Cécile Arellano

Tyrosine kinase inhibitors pazopanib and sunitinib are both used to treat advanced renal cell carcinoma but expose patients to an increased risk of hepatotoxicity. We have previously identified two aldehyde derivatives for pazopanib and sunitinib (P-CHO and S-CHO, respectively) in liver microsomes. In this study, we aimed to decipher their role in hepatotoxicity by treating HepG2 and HepaRG hepatic cell lines with these derivatives and evaluating cell viability, mitochondrial dysfunction, and oxidative stress accumulation. Additionally, plasma concentrations of P-CHO were assessed in a cohort of patients treated with pazopanib. Results showed that S-CHO slightly decreased the viability of HepG2, but to a lesser extent than sunitinib, and affected the maximal respiratory capacity of the mitochondrial chain. P-CHO decreased viability and ATP production in HepG2. Traces of P-CHO were detected in the plasma of patients treated with pazopanib. Overall, these results showed that P-CHO and S-CHO affect hepatocyte integrity and could be involved in the pazopanib and sunitinib hepatotoxicity.

Tyrosine kinase inhibitors (TKI) are targeted anticancer drugs that have been successfully developed over the past two decades. To date, many of them (around 70%) require warnings for liver injury and five of them, including pazopanib and sunitinib, have Black Box Warning (BBW) labels. Although TKI-induced hepatotoxicity is the first cause of drug failures in clinical trials, BBW labels and market withdrawals, the underlying mechanisms remain unclear. However, the recent discovery of new reactive metabolites (RM) with aldehyde structures during pazopanib and sunitinib metabolism offers new perspectives for investigating their involvement in the toxicity of these two TKI. These hard electrophiles have a high reactivity potential toward proteins and are thought to be responsible for cytochrome P450 inactivation, drug-drug interactions (DDI) and liver toxicity. We report here, for the first time, the presence of these aldehyde RM in human plasma samples obtained during drug monitoring. Docking experiments in the CYP3A4 active site were performed and showed that pazopanib and sunitinib fitting in the catalytic site are in accordance with their regioselective oxidation to aldehydes. They also suggested that aldehyde RM may react with lysine and arginine residues. Based on these results, we studied the reactivity of the aldehyde RM toward lysine and arginine residues as potential targets on the protein framework to better understand how these RM could be involved in liver toxicity and drug-drug interactions. Adduct formation with different hepatic and plasma proteins was investigated by LC-MS/MS and adducts between pazopanib or sunitinib aldehyde derivatives and lysine residues on both CYP3A4 and plasma proteins were indeed shown for the first time.

Tyrosine kinase inhibitors (TKI) are small heterocyclic molecules targeting transmembrane and cytoplasmic tyrosine kinases that have met with considerable success in clinical oncology. TKI are associated with toxicities including liver injury that may be serious and even life‐threatening. Many of them require warnings in drug labeling against liver injury, and five of them have Black Box Warning (BBW) labels. Although drug‐induced liver injury is a matter of clinical and industrial concern, little is known about the underlying mechanisms that likely involve reactive metabolites (RM). RM are electrophiles or radicals originating from the metabolic activation of particular functional groups, known as structural alerts or toxicophores. RM are able to covalently bind to proteins and macromolecules, causing cellular damage and even cell death. If the adducted protein is the enzyme involved in RM formation, time‐dependent inhibition of the enzyme—also called mechanism‐based inhibition (MBI) or inactivation—can occur and lead to pharmacokinetic drug‐drug interactions. To mitigate RM liabilities, common practice in drug development includes avoiding structural alerts and assessing RM formation via RM trapping screens with soft and hard nucleophiles (glutathione, potassium cyanide, and methoxylamine) in liver microsomes. RM‐positive derivatives are further optimized to afford drug candidates with blocked or minimized bioactivation potential. However, different structural alerts are still commonly used scaffolds in drug design, including in TKI structures. This review focuses on the current state of knowledge of the relations among TKI structures, bioactivation pathways, RM characterization, and hepatotoxicity and cytochrome P450 MBI in vitro.

