Unique depot formed by an oil based vaccine facilitates active antigen uptake and provides effective tumour control.

BACKGROUND: Oil emulsions are commonly used as vaccine delivery platforms to facilitate slow release of antigen by forming a depot at the injection site. Antigen is trapped in the aqueous phase and as the emulsion degrades in vivo the antigen is passively released. DepoVax™ is a unique oil based delivery system that directly suspends the vaccine components in the oil diluent that forces immune cells to actively take up components from the formulation in the absence of passive release. The aim of this study was to use magnetic resonance imaging (MRI) with additional biological markers to evaluate and understand differences in clearance between several different delivery systems used in peptide-based cancer vaccines. METHODS: C57BL/6 mice were implanted with a cervical cancer model and vaccinated 5 days post-implant with either DepoVax (DPX), a water-in-oil emulsion (w/o), a squalene oil-in-water emulsion (squal o/w) or a saponin/liposome emulsion (sap/lip) containing iron oxide-labeled targeted antigen. MRI was then used to monitor antigen clearance, the site of injection, tumour and inguinal lymph node volumes and other gross anatomical changes. HLA-A2 transgenic mice were also vaccinated to evaluate immune responses of human directed peptides. RESULTS: We demonstrated differences in antigen clearance between DPX and w/o both in regard to how quickly the antigen was cleared and the pattern in which it was cleared. We also found differences in lymph node responses between DPX and both squal o/w and sap/lip. CONCLUSIONS: These studies underline the unique mechanism of action of this clinical stage vaccine delivery system.

Type III hypersensitivity reactions to a B cell epitope antigen are abrogated using a depot forming vaccine platform.

Peptide antigens are combined with an adjuvant in order to increase immunogenicity in vivo. The immunogenicity and safety of a RSV vaccine formulated in a novel oil-based platform, DepoVax™ (DPX), was compared to an alum formulation. A peptide B cell epitope derived from RSV small hydrophobic ectodomain (SHe) served as the antigen. Both vaccines induced SHe-specific antibodies after immunization of mice. A single dose of the DPX-based formulation resulted in anti-SHe titres for up to 20 weeks. Boosting with Alum-SHe, but not with DPX-SHe, led to unexpected clinical signs such as decreased activity, cyanosis and drop in body temperature in mice but not in rabbits. The severity of adverse reactions correlated with magnitude of SHe-specific IgG immune responses and decreased complement component 3 plasma levels, indicating a type III hypersensitivity reaction. By RP-HPLC analysis, we found that only 8-20% of the antigen was found to be adsorbed to alum in vitro, indicating that this antigen is likely released systemically upon injection in vivo. Clinical signs were not observed in rabbits, indicating the response correlates with peptide dose relative to size of animal. These results suggest that peptide antigens targeted to produce B cell mediated response may result in increased incidence of type III hypersensitivity reactions when delivered in non-depot forming vaccines. The DPX formulation induced strong antibody titres to the antigen without causing adverse events, likely due to the strength of the depot in vivo, and demonstrates the potential safety and immunogenicity of this platform for B cell peptide antigens.

Survivin-targeted immunotherapy drives robust polyfunctional T cell generation and differentiation in advanced ovarian cancer patients.

DepoVax™ is an innovative and strongly immunogenic vaccine platform. Survivin is highly expressed in many tumor types and has reported prognostic value. To generate tumor-specific immune response, a novel cancer vaccine was formulated in DepoVax platform (DPX-Survivac) using survivin HLA class I peptides. Safety and immune potency of DPX-Survivac was tested in combination with immune-modulator metronomic cyclophosphamide in ovarian cancer patients. All the patients receiving the therapy produced antigen-specific immune responses; higher dose vaccine and cyclophosphamide treatment generating significantly higher magnitude responses. Strong T cell responses were associated with differentiation of naïve T cells into central/effector memory (CM/EM) and late differentiated (LD) polyfunctional antigen-specific CD4+ and CD8+ T cells. This approach enabled rapid de novo activation/expansion of vaccine antigen-specific CD8+ T cells and provided a strong rationale for further testing to determine clinical benefits associated with this immune activation. These data represent vaccine-induced T cell activation in a clinical setting to a self-tumor antigen previously described only in animal models.

Clearance of depot vaccine SPIO-labeled antigen and substrate visualized using MRI.

