Biological evaluation of PEG-based conjugates offering localisation of conjugated drugs at the desired compartment

Polymer-drug conjugates (PDCs) are drug delivery systems in which drug molecules are covalently linked to hydrophilic polymers. These systems have initially been developed to improve the anticancer efficacy and safety of chemotherapeutic agents based on the enhanced permeability and retention (EPR) effect. In these applications, the PDCs require the release of the conjugated drugs within the tumour tissues. However, there are few cases in which the release of the drug is not the aim. For example, Movantik®, the only available PDC on the market, was developed to prevent the unfavourable penetration of naloxol across the bloodbrain barrier (BBB) while retaining its effect in the intestines. This system was designed as a non-prodrug employing PEG (as a polymer) and an ether bond as a linker. The current project focuses on the biological evaluation of utilising the PEGylation strategy to develop non-prodrug PDCs of haloperidol providing compartmentalisation of haloperidol at the intended site of action, which could allow potential non-CNS applications of the conjugated haloperidol. In Chapter 1, a general introduction to the biological barriers, PDCs and their applications are discussed. The rationale behind this project is also provided. Chapter 2 identifies the various non-cancer applications of PDCs and key features influencing the design of such systems for specific diseases are recognised. Chapter 3 represents a systematic analysis of clinical studies of nanomedicines (including PDCs) used to treat solid tumours of different origins based on the EPR effect. From all studied cancers, ovarian, brain, stomach, breast, colon and colorectal, and pancreatic cancers showed the highest levels(up to >8-fold) of accumulation of nanomedicines compared to other tumours. Moreover, tumour size was another factor that impacted the accumulation of nanomedicines, with high levels of accumulation observed in large tumours (~5-fold) compared to medium or very large tumours. Other parameters such as perfusion levels, the presence of angiogenesis and inflammation in tumour tissues were identified as factors that might influence the magnitude of the EPR effect and, as a consequence, the accumulation of nanomedicines within tumour tissues. The chapter proposes two strategies to select patients who could potentially benefit from the increased accumulation of nanomedicines based on the EPR effect, which might enhance the clinical outcomes of using nanomedicines as personalised anticancer agents. In Chapter 4, the feasibility of utilising the PEGylation strategy to prevent haloperidol diffusing through the BBB is demonstrated using different in silico, in vitro and in vivo approaches. The synthesis of a PEG-haloperidol conjugate was carried out (using PEG 6000 Da) by applying a protocol slightly modified based on our previously reported protocol. The in vitro binding assay indicated that the PEG-haloperidol conjugate had a retained activity through D2 receptors, however, this was lower than that of the free drug (~18-fold at 10 nM). Molecular docking (MD) studies indicated that the conjugates exhibited a retained binding affinity for the D2 receptors, and the binding pattern of the conjugate in the binding pocket explained the loss of the biological activity of the conjugates compared to the free haloperidol. In vivo studies on rats revealed that rats treated with PEG-haloperidol were not cataleptic in contrast to the free haloperidol treated rats, which indicated the prevented crossing of PEG-haloperidol into the CNS. Chapter 5 describes potential applications of PEG-haloperidol conjugates in the field of cancer (acting via s receptors) assessed using in vitro and in silico approaches. PEGs of two MWs (2000 and 6000 Da) were synthesised. PEG (2000 Da) enhanced the haloperidol's loading in the conjugate (~25% w/w) by ~3-fold compared with the loading of haloperidol in PEG (6000 Da) conjugate. The cytotoxicity of the conjugates was evaluated using breast cancer cell lines (MCF-7 and MDA-MB 231). The application of the conjugates as potential antiproliferative agents was limited as their IC50 values were > 100 µM (compared to ~50 µM for the free haloperidol) for both cell lines. The conjugates were also tested for potential antimigratory activity in vitro on vascular endothelial cells (HUVECs). The conjugates significantly inhibited the VEGF-stimulated migration of HUVECs (> 65% inhibition) although at a lower level compared to the free haloperidol (91% of inhibition). MD studies were performed and explained the loss of the biological activity of the conjugates compared to free haloperidol. Chapter 6 indicates a preliminary evaluation of potential cardiovascular applications of PEG-haloperidol by studying its effects on human platelets' aggregation induced by CRP-XL or ADP. The results indicated that free and conjugated haloperidol (at all tested concentrations) did not significantly abrogate the platelets aggregation stimulated by the CRP-XL (mediated by GPVI receptors). Moreover, haloperidol and PEG-haloperidol inhibited, however, not significantly, ADP-induced aggregation of human platelets at concentrations ³12.5 µM haloperidol equivalent, probably through P2Y1 receptors. However, further studies by employing other agonists and/or increasing the incubation time, and using different methodologies are required to identify the final conclusion. Chapter 7 represents the feasibility of using PEG-based PDC (designed as a non-prodrug system) to decrease or avoid the transfer of conjugated drugs through the human placenta. PEG, as a polymeric carrier, did not significantly affect the apoptosis or proliferation rates within placental explants when incubated up to 48 h, as indicated via immunohistochemistry staining. No signs of necrosis were observed when the explants were challenged with PEG as the released levels of lactate dehydrogenase from the explants did not significantly change. Treatment with PEG did not alter the normal function of the placental tissues where the secreted levels of hCG hormone from the explants were not significantly influenced by the polymer. Moreover, the cellular uptake studies of the PEG-Cy5.5 (dye) and PEG-haloperidol conjugates, used as model drugs, using fluorescent microscopy and RP-HPLC, respectively, showed complete absence of PEG-Cy5.5 from the placental tissues and limited uptake of PEG-haloperidol by the tissues compared to the free Cy5.5 and haloperidol, respectively. This indicated the potential efficiency of PEGylation strategy to design non-prodrug systems to treat illnesses during pregnancy without inducing negative effects on the developing fetus. In Chapter 8, the key findings of this PhD project are summarised, critical evaluation of work-related aspects and potential future work is suggested. Specifically, taken together, the data presented in this thesis demonstrated the feasibility of using PEGylated macromolecules (designed as non-prodrug systems) to reduce or prevent the transfer of conjugated drugs across biological barriers while retaining their activity. This strategy would form a platform to design drug delivery systems for applications where specific compartmentalisation of the effects of drugs is required. Future work will look to investigate this further by exploring PEGylated systems of therapeutic agents of different classes for their potential clinical applications.