Practical fluorimetric assay for the detection of anticancer drug SN-38 in human plasma
Graphical abstract
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”.
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