Immunotherapies, including peptide-based vaccines, are a growing area of cancer research, and understanding their mechanism of action is crucial for their continued development and clinical application. Exploring the biodistribution of vaccine components may be key to understanding this action. This work used magnetic resonance imaging (MRI) to characterize the in vivo biodistribution of the antigen and oil substrate of the vaccine delivery system known as DepoVax(TM). DepoVax uses a novel adjuvanted lipid-in-oil based formulation to solubilise antigens and promote a depot effect. In this study, antigen or oil were tagged with superparamagnetic iron oxide (SPIO), making them visible on MR images. This enables tracking of individual vaccine components to determine changes in biodistribution. Mice were injected with SPIO-labeled antigen or SPIO-labeled oil, and imaged to examine clearance of labeled components from the vaccine site. The SPIO-antigen was steadily cleared, with nearly half cleared within two months post-vaccination. In contrast, the SPIO-oil remained relatively unchanged. The biodistribution of the SPIO-antigen component within the vaccine site was heterogeneous, indicating the presence of active clearance mechanisms, rather than passive diffusion or drainage. Mice injected with SPIO-antigen also showed MRI contrast for several weeks post-vaccination in the draining inguinal lymph node. These results indicate that MRI can visualize the in vivo longitudinal biodistribution of vaccine components. The sustained clearance is consistent with antigen up-take and trafficking by immune cells, leading to accumulation in the draining lymph node, which corresponds to the sustained immune responses and reduced tumor burden observed in vaccinated mice.

Potent and orally efficacious benzothiazole amides as TRPV1 antagonists.

Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.

Development and validation of an HPLC/UV assay for separation and quantification of peptide antigens from a liposomal vaccine delivery platform.

The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax™, a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907.

Differential binding of phenothiazine urea derivatives to wild-type human cholinesterases and butyrylcholinesterase mutants.

A series of N-10 urea derivatives of phenothiazine was synthesized and each compound was evaluated for its ability to inhibit human cholinesterases. Most were specific inhibitors of BuChE. However, the potent inhibitory effects on both cholinesterases of one sub-class, the cationic aminoureas, provide an additional binding mechanism to cholinesterases for these compounds. The comparative effects of aminoureas on wild-type BuChE and several BuChE mutants indicate a binding process involving salt linkage with the aspartate of the cholinesterase peripheral anionic site. The effect of such compounds on cholinesterase activity at high substrate concentration supports ionic interaction of aminoureas at the peripheral anionic site.

Structure-activity relationships for inhibition of human cholinesterases by alkyl amide phenothiazine derivatives.

Several lines of evidence indicate that inhibition of butyrylcholinesterase (BuChE) is important in the treatment of certain dementias. Further testing of this concept requires inhibitors that are both BuChE-selective and robust. N-alkyl derivatives (2, 3, 4) of phenothiazine (1) have previously been found to inhibit only BuChE in a mechanism involving pi-pi interaction between the phenothiazine tricyclic ring system and aromatic residues in the active site gorge. To explore features of phenothiazines that affect the selectivity and potency of BuChE inhibition, a series of N-carbonyl derivatives (5-25) was synthesized and examined for the ability to inhibit cholinesterases. Some of the synthesized derivatives also inhibited AChE through a different mechanism involving carbonyl interaction within the active site gorge. Binding of these derivatives takes place within the gorge, since this inhibition disappears when the molecular volume of the derivative exceeds the estimated active site gorge volume of this enzyme. In contrast, BuChE, with a much larger active site gorge, exhibited inhibition that increased directly with the molecular volumes of the derivatives. This study describes two distinct mechanisms for binding phenothiazine amide derivatives to BuChE and AChE. Molecular volume was found to be an important parameter for BuChE-specific inhibition.

(Z)-tamoxifen and tetrasubstituted alkenes and dienes via a regio- and stereospecific three-component magnesium carbometalation palladium(0) cross-coupling strategy.

[reaction: see text] The synthesis of various tetrasubstituted alkenes and dienes in a regio- and stereocontrolled manner is described. This three-component coupling strategy involves the addition of Grignard reagents to propargyl alcohols followed by palladium(0)-mediated cross-coupling with aryl or vinyl halides. This protocol has been applied to the synthesis of (Z)-Tamoxifen and related mimics.