Total, renal and hepatic clearances of doxorubicin and formation clearance of doxorubicinol in patients with breast cancer: Estimation of doxorubicin hepatic extraction ratio
Graphical abstract
Introduction
Doxorubicin (DOX), an anthracycline derived from daunorubicin, has remained in the past 50 years as an essential component of the chemotherapy regimen of different types of cancer, including leukemia, small cell lung cancer, osteosarcomas, Kaposi’s sarcoma, Hodgkin’s and non- Hodgkin’s lymphomas, breast, and ovarian cancers. DOX is a topoisomerase II inhibitor, as well as a DNA intercalator agent, further inhibiting DNA- and RNA polymerases. Besides being a cytotoxic drug, the main adverse effect of the treatment with DOX is cardiomyopathy due to its main metabolite doxorubicinol (DOXOL) accumulation in the cardiac tissue, where it is believed to inhibit ion pumps in cardiomyocytes [[1], [2], [3], [4]].
The hepatobiliary route predominantly mediates DOX clearance, and more than 50 % of the unchanged or metabolized drug is excreted into bile within seven days. DOX undergoes phase I reduction to its main metabolite DOXOL. This stage is mostly dependent on carbonyl reductases 1 and 3 (CBR1, CBR3) and, to a lesser extent, aldo-keto reductase (AKR1C). Although the metabolite DOXOL in mice has been associated with increased cardiac toxicity, it is 1.5-fold less potent than the unchanged drug in growth inhibition of tumor cell lines and 10-fold less in growth inhibition human marrow erythroid and myeloid progenitors cells. Subsequent metabolism of DOX and DOXOL comprises glycosylation, O-demethylation and O-glucuronidation. The renal clearance of DOX is low, with approximately 12 % of the dose excreted in the urine for six days [5].
The pharmacokinetics of DOX shows high interindividual variability and is dependent on the polymorphisms of a variety efflux and influx transporters, receptors, and metabolizing enzymes, such as CBR1 and CBR3. It is suggested that pharmacogenetics accounts for 20–95 % of DOX efficacy and toxicity in treated patients [[5], [6], [7]].
DOX pharmacokinetics is linear in the range of 20–60 mg⋅m−2 [2]. Due to DOX wide distribution to tissue, it exhibits a three phases decay profile, and a total body clearance reported as 62.4 L⋅h−1 [4]. DOX distribution (t½ α), rapid elimination (t½ β), and terminal elimination half-lives (t½ γ) have been reported to be 12 min, 3.3 h, and 29.6 h, respectively [8].
DOX pharmacokinetics has been described in populations of patients treated for different sorts of tumors, including breast cancer [[1], [2], [3], [4],9,10]. However, these studies usually report the pharmacokinetics of DOX alone in plasma, and some include its metabolite DOXOL. Our study assessed DOX and DOXOL pharmacokinetics in a homogeneous population of young female adults making use of a standardized drug association, evaluated in the very first cycle the chemotherapy treatment, in order to avoid inducing effects on cell membrane drug transporters or enzymatic systems. Since the variability in the pharmacokinetics of DOX and its metabolite DOXOL have been considered to contribute to the wide inter-individual variability in DOX efficacy and toxicity [[5], [6], [7]], the detailed pharmacokinetic data in the present study allowed us to calculate total, hepatic and renal clearances of DOX, formation clearance of DOXOL, the fraction of the dose excreted into the urine as its unchanged form and as the main metabolite, and the unbound fractions of DOX and DOXOL, as well as to estimate the hepatic extraction ratio of DOX. Previous studies assessed DOX hepatic extraction ratio (Ehep) in rats [11,12], pigs [13], or in humans with breast adenocarcinoma metastatic to the liver [14], indicating the need to obtain Ehep reflecting a human liver in normal conditions. To our knowledge, the complete DOX pharmacokinetics has not yet been investigated but remains as an essential contribution to dosage adjustment.
The protein binding of DOX widely varies from 50 to 85 % [2]. DOX and DOXOL protein binding has been assessed in the past by equilibrium dialysis [15]. Other studies applied the ultrafiltration technique, but assessed solely the unchanged drug, not its metabolite, in artificial buffers [16] or purchased human plasma [17], rather than in real patients’ samples. To the best of our knowledge, this is the first report of ultrafiltration procedure, and liquid chromatography coupled to mass spectrometry (LC–MS/MS) was applied to quantify the unbound fraction of both DOX and DOXOL in clinical pharmacokinetics study samples.
The present study reports the pharmacokinetics of DOX and DOXOL as total and unbound concentrations in plasma and urine of twelve patients with breast cancer. The present study also provides data of protein binding for both DOX and DOXOL, as well as the determination of total, renal and hepatic clearances, in addition to the estimation of the hepatic extraction ratio of DOX.
Section snippets
Standard solutions and reagents
Water was obtained from Synergy® Water Purification System (Millipore, Burlington, MA, USA.). Methanol and acetonitrile were obtained from J. T. Baker (Phillipsburg, NJ, USA) and isopropanol from Fischer-Scientific (Fair Lawn, NJ, USA) as chromatographic grade. Formic acid (90,1%) was obtained from J. T. Baker as analytical grade.
Stock DOX (doxorubicin hydrochloride 98 %, Sigma-Aldrich, St. Louis, MO, USA) and DOXOL (doxorubicinol hydrochloride 90 %, Toronto Research Chemicals Inc., North York,
Results
Fig. 1 shows the protonated ions [M+H]+ and their respective ion products of DOX (m/z 544.3 → 397.1), DOXOL (m/z 546.5 → 399.1) and IS (m/z 528.4 → 321.1) infused into the mass spectrometer system. The chromatograms of DOX and DOXOL Sequential analyses in plasma, plasma ultrafiltrate and urine are presented in Fig. 2, Fig. 3, Fig. 4, respectively. The anthropometric, biochemical and hematological parameters are presented in Table 1, and all values are within the reference ranges. The confidence
Discussion
The present study reports the pharmacokinetics of both DOX and DOXOL for the first time in a homogeneous population of young female adults making use of a standardized drug association, evaluated in the very first cycle the chemotherapy, using plasma and urine data that allowed the calculation of the renal clearance of DOX, the formation clearance of DOXOL and the hepatic clearance of DOX. The extensive data availability also made it possible to estimate the Ehep of DOX for the investigated
Conclusions
The pharmacokinetics profile of both DOX and DOXOL, evaluated in the first cycle of chemotherapy for breast cancer treatment, showed high variability (AUC0–∞ values with a geometric coefficient of variation of approximately 215 %). The geometrics means were 0.26 for DOXOL/DOX AUC ratio, 15.42 % and 17.07 % for unbound fractions of DOX and DOXOL, respectively, 30.70 L⋅h−1 for total clearance, 0.66 L⋅h−1 for renal clearance, 29.97 L⋅h−1 for hepatic clearance and 0.39 L⋅h−1 for the formation
CRediT authorship contribution statement
Leandro Francisco Pippa: Conceptualization, Investigation, Formal analysis, Writing - original draft. Milena Locci de Oliveira: Investigation. Adriana Rocha: Investigation. Jurandyr Moreira de Andrade: Conceptualization, Resources. Vera Lucia Lanchote : Conceptualization, Writing - original draft, Supervision, Funding acquisition.
Acknowledgements
The authors would like to thank Danilo Augusto Alves Pereira (Mass Spectrometry Applications Specialist, Waters Technologies do Brasil) for his valuable support. The present study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES, Finance Code 001), by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 304209/2011-2), and by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process 2014/06846-1).
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