Chemical, metabolic and structure-activity relationships to probe abacavir toxicity

• Main Author: Yang, Emma
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2014
• Subjects: Design; Metabolism; Metabolites

Adverse drug reactions (ADRs) are responsible for an increasing number of hospitalised patients, with the large majority of these ADRs classed as either type A or type B. Drug hypersensitivity reactions fall within the type B category and one such drug responsible for this form of ADR is abacavir (ABC). ABC, a nucleoside reverse transcriptase inhibitor, is used to treat the HIV-1 virus. It is responsible for a potentially life-threatening type IV hypersensitivity reaction which occurs in patients bearing the HLA-B*57:01 allele. Although many mechanisms have been proposed, it was the objective of this research to examine all these previous proposals to further extend and develop the mechanism of ABC toxicity. In Chapter 2, deuterated-ABC (D2-ABC) was designed and synthesised where the two 5'-H atoms were replaced with two 5'-D atoms. The design of this analogue was intended to retard the oxidative metabolism of ABC to its aldehyde and carboxylic acid metabolites. The synthesis of this compound was paramount to investigating this metabolism and through a series of metabolic experiments, described in Chapter 3, a kinetic isotope effect between ABC and D2-ABC was determined, ultimately showing an altered metabolism between the two compounds. To investigate binding of ABC within the HLA-B*57:01 protein, analogues of ABC, with alterations at varying positions within the molecule, were required. Using a racemic method, ABC enantiomers were synthesised and ABC’s enantiomer failed to stimulate T-cells in vitro. The creation of further analogues required the development of an asymmetric synthetic route. A total synthetic method was desired to synthesise intermediates to be used in future analogue synthesis. Finally, as described in Chapter 5, a range of 6-position analogues were designed, using a structure-activity relationship method, and synthesised, to further investigate the altered repertoire mechanism. These analogues, consisting of primary and secondary amine and oxy moieties, were subjected to in vitro immunological assays to determine their stimulation of T-cells. Additionally, the synthesised analogues were modelled in silico using molecular docking within the HLA protein. The in silico results assisted in explaining the basis of such T-cell activation/inactivation and will direct future analogue design. IC50 and EC50 values were determined for analogues that presented a negative T-cell response and a compound showing positive values was subjected to further pharmacokinetic testing. The oxidative metabolism of ABC was affected by isotopic substitution, but initial results have shown no altered T-cell stimulation of D2-ABC compared to ABC. This mechanism cannot be discarded, with further investigational work required. However, the synthesised 6-position analogues have assisted in further examining the altered repertoire mechanism and initial findings have enabled further understanding of the binding of ABC within HLA-B*57:01. This mechanism of ABC toxicity appears paramount to others proposed and the results presented in this thesis support this. Additional analogue synthesis and in vivo experiments will assist in confirming this further.