired immune responsiveness is a significant barrier to vaccination of neonates. By way of example, the low seroconversion observed following influenza vaccination has led to restriction of its use to infants over 6 months of age, leaving younger infants vulnerable to infection. Our previous studies using a non-human primate neonate model demonstrated that the immune response elicited following vaccination with inactivated influenza virus could be robustly increased by inclusion of the Toll-like receptor agonist flagellin or R848, either delivered individually or in combination. When delivered individually, R848 was found to be the more effective of the two. To gain insights into the mechanism through which these adjuvants functioned in vivo, we assessed the initiation of the immune response, i.e. at 24 hr, in the draining lymph node of neonate non-human primates. Significant up-regulation of co-stimulatory molecules on dendritic cells could be detected, but only when both adjuvants were present. In contrast, R848 alone could increase the number of cells in the lymph node, presumably through enhanced recruitment, as well as B-cell activation at this early time-point. These changes were not observed with flagellin and the dual adjuvanted vaccine did not promote increases beyond those observed with R848 alone. In vitro studies showed that R848 could promote B-cell activation, supporting a model wherein a direct effect on neonate B-cell activation is an important component of the in vivo potency of R848 in neonates.

A Novel R848-Conjugated Inactivated Influenza Virus Vaccine Is Efficacious and Safe in a Neonate Nonhuman Primate Model.

Influenza virus infection of neonates poses a major health concern, often resulting in severe disease and hospitalization. At present, vaccines for this at-risk population are lacking. Thus, development of an effective vaccine is an urgent need. In this study, we have used an innovative nonhuman primate neonate challenge model to test the efficacy of a novel TLR 7/8 agonist R848-conjugated influenza virus vaccine. The use of the intact virus represents a step forward in conjugate vaccine design because it provides multiple antigenic targets allowing for elicitation of a broad immune response. Our results show that this vaccine induces high-level virus-specific Ab- and cell-mediated responses in neonates that result in increased virus clearance and reduced lung pathology postchallenge compared with the nonadjuvanted virus vaccine. Surprisingly, the addition of a second TLR agonist (flagellin) did not enhance vaccine protection, suggesting that combinations of TLR that provide increased efficacy must be determined empirically. These data support further exploration of this new conjugate influenza vaccine approach as a platform for use in the at-risk neonate population.

Ring expansions of acyloxy nitroso compounds.

Treatment of cyclopentanone and cyclobutanone-derived oximes with lead (IV) tetraacetate gives the bright blue acyloxy nitroso compounds, which upon basic hydrolysis yields the ring expansion product cyclic hydroxamic acids in 12-81% yield. Reactions of substituted cyclopentanones provide ring expanded products where the -NOH group regioselectively inserts to the more substituted position and gives a better yield compared to the treatment of the same ketone with a basic solution of Piloty's acid. Reaction of phosphines with acyloxy nitroso compounds generally generates a ring-expanded Beckmann rearrangement product that can be hydrolyzed to the corresponding lactam. Acyloxy nitroso compounds that undergo rapid hydrolysis to HNO do not show this ring expansion reactivity. These results further demonstrate the versatility of acyloxy nitroso compound to yield structurally complex materials.

Synthesis of a 2-aryl-3-aroyl indole salt (OXi8007) resembling combretastatin A-4 with application as a vascular disrupting agent.

The natural products colchicine and combretastatin A-4 are potent inhibitors of tubulin assembly, and they have inspired the design and synthesis of a large number of small-molecule, potential anticancer agents. The indole-based molecular scaffold is prominent among these SAR modifications, leading to a rapidly increasing number of agents. The water-soluble phosphate prodrug 33 (OXi8007) of 2-aryl-3-aroylindole-based phenol 8 (OXi8006) was prepared by chemical synthesis and found to be strongly cytotoxic against selected human cancer cell lines (GI50 = 36 nM against DU-145 cells, for example). The free phenol, 8 (OXi8006), was a strong inhibitor (IC50 = 1.1 µM) of tubulin assembly. The corresponding phosphate prodrug 33 (OXi8007) also demonstrated pronounced interference with tumor vasculature in a preliminary in vivo study utilizing a SCID mouse model bearing an orthotopic PC-3 (prostate) tumor as imaged by color Doppler ultrasound. The combination of these results provides evidence that the indole-based phosphate prodrug 33 (OXi8007) functions as a vascular disrupting agent that may prove useful for the treatment of cancer.

