Practical fluorimetric assay for the detection of anticancer drug SN-38 in human plasma

https://doi.org/10.1016/j.jpba.2018.06.032Get rights and content

Highlights

  • SN-38 is the active metabolite of anticancer drug irinotecan and displays a strong fluorescence emission.

  • A fluorimetric assay for the quantification of SN-38 in denatured human plasma was developed.

  • A linear range between 10 and 500 ng mL−1 with intra- and inter-day precision and accuracy within the 15% tolerance range were obtained.

  • The comparison of the fluorescent assay for SN-38 with HPLC-MS showed a good agreement between the two techniques.

Abstract

The implementation of therapeutic drug monitoring in the routine clinical practice in oncology is mainly limited by the lack of therapeutic indexes for the majority of the anticancer drugs, and by the absence of suitable analytical tools, which can accurately quantify in real time the concentration of the administered drugs and their relevant metabolites in biological fluids. In this work, a simple and efficient fluorimetric determination of SN-38, the active metabolite of the anticancer drug irinotecan, was developed and applied to human plasma samples. The intrinsic fluorescence of SN-38 allowed its quantification in the range 10–500 ng mL−1 with a LOQ of 5.0 ng mL−1 and a LOD of 1.5 ng mL−1. Low interferences due to main metabolites of irinotecan and comedications, commonly associated with administration of irinotecan, were observed. A validation study, according to FDA and EMA guidelines for bioanalytical method validation, was carried out and, finally, blind samples were analyzed in parallel with a HPLC-MS method obtaining an excellent agreement between the two techniques.

Introduction

Irinotecan (CPT-11) is an anticancer drug that belongs to the class of camptothecins, and is widely used for the treatment of metastatic colorectal cancer [1,2]. CPT-11 is often used in combination with other chemotherapeutic drugs (i.e. FOLFIRI: infusional 5-fluorouracil (5-FU), leucovorin, and CPT-11) [3,4] or monoclonal antibodies (i.e. cetuximab and bevacizumab) [5,6].

Water soluble camptothecins show a broad spectrum of anti-neoplastic activity against various cancers due to their action as inhibitors of DNA topoisomerase-I [7,8]. The death of cancer cells results from stabilization of complexes between topoisomerase I and DNA, which are usually cleaved during replication, transcription, and repair of DNA [9,10]. Consequently, neoplastic cells decrease the ability to use genetic information to synthesize proteins for metabolic processes.

The pharmacological profile of CPT-11 (1) depends on several enzymes and active transport proteins involved in the regulation of intestinal absorption and hepatobiliary secretion mechanisms, as summarized in Scheme 1. After intravenous administration, the cleavage of the bulky side (1,4’-bipiperidine) chain from the camptothecin core structure by carboxylesterase enzymes converts 1 to its active metabolite SN-38 (2) [11], which displays a stronger cytotoxic activity (40- to 3000-fold) with respect to 1 in vitro models [12].

According to the metabolic pathway, other metabolites of 1 have been also identified, which include APC (3) and NPC (4), generated by cytochrome P-450 3A4 (CYP3A4). 2 is converted into SN-38 glucoronide (5) by hepatic UDP-glucuronyltransferase, and finally excreted through urine (Scheme 1) [13].

In humans, the maximum plasma concentration of 2 was found at about 1 h after a short (30 min) infusion [14] and 3 h after a 2 h continuous intravenous infusion [15]. Moreover, a high peak concentration and the AUC (area under the curve) of this metabolite in plasma correlate significantly with severe toxicity and is associated to the administered dose of 1 [16].

Therapeutic drug monitoring (TDM) represents a potential strategy to personalize the drug dosage, but its application in oncology is still very limited or absent, mainly due to the lack of plasma concentration range for many drugs, that should lead to the optimal therapeutic effects while reducing toxicity events. Point-of-care testing (PoCT) could provide a new and convenient strategy to apply TDM, to a wide number of patients with the aim of adjusting standard doses, if needed, and reducing adverse effects, which would not immediately manifest during the administration of drugs, especially neutropenia and diarrhea in the case of 1 [1,[17], [18], [19]].

Several analytical methods for the quantification of 1 and its main metabolites rely on their simultaneous determination by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with fluorescence [[20], [21], [22], [23]] or mass spectrometry detectors [15,[24], [25], [26]].

