Joanne T. Blanchfield

Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model

Background:  Echinacea is composed of three major groups of compounds that are thought to be responsible for stimulation of the immune system – the caffeic acid conjugates, alkylamides and polysaccharides. This study has focussed on the former two classes, as these are the constituents found in ethanolic liquid extracts.

Mucosal Immunisation: Adjuvants and Delivery Systems

The mucosal administration of vaccines is an area currently receiving a high level of interest due to potential advantages offered by this technique. These advantages include the ability to administer vaccines without need for needles, thus improving patient compliance with vaccination schedules, and the capacity to induce immune responses capable of preventing infections at the site of acquisition. Despite these advantages a number of limitations exist which currently inhibit our ability to successfully develop new mucosal vaccines. As such, much research is currently focused on developing new adjuvants and delivery systems to overcome these difficulties. However, despite high levels of interest in this area, relatively few mucosal vaccine candidates have successfully progressed to human clinical trials. In the review that follows, we aim to provide the reader with an overview of the immune system with respect to induction of mucosal immune responses. Furthermore, the review provides an overview of a number of microbial (bacterial toxins, CpG DNA, cytokines/chemokines, live vectors, and virus like particles) and synthetic (microspheres, liposomes, and lipopeptides) strategies that have been investigated as adjuvants or delivery systems for mucosal vaccine development, with a focus on the delivery of vaccines via the oral route.

Particulate systems as adjuvants and carriers for peptide and protein antigens.

The most common feature for antigen-delivery systems is their particulate nature. Together with a certain depot effect, it is the particulate nature that primarily dictates whether the antigen-delivery system will be successful in inducing a certain type and strength of immune response. In this article, we will summarize recent data on particulate delivery systems for peptide and protein antigens with a main focus on lipid or polymer-based particles, all of which possess high potential as both preventive and therapeutic vaccines for parenteral, nasal, and possibly oral administration.

Synthesis and biological evaluation of an orally active glycosylated endomorphin-1

The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability and membrane permeability of 2 while retaining μ-opioid receptor binding affinity and agonist activity. Analogue 2 produced dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of neuropathic pain with an ED(50) of 8.3 (± 0.8) μmol/kg. The corresponding ED(50) for morphine was 2.6 (± 1.4) μmol/kg. Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED(50) = 19.6 (± 1.2) μmol/kg), which was comparable with that of morphine (ED(50) = 20.7 (±3.6) μmol/kg). Antineuropathic effects of analogue 2 were significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents, are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetrahydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculeata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous 1H and 13C NMR data are presented for these alkaloids.

Identification of Bacterial Protein O-Oligosaccharyltransferases and Their Glycoprotein Substrates

O-glycosylation of proteins in Neisseria meningitidis is catalyzed by PglL, which belongs to a protein family including WaaL O-antigen ligases. We developed two hidden Markov models that identify 31 novel candidate PglL homologs in diverse bacterial species, and describe several conserved sequence and structural features. Most of these genes are adjacent to possible novel target proteins for glycosylation. We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site. For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated. Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

A novel synthetic adjuvant enhances dendritic cell function

The lipid core peptide (LCP) is a novel, synthetic, self‐adjuvanted vaccine delivery system that neatly incorporates the adjuvant, carrier and antigenic peptides of a vaccine into a single molecular entity. This system has been previously shown to efficiently deliver vaccines and induce immunity. Because adjuvants target sentinels of the immune response, such as dendritic cells (DCs), that are widely distributed throughout the body to initiate specific immune responses, we investigated the effects of the adjuvant on DCs. Here we show that LCP targets vaccines to DCs and induces their activation.

Bile pigment pharmacokinetics and absorption in the rat: therapeutic potential for enteral administration

BACKGROUND AND PURPOSE Bilirubin and biliverdin possess antioxidant and anti‐inflammatory properties and their exogenous administration protects against the effects of inflammation and trauma in experimental models. Despite the therapeutic potential of bile pigments, little is known about their in vivo parenteral or enteral absorption after exogenous administration. This study investigated the absorption and pharmacokinetics of bile pigments after i.v., i.p. and intraduodenal (i.d.) administration in addition to their metabolism and routes of excretion.

Lipo-endomorphin-1 derivatives with systemic activity against neuropathic pain without producing constipation.

To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (1 = Tyr-Pro-Trp-Phe-NH2), the N-terminus of the peptide was modified with 2-aminodecanoic acid, resulting in compound 3. Tyr in compound 1 was replaced with 2,6-dimethyltyrosine yielding compound 2. Derivative 2 was also substituted with 2-aminodecanoic acid producing compound, 4. Lipoamino acid-modified derivatives showed improved metabolic stability and membrane permeability while maintaining high μ-opioid (MOP) receptor binding affinity and acting as a potent agonist. In vivo studies showed dose-dependent antinociceptive activity following intravenous (i.v.) administration of compounds 3 and 4 in a chronic constriction injury (CCI)-rat model of neuropathic pain with ED50 values of 1.22 (±0.93) and 0.99 (±0.89) µmol/kg, respectively. Pre-treatment of animals with naloxone hydrochloride significantly attenuated the anti-neuropathic effects of compound 3, confirming the key role of opioid receptors in mediating antinociception. In contrast to morphine, no significant constipation was produced following i.v. administration of compound 3 at 16 µmol/kg. Furthermore, following chronic administration of equi-potent doses of compound 3 and morphine to rats, there was less antinociceptive tolerance for compound 3 compared with morphine.

In vitro stability and permeability studies of liposomal delivery systems for a novel lipophilic endomorphin 1 analogue

We have previously shown that the stability and permeability of peptides can be greatly improved by conjugation with lipoamino acids such as 2-aminododecanoic acid (C12Laa). However, the increase in lipophilicity which this conjugation provides can also cause a significant decrease in the compounds water solubility. In this study, we coupled C12Laa to the N-terminus of endomorphin1 (Endo-1, Tyr-Pro-Trp-Phe-NH(2)), and addressed its solubility issue by formulating C12Laa-Endo-1 into phosphatidylcholine liposomes. The aqueous solubility of the lipidic analogue was greatly improved, facilitating the accurate analysis of the compound in in vitro assays. The metabolic stability and in vitro endothelial permeability of the C12Laa-Endo-1 liposomal formulation was assessed using Caco-2 cells, and compared with the formulation of the parent peptide Endo-1. The liposome-encapsulated C12Laa-Endo exhibited significant increases in both stability and permeability. These results suggest that the combination of chemical modification and liposome formulation has great potentials in improving the bioavailability of neuroactive peptides.