THE PERIPHERAL MECHANISMS OF ANALGESIA AND THE INVOLVEMENT OF ADHESION MOLECULES - Development of a Novel Targeted Drug Delivery System to Sites of Inflammation and Application to Peripheral Analgesia

Peripheral mechanisms of endogenous pain control are significant. Increasing studies have clearly produced evidence for the clinical usefulness of opioids in peripheral analgesia. The immune system uses mechanisms of cell migration not only to fight pathogens but also to control pain within injured tissue. It has been demonstrated that peripheral inflammatory pain can be effectively controlled by an interaction of immune cell derived opioid peptides with opioid receptors on peripheral sensory nerve terminals. Animal and clinical studies have clearly shown that activation of peripheral opioid receptors with exogenous opioid agonists and endogenous opioid peptides are able to produce significant antinociception. Although studies on topical opioid application are promising, the goal remains to develop peripherally selective opioid compounds, suitable for oral and/or intravenous route of administration to improve clinical pain relief. The use of drug delivery and targeting may be the key to effective development of many novel and existing therapeutics to enable optimal therapeutic use of such molecules. Drug targeting using liposomes as carriers holds much promise, especially in reducing toxicity and targeting delivery to pathological sites of inflammation. Adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) is locally induced or enhanced in a multitude of clinical diseases where inflammation and immune cells are involved. In particular, ICAM-1 plays an important role in peripheral analgesia at both the endothelial and immune cell–neuronal level. This peripheral analgesic pathway has yet to be fully exploited in pharmaceutical formulation and clinical pain management. In this thesis, long circulating immunoliposomes incorporating ICAM-1 monoclonal antibodies were prepared to create a delivery system with the unique properties of binding to adhesion molecules that are increased only at the site of tissue injury with the intent for further use as drug carriers. Loperamide hydrochloride (loperamide HCl), a peripherally selective mu-opioid receptor (MOR) agonist, was encapsulated in the liposome formulation. Immunoliposomes were prepared using an optimised method for the coupling of low concentrations of antibody to liposomes, thereby preventing the loss of antibody through the derivatisation, extraction and activation process. This is especially suitable for limiting ligand conjugates that are isolated or synthesised in small quantities such as monoclonal antibodies. The optimised method has been demonstrated to provide versatile, reproducible and efficient conjugation, with high antibody conjugation efficiency and high drug entrapment efficiency. The ICAM-1 targeted immunoliposomes taken into in vitro and in vivo studies had a mean particle size of 296 nm +/- 6.8 nm and a mean loperamide HCl encapsulation efficiency of 3.981 +/- 0.078 mg/ml of liposome suspension. Targeting efficacy of the immunoliposomes was assessed on primary high endothelial venules (HEV) cells, which were isolated and cultured from rat lymph nodes. Specificity of binding and cellular uptake was assessed by fluorescence microplate spectroscopy on tumor necrosis factor-alpha (TNF-alpha) activated and non-activated HEV cells at 4oC and 37oC. Results indicate that ICAM-1-mediated targeting of liposomes to ICAM-1 expressing cells in vitro is rapid and specific, with minimal cellular internalisation. The degree of anti-ICAM-1 immunoliposomes bound was significantly increased following pre-treatment of HEV cells with the inflammatory cytokine TNF-alpha. Negative control binding experiments resulted in low binding values. Pre-incubation with excess anti-ICAM-1 monoclonal antibodies significantly inhibited the binding of ICAM-1-targeted immunoliposomes to TNF-alpha activated HEV cells, further demonstrating the specific nature of the liposome-cell interaction. In addition, the liposome formulation was shown to be stable against leakage of encapsulated drug in both PBS and serum using in vitro dialysis. In vivo studies in rats with Freund’s Complete Adjuvant (FCA)-induced inflammation of the paw demonstrated significant and prolonged antinociceptive and anti-inflammatory efficacy over the 48 h study period following intravenous administration of loperamide HCl-encapsulated anti-ICAM-1 immunoliposomes. Evaluation of paw pressure threshold (PPT) of the loperamide HCl-encapsulated anti-ICAM-1 immunoliposome group demonstrated a peak antinociceptive effect between 7 h to 10 h following liposome administration. PPTs and paw displacement volumes of control groups (empty conventional liposomes, loperamide HCl-encapsulated conventional liposomes and loperamide HCl solution) were not significantly different across all time points over the 48 h period. Incorporation of ICAM-1 monoclonal antibody targeting ligands to the surface of the liposomes labelled with tritiated cholesteryl hexadecyl ether ([3H]-CHE) in biodistribution studies showed increased localisation to peripheral inflammatory tissue, with no significant distribution into the central nervous system (CNS). In conclusion, the work presented in this thesis provides a novel drug delivery system that is capable of targeting pathological sites of inflammation. Developing a drug delivery system to provide site-directed analgesia by delivering exogenous opioid agonists to peripheral opioid receptors within inflamed tissue may provide an effective alternative for the management of chronic arthritic and other inflammatory conditions, whist simultaneously decreasing distribution of the drug to other sites in the body. In this way diminishing central opioid mediated side effects (e.g. sedation, respiratory depression, tolerance, dependence) and of typical side effects produced by nonsteroidal and steroidal anti-inflammatory drugs