Clozapine metabolism and cardiotoxicity: A prospective longitudinal study.

BACKGROUND: Clozapine-induced myocarditis and cardiomyopathy are difficult to detect clinically and may be fatal if not detected early. The current/routine biomarkers for clozapine-induced myocarditis are non-specific indicators of inflammation (C-reactive protein) or cardiomyocyte damage (troponins I and T) that lack sensitivity, and for which changes often arise too late to be clinically useful. METHODS: The Clozapine Safety Study was a prospective, longitudinal, observational study to determine what, if any, the plasma concentrations of clozapine, N-desmethylclozapine, and clozapine-N-oxide in patients contribute to cardiotoxicity. Samples were collected and analysed using liquid chromatography mass spectrometry over a 41-month period from patients in the Auckland District Health Board. RESULTS: Sixty-seven patients were included. Six patients were diagnosed with myocarditis; none were diagnosed with cardiomyopathy in the study period. In patients not undergoing dose titration, clozapine biotransformation may shift to the N-oxide pathway rather than the N-desmethyl pathway with increasing dose. During dose titration, the timeframe in which myocarditis occurs, the rate of increase in the plasma concentration of clozapine-N-oxide, as well as the ratio of N-oxidation relative to N-desmethylation, were significantly higher in patients diagnosed with myocarditis. CONCLUSIONS: The assessment of clozapine-N-oxide formation, and N-oxidation relative to N-desmethylation ratios during treatment, may help identify a biomarker to aid the early detection of patients at risk of developing clozapine-induced cardiotoxicity.

Ethnic disparity in clozapine dosing and cardiotoxicity in New Zealand.

AIMS: To investigate ethnic disparities in the treatment and incidence of cardiotoxicity for patients prescribed clozapine in New Zealand. METHODS: A post hoc analysis was undertaken using data from four studies investigating clozapine cardiotoxicities in New Zealand: two population studies (one prospective, one retrospective) conducted in the Auckland District Health Board (2011-2017), and two studies of coronial autopsy records (2001-2016). The relationship between ethnicity and cases (N=26) of myocarditis and/or cardiomyopathy was examined in comparison to non-cases in the rest of the study population (N=161). Patient demographics, comorbidities, and risk factors were investigated for any associations with ethnicity, where data was available. RESULTS: Maori and Pacific patients were over-represented in the population studies. Moreover, across the cohorts investigated 46% of myocarditis and cardiomyopathy cases were Maori. In contrast, only one case (4%) of cardiomyopathy was identified in a patient of Pacific descent. Where clozapine titration data was available, the rate of dose escalation was higher in Maori and Pacific peoples, as was the cumulative dose received before the first case of cardiotoxicity (day 13 of dose titration). Maori patients were more likely to be co-medicated with sodium valproate than others during clozapine titration, and sodium valproate was also significantly associated with myocarditis in these patients. CONCLUSIONS: The factors underpinning the more rapid titration of Maori and Pacific patients onto clozapine and the increased use of concomitant sodium valproate in Maori are unclear. While the latter may explain the heightened risk of clozapine-induced myocarditis in Maori, further work is required to mitigate the effects of this inequity on the safe use of clozapine in New Zealand.

A systematic review of inter-individual differences in the DNA repair processes involved in melphalan monoadduct repair in relation to treatment outcomes.

PURPOSE: Melphalan is a bifunctional alkylating agent that elicits its cytotoxic activity by rapidly forming an initial DNA monoadduct, which then produces an inter-strand crosslink. Most studies exploring the role of inherited differences in DNA repair and melphalan outcomes focus on inter-strand crosslink repair, however, monoadduct repair likely plays a key role since it minimises the ultimate production of these crosslinks. The purpose of this systematic review was to assess evidence of an association between variation in monoadduct repair pathways and melphalan response. METHODS: A literature search was undertaken using Medline, Embase, Scopus and PubMed databases. Duplicates were removed and only full-text articles were included. To be included for critique in this systematic review, articles were assessed for relevance using strict inclusion/exclusion criteria. RESULTS: Fourteen studies were identified that involved patients treated with melphalan, however, in 3, only a minority of the cohort received melphalan. Across the remaining 11 studies, 61 genes/proteins in DNA monoadduct repair pathways were assessed. Both germline SNP (CDKN1A, ERCC1, ERCC2, ERCC4, ERCC6, EXO1, MLH1, MNAT1, MUTYH, PARP4, PCNA, POLE, POLR1G, RAD23B, RFC1, RFC3, RPA1, RPA3, TREX1, UNG, XPC, XRCC1) and somatic expression (CDKN1A, PARP1, PCNA, MGMT, RECQL, RFC5) were associated with melphalan outcomes in ≥ 1 study. CONCLUSION: It appears that inherited germline differences in monoadduct repair genes may be a risk factor for poor outcomes. However, the diversity of study design, patient cohorts, genes assessed and lack of replication, preclude any meta-analysis. Further prospective studies are required to validate these findings.

Comparison of a thymine challenge test and endogenous uracil-dihydrouracil levels for assessment of fluoropyrimidine toxicity risk.

PURPOSE: Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. METHODS: Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC-MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. RESULTS: There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). CONCLUSIONS: The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.

Circulating microRNA as biomarkers of clozapine-induced cardiotoxicity.

