ABSTRACT
Antibody drug conjugates (ADCs) are one of the most promising technologies to treat cancer as they combine the specificity of an antibody with the high potency of a cytotoxic molecule such as tomaymycin derivatives, which are DNA-interactive antitumor antibiotics previously isolated from bacterial broth. The multistep chemical synthesis of some tomaymycin derivatives is complicated because their structures contain a reactive imine bond. Therefore, we turned to biosynthesis to obtain 14 C radiolabelled tomaymycin derivative to support ADME studies. Following Hurley's work (J. Antibiotics 1977, 30, 349-370; Antimicrob. Agents Chemother. 1979, 15, 42-45; Acc. Chem. Res. 1980, 13, 263-269), the 14 C radiolabel was incorporated efficiently in one step from radiolabelled tyrosine using the strain Streptomyces sp. FH6421. This process has been further optimized by using anthranilic acid as radiolabelled precursor, leading to one of the highest incorporation levels of radiochemical precursors published to date. This biosynthetic strategy is the fastest way to access such radiolabelled precursors.
Subject(s)
ImmunoconjugatesABSTRACT
Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of [³³P]-2-methylthio-ADP on P2Y12-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC50: CHO-P2Y12: 0.12 µM; PRP: 0.97 µM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.