Development of synthetic methodology suitable for the radiosynthesis of combretastatin A-1 (CA1) and its corresponding prodrug CA1P.

Synthetic methodology has been established suitable for the preparation of combretastatin A-1 (CA1) and its corresponding phosphate prodrug salt (CA1P) in high specific activity radiolabeled form. Judicious selection of appropriate phenolic protecting groups to distinguish positions on the A-ring from the B-ring of the stilbenoid was paramount for the success of this project. Methylation of the C-4' phenolic moiety by removal of the tert-butyldimethylsilyl protecting group in the presence of methyl iodide was accomplished in excellent yield without significant Z to E isomerization. This step (carried out with (12)C-methyl iodide as proof of concept in this study) represents the process in which a (14)C radioisotope could be incorporated in an actual radiosynthesis. CA1 is a natural product isolated from the African bush willow tree (Combretum caffrum) that has important medicinal value due, in part, to its ability to inhibit tubulin assembly. As a prodrug, CA1P (OXi4503) is in human clinical trials as a vascular disrupting agent.

Design, synthesis, biochemical, and biological evaluation of nitrogen-containing trifluoro structural modifications of combretastatin A-4.

A new trifluorinated amino-combretastatin analogue, (Z)-2-(4'-methoxy-3'-aminophenyl)-1-(3,4,5-trifluorophenyl)ethene, prepared by chemical synthesis, was found to be a potent inhibitor of tubulin assembly (IC(50)=2.9 microM), and cytotoxic against selected human cancer cell lines. This new lead compound is among the most active from a group of related structural modifications.

Adjuvanting an inactivated influenza vaccine with conjugated R848 improves the level of antibody present at 6months in a nonhuman primate neonate model.

Generation of a potent antibody response that can be sustained over time is highly challenging in young infants. Our previous studies using a nursery-reared nonhuman primate model identified R848 conjugated to inactivated influenza virus as a highly immunogenic vaccine for neonates. Here we determined the effectiveness of this vaccine in mother-reared infants as well as its ability to promote improved responses at 6months compared to vaccination in the absence of R848. In agreement with our nursery study, R848 conjugated to influenza virus induced a higher antibody response in neonates compared to the non-adjuvanted vaccine. Further, the increase in the response relative to that induced by the non-adjuvanted vaccine was maintained at 6months suggesting the increased antibody secreting cells that resulted from inclusion of conjugated R848 production were capable of surviving long term. There was no significant difference in quality of antibody (i.e. neutralization or affinity), suggesting the beneficial effect of conjugated R848 during vaccination of neonates with inactivated influenza virus is likely manifest during the early generation of antibody secreting cells.

The vascular disrupting activity of OXi8006 in endothelial cells and its phosphate prodrug OXi8007 in breast tumor xenografts.

This study describes the vascular disrupting ability and the mechanism of action of the indole-based tubulin-binding compound, OXi8006, and its water-soluble phosphate prodrug OXi8007. Treatment of rapidly proliferating human umbilical vein endothelial cells (HUVECs), used as a model for the tumor vasculature, with OXi8006 or OXi8007, caused potent microtubule disruption followed by extensive reorganization of the cytoskeletal network. The mechanism of action involved an increase in focal adhesion formation associated with an increase in phosphorylation of both non-muscle myosin light chain and focal adhesion kinase. These effects were dramatically diminished by an inhibitor of RhoA kinase, a downstream effector of RhoA. Cell cycle blockade at G2/M and cytotoxicity toward rapidly proliferating HUVECs were also observed. Capillary-like networks of HUVECs were disrupted by the action of both OXi8006 and OXi8007. The prodrug OXi8007 exhibited potent and rapid dose-dependent antivascular activity assessed by dynamic bioluminescence imaging (BLI) in an MDA-MB-231-luc breast cancer xenograft mouse model. By 6 hours post treatment, over 93% of the BLI signal was abolished with only a slight recovery at 24 hours. These findings were confirmed by histology. The results from this study demonstrate that OXi8007 is a potent vascular disrupting agent acting through an anti-microtubule mechanism involving RhoA.