In addition to the effect of cytochrome P450 (CYP) 2D6 genetic polymorphisms, the metabolism of tamoxifen may be impacted by other factors with possible consequences on therapeutic outcome (efficacy and toxicity). This analysis focused on the pharmacokinetic (PK)‐pharmacogenetic evaluation of tamoxifen in 730 patients with adjuvant breast cancer included in a prospective multicenter study. Plasma concentrations of tamoxifen and six major metabolites, the genotype for 63 single‐nucleotide polymorphisms, and comedications were obtained 6 months after treatment initiation. Plasma concentrations of endoxifen were significantly associated with CYP2D6 diplotype (P < 0.0001), CYP3A4*22 genotype (P = 0.0003), and concomitant intake of potent CYP2D6 inhibitors (P < 0.001). Comparison of endoxifen levels showed that the CYP2D6 phenotype classification could be improved by grouping intermediate metabolizer (IM)/IM and IM/poor metabolizer diplotype into IM phenotype for future use in tamoxifen therapy optimization. Finally, the multivariable regression analysis showed that formation of tamoxifen metabolites was independently impacted by CYP2D6 diplotype and CYP3A4*22, CYP2C19*2, and CYP2B6*6 genetic polymorphisms.

Oxidation of two tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib, using a chemical catalytic system able to mimic the cytochrome P450 type oxidation, allowed us to prepare and evidence putative reactive/toxic metabolites of these anticancer drugs. Among these metabolites, aromatic aldehyde derivatives were unambiguously characterized. Such biomimetic oxidation of TKI-type drugs was essential to facilitate the identification of low amounts of aldehydes generated from these TKIs when incubated with human liver microsomes (HLM), which are classical models of human hepatic metabolism. These TKI derivative aldehydes quickly react in vitro with amines. A similar reaction is expected to occur in vivo, and may be at the origin of the potentially severe hepatotoxicity of these TKIs.

Supported by a PHRC grant (#09-18-005) Background: The role of CYP2D6 genetic polymorphisms and plasma levels of active metabolites of tamoxifen (TAM) on clinical response and occurrence of side effects remains controversial. We conducted a prospective, adjuvant, multicentre, 3-year follow-up study of breast cancer patients in order to evaluate the relationships between pharmacogenetics, pharmacokinetics and toxicity of TAM and its metabolites (n=879) or aromatase inhibitors (AI, n=1098). The present report focuses on the evaluation at 6 months after inclusion of 864 patients treated with 20 mg/day TAM.The clinical results and the AI PG/PK analyses are described elsewhere (abstracts #851544 and #851525). Methods: Residual plasma concentrations for tamoxifen and its 6 major metabolites (endoxifen ENDO, 4-hydroxy-tamoxifen 4-OH-TAM, N-desmethyl TAM, TAM-N-oxyde, 4'-OH-TAM and Z'ENDO) at 6 months after start of treatment were measured by UPLC-MS/MS in 789 patients. Nine patients with TAM concentrations below the limit of quantification were excluded for non-compliance. SNP genotyping of 95 selected SNPs was performed on the Biomark (Fluidigm) in a microfluidic multiplex 96 dynamic array chip with Taqman assays and was available for 857 patients. Patients were classified according to their CYP2D6 metaboliser status (MS) (PM, IM, EM and UM) based on presence of functional, decreased function or no functional alleles (*4, *6, *7, *9, *10, *17, *41) and number of CYP2D6 copies (*5 or duplication). Metabolic ratios (MR) were calculated for TAM/4-OH-TAM, TAM/N-desmethyl tamoxifen (NDT), NDT/ENDO and 4-OH-TAM/ENDO. Anti-estrogenic activity score (AAS) was calculated according to a recently proposed algorithm (De Vries Schultink et al.,Breast Cancer Res Treat. 2017).Toxicity was measured as a binary outcome (first occurrence or worsening of hot flushes, fatigue, depression, pain, arthralgia, vaginal dryness). All genetic associations were adjusted for multiple testing. Results: ENDO concentration and AAS increased significantly with CYP2D6 MS (p=1798 (i.e., threshold previously associated with recurrence-free survival RFS by De Vries et al. 2017) was 6%, 50%, 84% and 91% of patients respectively classified as PM, IM, EM and UM. Side effects were not significantly associated with higher levels of TAM metabolites concentrations. After correction for multiple testing, SNPs or CYP2D6 MS were not significantly associated with occurrence or worsening of adverse events, premature treatment discontinuations or switch due to toxicity within the first 6 months. Conclusions: In this large prospective study, we quantified the impact of PG on TAM PK and AAS, previously shown to predict RFS. Although the toxicity observed after 6 months of TAM does not seem correlated with PK or PG, these relationships need to be re-evaluated during the 3-year follow-up. Citation Format: White-Koning M, Arellano C, Le Morvan V, Evrard A, Puzskiel A, Vachoux C, Dauba J, Houyau P, Poublanc M, Robert J, Boyer J-C, Roché H, Thomas F, Chatelut E. Impact of genetic polymorphisms on plasma levels of tamoxifen and its metabolites and toxicity: 6-months results of the adjuvant breast cancer longitudinal PHACS study (NCT01127295) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-12-03.