However, there are no fast and efficient diagnostic tools available in the market, nor described in the literature, which are capable of detecting small molecules (i.e. therapeutic drugs) in biological fluids within small time intervals [27]. Sensing technologies are very attractive in this context, since several examples of spectroscopic, electrochemical and mass-based techniques were applied to this aim in environmental [28,29] food [30,31] and clinical [32,33] samples. However, the main drawback of these techniques is the absence of a separation system before the detector, which could eventually reduce their applicability, as the matrix components very often contribute with strong interferences to the analysis of the targets in samples.

In this work, we investigated the fluorescence properties of SN-38 (2) in denatured plasma to develop a fast and selective fluorimetric assay capable of detecting and quantifying 2 in human plasma samples in the presence of CPT-11 (1), APC (3), NPC (4) and SN-38 G (5) at those concentration levels commonly found in clinical samples.

Section snippets

Chemicals

Analytical reference standards of 1 (CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin, purity 97%), 2 (SN-38, 7-ethyl-10-hydroxycamptothecin, purity 98%) were purchased from Sigma-Aldrich Co. (Milan, Italy). 3 (APC, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin, purity 95%), 4 (NPC, 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin, purity 98%) and 5 (SN-38G, 7-ethyl-10-[3,4,5-trihydroxy-pyran-2-carboxylic acid]-camptothecin,

Results and discussion

Camptothecins display an intrinsic fluorescence, which was investigated in simple buffered aqueous media and organic solvents [[38], [39], [40], [41]]. In this work, we studied the fluorescence of 1 and its main metabolites in denatured plasma to develop a fast and simple analytical method that could be applied for the rapid determination of 2 in human plasma samples.

In preliminary experiments, the fluorescence of 1 at clinically relevant concentrations in human plasma was either covered by the

Conclusions

In conclusion, we described the development of a fast fluorimetric assay with consistent analytical validation data capable of detecting SN-38 (2) in human plasma. Once investigated the physico-chemical properties of 2 in denatured plasma, we determined and chose specific experimental conditions to selectively quantify 2 with minimal interference by 1 and its metabolites. The method was then validated and tested on unknown plasma samples with very promising results. The implementation of the

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgements

We acknowledge financial support from the grant AIRC 5X1000 (Rif. 12214) “Application of advanced nanotechnology in the development of innovative cancer diagnostics tools”.

References (44)

  • F. Ricci et al.

    A review on novel developments and applications of immunosensors in food analysis

    Anal. Chim. Acta

    (2007)
  • C.I.L. Justino et al.

    Review of analytical figures of merit of sensors and biosensors in clinical applications

    Trends Anal. Chem.

    (2010)
  • I. Chourpa et al.

    Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy

    Biochim. Biophys. Acta - Gen. Subj.

    (1998)
  • M.I. Rodríguez Cáceres et al.

    Spectrofluorimetric determination of irinotecan in the presence of oxidant agents and metal ions

    Talanta

    (2008)
  • M.L. Rothenberg et al.

    Phase II trial of irinotecan in patients with progressive or rapidly recurrent colorectal cancer

    J. Clin. Oncol.

    (1996)
  • J. a Conti et al.

    Irinotecan is an active agent in untreated patients with metastatic colorectal cancer

    J. Clin. Oncol.

    (1996)
  • C. Tournigand et al.

    FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: a randomized GERCOR study

    J. Clin. Oncol.

    (2004)
  • K. Chen et al.

    Efficacy and safety of addition of bevacizumab to FOLFIRI or irinotecan/bolus 5-FU/LV (IFL) in patients with metastatic colorectal cancer

    Medicine (Baltim.)

    (2016)
  • M. Kotaka et al.

    Study protocol of the Asian XELIRI ProjecT (AXEPT): a multinational, randomized, non-inferiority, phase III trial of second-line chemotherapy for metastatic colorectal cancer, comparing the efficacy and safety of XELIRI with or without bevacizumab versus

    Chin. J. Cancer

    (2016)
  • Y. Pommier

    Drugging topoisomerases: lessons and challenges

    ACS Chem. Biol.

    (2013)
  • Y.H. Hsiang et al.

    DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues

    Cancer Res.

    (1989)
  • L.F. Liu

    DNA topoisomerase poisons as antitumor drugs

    Annu. Rev. Biochem.

    (1989)
  • Cited by (7)

    View all citing articles on Scopus
    View full text