BACKGROUND AND PROBLEM DEFINITION

Of late, much interest has been placed on the biological activity of selective cyclic dipeptides (CDPs). In a study by Milne and colleagues (1998), it was noted that some of these compounds containing a tryptophan moiety showed significant tumour inhibition. Subsequent studies (Lucietto et al., 2006; Van der Merwe et al., 2008) have confirmed these results, identifying cyclic dipeptides as potential chemotherapeutic agents. Other studies (Graz et al., 1999; Rhee, 2004) have also outlined the potential therapeutic usefulness of these compounds, exhibiting effects such as ion channel modulation, antibacterial and even antifungal properties. In a recent study (Brauns et al., 2004), selected cyclic dipeptides were evaluated against a number of cell lines, namely MCF-7 (breast carcinoma), HeLa (cervical carcinoma) and HT-29 (colon carcinoma), indicating that one of the compounds tested, cyclo(Phe-Pro), showed inhibition of more than 50% in some cell lines, and in addition induced apoptosis in the HT-29 cell line. More recently, significant inhibition of the same cell lines was demonstrated with the histidine-containing cyclic dipeptides, cyclo(His-Gly) and cyclo(His-Ala) (Lucietto et al., 2006).

A parameter routinely used to assess the ability of drug molecules to be absorbed and permeate through biological membranes to their site of action is the octanol/water partition coefficient or Log P. An ideal Log P for a drug molecule would be approximately 2, indicating that it has a slightly lipophilic character, enabling it to pass through lipid membranes. Drug molecules are able to pass through membranes through several mechanisms, but three of the primary mechanisms include passive diffusion (transcellular), passage through pores and junctions (paracellular) and transporter-mediated (active transport) as shown in Figure 1.1. Lack of active

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transporters and large molecular weight requires that drugs are able to passively diffuse through cells as their primary means of absorption (Smith et al., 2006).

Figure 1.1: Routes of transport of drugs across membranes (adapted from Smith et al.

(2006)).

The line between good permeability and poor solubility is a fine one and a balance between the hydrophilic and lipophilic nature of drug is important as shown in Figure 1.2 (Kerns and Di, 2008).

Figure 1.2: Hypothetical influence of Log P on oral bioavailability of a drug (Kerns and Di,

2008). TRANSCELLULAR (Passive diffusion) PARACELLULAR (via pores) ACTIVE TRANSPORTERS

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The theoretical Log P for cyclo(His-Gly) and cyclo(His-Ala) (Section 3.1.1) were calculated to be -1.093 and -1.264 respectively, which, according to Figure 1.2, would classify them as having poor membrane permeability. Despite this, Lucietto and colleagues (2006) found these compounds to possess significant antineoplastic activity.

Log D, similar to Log P but taking pH into consideration, can evaluate the permeability of a substance at a physiological pH of 7.4. Table 1.1 illustrates the impact the Log D parameter could have on drug-like properties of a substance (Kerns and Di, 2008).

Table 1.1: Influence of Log D of drugs on drug-like properties such as solubility,

permeability and metabolism (Adapted from Kerns and Di, 2008).

Log D Impact on Drug-like Properties

<1

Solubility high

Permeability low by passive transcellular diffusion Permeability possible via paracellular diffusion if MW < 200 1 – 3 Solubility moderate Permeability moderate Metabolism low 3 – 5 Solubility moderate Permeability high

Metabolism moderate to high >5

Solubility low Permeability high Metabolism high

Physicochemical properties of these molecules indicate limitations with respect to their solubility in biological solutions as well as limited cell permeation (Prasad, 1995). The potential tumour suppressive properties as well as the physicochemical

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limitations of this group of molecules make them ideal test candidates in the development of a targeted liposomal drug delivery system.

Liposomes can be characterized as colloidal particles comprising mainly of phospholipids and cholesterol. They form spherical vesicles consisting of a phospholipid bilayer, much like that of normal cell membranes (Malam et al., 2009). These particles have a myriad of possibilities with respect to pharmaceutical applications due to their variation in composition, size and structure (Endruschat and Henschke, 2000). Incorporation of many different types of therapeutic molecules such as simple organic drug compounds as well as protein-based and gene therapeutics aim to enhance their actions through the attainment of a number of goals, including the enhancement of their pharmacokinetics, decreased metabolic degradation and improved targeting, thereby enhancing their efficacy and decreasing potential side effects (Vemuri and Rhodes, 1995).

The properties of the liposomes can be vastly altered by changing the composition of the phospholipids, improving their pharmacokinetics. For instance, the incorporation of polyethylene glycol (PEG) modified lipids in the synthesis of liposomes has been shown to decrease antibody-mediated elimination of these particles, hence increasing circulation time of the liposomes (Li et al., 2002). The production of liposomes can take many routes, resulting in a final product that shows significantly different characteristics with respect to vesicle size, number of bilayers and their morphology, bilayer characteristics and surface charge characteristics. A few basic methodologies for liposome production include thin-film hydration of a lipid layer to form multilamellar vesicles, extrusion techniques to form large unilamellar vesicles, preparation by sonication to form small unilamellar vesicles as well as the formation of giant unilamellar vesicles through reverse-phase evaporation. Most of these methods are described as being laboratory bench scale production methods, however, scale-up to production scale is possible for most methodologies

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(Drummond et al., 1999; Endruschat and Henschke, 2000; Agarwal et al., 2001; Malam et al., 2009). These liposomal particles may vary considerably with respect to the degree of drug encapsulation, stability, drug release, interaction with target sites and circulation time (Vemuri and Rhodes, 1995). These variations in properties are cause for concern in the pharmaceutical industry where standardization is essential. It would therefore seem logical to apply some sort of statistical methodology, such as experimental design in order to better optimize formulations while gaining a deeper understanding of the parameters that may affect their properties.

Another major advancement in liposome technology has been the development of immunoliposomes that contain surface monoclonal antibodies (mAb) specific to proteins over expressed in the target cell. Examples of such proteins include HER2, an oncogene that is found to be over expressed in tumour cells. Efforts have shown that the incorporation of anti-HER2 mAb fragments into the lipid bilayer of sterically stabilized liposomes (forming immunoliposomes) has shown selective uptake by tumour cells (Park et al., 2001). This concept of sterically stabilized, long circulating immunoliposomes that are specific to their site of action may prove extremely beneficial in the treatment of malignancies, where current therapeutic options result in severe debilitating side effects. Besides tethering antibodies to liposomes, any molecule that can target proteins that are over expressed in tumour cells could prove beneficial. One such molecule is folic acid, as folate receptors are often found to be over expressed in tumours and this approach has been used in assisting with the targeting of liposomes (Lee and Low, 1995; Gabizon et al., 1999; Lu and Low, 2003).

1.1 AIMS AND OBJECTIVES