Many liposome formulations for parenteral applications are intended to alter the bio disposition of biologically active compounds. The parenteral route has been concentrated upon, because this route bypasses the natural barriers of the body. Within parenterals, although many different routes have been examined, most effort has been devoted to the intravenous route (Ostro and Cullis, 1989). From the very beginning, liposomes have been advocated as “magic bullets”, in order to target biologically active materials. Hence, work has focused upon extending the circulation time of liposomes in vivo with the aqueous drug stably entrapped and targeting these vesicles to specific sites. In the early 1980s extensive work was carried out employing liposomes as a targeted carrier system. It was believed that once injected, they would circulate around the bloodstream unnoticed, targeting and delivering the encapsulated drug to the desired site. However, it was discovered that if unsaturated phosphatidylcholine vesicles are employed, the liposomes not only immediately leaked their entrapped material but also rapidly disintegrated in vivo (Gregoriadis, 1988). In order to decrease this leakage and disintegration, two difficulties had to be overcome. Firstly, the stability and circulation time o f liposomes in the bloodstream had to be improved. Secondly, suitable biologically active materials had to be selected which could be effectively entrapped and retained inside the liposome.
Bilayer integrity was greatly aided by the addition of membrane stabilising components, such as the sterol cholesterol (Guo et al., 1980; Allen et al., 1981). Further improvements based upon the same principle, i.e. reducing the membrane mobility and increasing the rigidity, were made by employing saturated phosphatidylcholines. These types o f liposomes with hydrogenated phospholipids and cholesterol are being used to entrap daunorubicin, a toxic cytotoxic agent, in a commercially available product “Daunoxome®” (NeXstar, USA). Although such modifications greatly aid the integrity of the liposome in vivo, they do not confer the liposome with the ability to evade the host’s defence system. The liposome is still viewed as foreign by the immune system: once in the bloodstream, most liposomes are rapidly recognised and engulfed by the
MPS. This efficient clearance system may severely limit the half life of the liposome and the drug entrapped within the liposome.
In order to extend the circulation time of the liposome it was realised the size had to be carefully controlled. It was believed that a liposome diameter o f 100 nm or less maximised the circulation time of the vesicles (Hwang et al., 1980; Allen and Everest, 1983; Proffitt et al., 1983). Further improvements in circulation time of the liposome were made as different surface properties of liposomes were explored. Work in the 1980s employed phosphatidylinositol as a means of altering the properties of the liposome (Kao and Loo, 1980; Gabizon and Papahadjopoulos, 1988). This negatively charged phospholipid was incorporated into the bilayer, thereby projecting inositol groups at the surface of the bilayer, which significantly prolonged the retention of the liposome in the circulation. The mechanism of this prolongation has been attributed to ste fie stabilisation. By reducing liposome opsonisation by the plasma proteins, the liposome is not as quickly detected by the MPS. However, due to the high cost and potential toxicity, this approach was not adopted for commercial applications. Nevertheless, it demonstrated the principle that it was possible to increase the half life of liposomes in the bloodstream. The next major advance in circulation prolongation was the development of liposomes incorporating lipids covalently attached to polymers (Blume and Cevc, 1990; Woodle and Lasic, 1992). Although a variety of different polymers can be employed, the liposomes incorporating PEG-ylated phosphatidylethanolamine were selected for further development. Selection of a specific PEG size and incorporation of PEG-ylated phospholipid between 5-10% greatly improved the circulation time of these liposomes.
In terms o f selecting the appropriate drug, due to the relative ease of entrapping water soluble compounds stably, almost all liposome systems have employed hydrophilic compounds. Lipophilic hydrophobes, as explained in section 1.3.4.1, have a tendency to leak from the bilayer and therefore have been sidelined for targeting purposes. Originally, aqueous materials were entrapped passively. This meant the entrapment of non-membrane interacting hydrophilic drugs was directly proportional to the volume of aqueous spaces inside the liposomes. Hence entrapping more than 30-40% of the material was generally considered difficult. In order to achieve 100% entrapment, i.e. all of the drug inside the liposome, it was necessary to separate out the liposomes from
Chapter one- Introduction
unentrapped material by column chromatography or centrifugation. This was an inefficient and a wasteful means of entrapping material. Various techniques, such as pH gradient active loading and remote loading (Bally et al., 1985; Hope et al., 1985; Mayer et al., 1990), have greatly improved the loading efficiency of liposomes. These methods exploited the properties of weak bases: by creating a pH gradient between the liposome interior and exterior, it was possible to direct some drugs towards the aqueous interior of the liposome. The uncharged molecule diffused across the membrane, but once inside the aqueous channels of the liposome, the molecule became charged as a result of the change in pH. The charge prevented diffusion of the molecule out of the liposome, thereby entrapping the charged material within the liposome. Further refinements were made using an elegant ammonium sulphate gradient for amphiphiles, such as doxorubicin. This enabled up to 90% of the active material to be entrapped inside the liposome without further purification (Cohen, 1991; Haran et al., 1993).
Although these systems have developed considerably since the first description of the classical liposome in 1965, the challenge, however, of true site specific delivery still remains. Currently the most advanced system being evaluated clinically are the liposomes incorporating PEG-ylated phosphatidylethanolamine with ammonium gradient loaded doxorubicin (Doxil®, Sequus, USA). These liposomes are highly stable in vivo, and have prolonged circulation times. However, they still do not offer true targeting, even if tumour sites may be more permeable than healthy tissue and enable slightly higher amounts of cytotoxic to be delivered. The true specificity required for “targeting” has yet to be achieved. An added intelligent step, which dictates where and when systems should release their load is required, before the term “targeted” can be genuinely applied. Many ideas have been considered and proposed for this programming, e.g. magnetic, fusogenic, pH dependant release, thermo release and antibodies (Straubinger et al., 1988 and Matthay et al., 1989). However, to date there has been little success with these approaches.
1.2 Parenteral administration