Purpose: This work investigated the utility of circulating microRNA (miRNA) as biomarkers of clozapine (CLZ)-induced cardiotoxicities: serious adverse events with an unusually high incidence in Australia and New Zealand.Methods: Global plasma miRNA expression was analysed by microarray in patients taking CLZ, to investigate differential expression between CLZ-induced cardiotoxicity cases (n = 6) and matched control patients (n = 12). The results were validated by RT-qPCR using a panel of 17 miRNA, and their expression was examined in both CLZ-naïve healthy volunteers (n = 12) and an expanded cohort of CLZ-taking patients (n = 21). Temporal changes were also examined in two healthy volunteers and two CLZ-induced cardiotoxicity patients.Results: No miRNA were differentially expressed between cases of CLZ-induced cardiotoxicity and control patients. Circulating levels of several miRNA were significantly altered in CLZ-taking patients compared to healthy volunteers, with miR-16-5p, miR-25-3p, miR-92a-3p, miR-320a-3p, and miR-486-3p upregulated and miR-22-3p, miR-126-3p, and miR-142-3p downregulated in the patients. Five of these (miR-16-5p, miR-22-3p, miR-92a-3p, miR-126-3p, miR-142-3p) were stably expressed over time in both CLZ-induced cardiotoxicity patients and CLZ-naïve healthy volunteers.Conclusions: Plasma miRNA are not useful biomarkers of CLZ-induced cardiotoxicity, however patients taking CLZ have significantly altered circulating miRNA compared to healthy volunteers.

A case-control study to assess the ability of the thymine challenge test to predict patients with severe to life threatening fluoropyrimidine-induced gastrointestinal toxicity.

AIMS: A previous study suggested that a thymine (THY) challenge dose could detect aberrant pharmacokinetics in known cases of fluoropyrimidine toxicity compared with healthy volunteers. The preliminary data suggested that urine sampling also could detect this aberrant disposition. The aim of this case-control study was to assess the ability of the urinary THY challenge test to discriminate cases of severe gastrointestinal toxicity in a cohort of patients treated with 5-fluorouracil or capecitabine. METHODS: Patients (n = 37) received a 250 mg (per os) dose of THY and a cumulative urine sample was collected for 0-4 h. The urinary amounts of THY and metabolite dihydrothymine (DHT) were determined by liquid chromatography/mass spectrometry. Genomic DNA was analysed for DPYD gene variants. Renal function was estimated from blood creatinine levels. Cases (n = 9) and noncases (n = 23) of severe (grade ≥ 3) gastrointestinal toxicity were defined based on Common Terminology Criteria for Adverse Events. RESULTS: The median THY/DHT ratios were 6.2 (interquartile range 2.9-6.4) in cases, including the 2 patients who were DPYD heterozygous carriers. However, this was not significantly different (P = .07) from the THY/DHT in noncases (median 2.6, interquartile range 2.8-4.2). Although creatinine clearance was lower (P = .001) in cases, renal function could not discriminate cases from noncases. However, logistic regression analysis using both of these explanatory variables could discriminate most cases (receiver operating characteristic area 0.8792, 95% confidence interval 0.72-1.00). CONCLUSIONS: The THY challenge test combined with a patient's renal function may be useful as a phenotypic diagnostic test to detect risk of life-threatening fluoropyrimidine gastrointestinal toxicity.

A higher throughput assay for quantification of melphalan-induced DNA damage in peripheral blood mononuclear cells.

Inter-individual differences in DNA adduct formation and repair influence the response to melphalan treatment, however, further clinical investigation of this variability requires a logistically feasible and reproducible bioassay. Our improved fluorescence-based QPCR-block assay is robust, has good precision, and improved throughput. It also incorporates direct PCR amplification from melphalan exposed PBMC using commercially available blood tubes and extraction kits to maximise the utility of this assay for future clinical studies. Using this assay we have demonstrated reproducible inter-individual differences in melphalan-induced QPCR-block across individual PBMC donors. As proof-of-principle we assessed nine healthy donors and found a 7.8 fold range in sensitivity following exposure of PBMC ex vivo. This likely reflects differences in melphalan transport into cells as well as differences in DNA adduct repair proficiency. This improved bioassay may be useful for assessment of these processes in patients about to receive melphalan treatment.

A simple ex vivo bioassay for 5-FU transport into healthy buccal mucosal cells.

PURPOSE: Fluorouracil (5-FU), a chemotherapeutic agent widely used in the treatment of numerous common malignancies, causes oral mucositis in a proportion of patients. The contribution of drug transport processes to the development of this toxicity is currently unknown. This work aimed to establish and optimise a simple phenotyping assay for 5-FU uptake into primary buccal mucosal cells (BMC). METHODS: The uptake kinetics of radiolabelled 5-FU were determined in pooled BMC freshly collected from healthy volunteers. The inter- and intra-individual variability in 5-FU uptake was then assessed across a cohort that included both healthy volunteers and cancer patients. RESULTS: 5-FU uptake into pooled primary BMC was both time and concentration dependent. An Eadie-Hofstee analysis suggested two components; a high-affinity (KM = 3.3 µM) low-capacity ([Formula: see text] = 57.8 pmol min-1 105 viable cells-1) transporter, and a high-capacity ([Formula: see text] = 1230 pmol min-1 105 viable cells-1) low-affinity (KM = 3932 µM) transporter. There was 180-fold variation in the rate of 5-FU uptake into BMC (0.10-17.86 pmol min-1 105 viable cells-1) across the 34 subjects (healthy participants N = 24, cancer patients N = 10). Notably, retesting of a subset of these participants (N = 16) multiple times over a period of up to 140 days demonstrated poor stability of the uptake phenotype within individuals. CONCLUSION: The uptake of 5-FU into healthy oral mucosal cells is a highly variable process facilitated by membrane transporters at pharmacologically relevant concentrations. This bioassay is simple, minimally invasive, and suitable for phenotypic analysis of drug transport in healthy primary cells.