Guaifenesin derivatives promote neurite outgrowth and protect diabetic mice from neuropathy.

In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of neurite outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the neurite outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy.

Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the SOS response in Escherichia coli.

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures.

Combretastatin dinitrogen-substituted stilbene analogues as tubulin-binding and vascular-disrupting agents.

Several stilbenoid compounds having structural similarity to the combretastatin group of natural products and characterized by the incorporation of two nitrogen-bearing groups (amine, nitro, serinamide) have been prepared by chemical synthesis and evaluated in terms of biochemical and biological activity. The 2',3'-diamino B-ring analogue 17 demonstrated remarkable cytotoxicity against selected human cancer cell lines in vitro (average GI 50 = 13.9 nM) and also showed good activity in regard to inhibition of tubulin assembly (IC 50 = 2.8 microM). In addition, a single dose (10 mg/kg) of compound 17 caused a 40% tumor-selective blood flow shutdown in tumor-bearing SCID mice at 24 h, thus suggesting the potential value of this compound and its corresponding salt formulations as new vascular-disrupting agents.

Design, synthesis, and biological evaluation of combretastatin nitrogen-containing derivatives as inhibitors of tubulin assembly and vascular disrupting agents.

A series of analogs with nitro or serinamide substituents at the C-2'-, C-5'-, or C-6'-position of the combretastatin A-4 (CA4) B-ring was synthesized and evaluated for cytotoxic effects against heart endothelioma cells, blood flow reduction to tumors in SCID mice, and as inhibitors of tubulin polymerization. The synthesis of these analogs typically featured a Wittig reaction between a suitably functionalized arylaldehyde and an arylphosphonium salt followed by separation of the resultant E- and Z-isomers. Several of these nitrogen-modified CA4 derivatives (both amino and nitro) demonstrate significant inhibition of tubulin assembly as well as cytotoxicity and in vivo blood flow reduction. 2'-Aminostilbenoid 7 and 2'-amino-3'-hydroxystilbenoid 29 proved to be the most active in this series. Both compounds, 7 and 29, have the potential for further pro-drug modification and development as vascular disrupting agents for treatment of solid tumor cancers and certain ophthalmological diseases.

2-(3-tert-Butyldimethylsiloxy-4-methoxyphenyl)-6-methoxy-3-(3,4,5-trimethoxybenzoyl)indole.

In the crystal structure of the title compound, C(32)H(39)NO(7)Si, all geometric parameters fall within experimental error of expected values. The analysis of molecular-packing plots reveals an infinite two-dimensional linear array running parallel to the b axis, formed by one N[bond]H...O intermolecular hydrogen-bonding interaction. Several potential C[bond]H...O interactions are also present.

Synthesis, in vitro, and in vivo evaluation of phosphate ester derivatives of combretastatin A-4.

Combretastatin A-4 disodiumphosphate (CA4P), a prodrug formulation of the natural product combretastatin A-4 (CA4), is currently in clinical investigation for the treatment of cancer. In vivo, CA4P is rapidly enzymatically converted to CA4, a potent inhibitor of tubulin polymerization (IC(50)=1-2 microM), and rapidly causes bloodflow shutdown in tumor tissues. A variety of alkyl and aryl di- and triesters of CA4P have been synthesized and evaluated as potential CA4 prodrugs and/or stable CA4P analogues.