Search

Il monitoraggio terapeutico dei farmaci (TDM) è la pratica clinica di misurazione di un farmaco specifico a intervalli di tempo definiti per mantenere le concentrazioni plasmatiche all'interno di una finestra terapeutica mirata, per massimizzare l'efficacia e ridurre al minimo la tossicità. Per rendere fattibile questo approccio sono necessari metodi analitici robusti, sensibili e riproducibili per quantificare i farmaci antitumorali nei fluidi biologici. Questo progetto di dottorato si è concentrato su quattro farmaci antitumorali orali utilizzati per il trattamento di pazienti con HCC avanzato: sorafenib (SORA), regorafenib (REGO), lenvatinib (LENVA) e idarubicina (IDA), la cui sicurezza ed efficacia è oggetto di indagine come opzione terapeutica di terza linea. SORA, REGO e LENVA sono stati inoltre coinvolti in uno studio di cross-validazione (CRO-2018-83) in corso presso il Centro di Riferimento Oncologico di Aviano, il cui obiettivo primario era la cross-validazione tra il metodo LC-MS/MS di quantificazione basato sul plasma e quello basato sul Dried Blood Spot (DBS) per dimostrare che il DBS può essere utilizzato come metodo di campionamento alternativo rispetto al campione plasmatico per la quantificazione di questi farmaci. Il DBS può migliorare l'applicabilità del TDM grazie alle sue procedure di raccolta del campione meno invasiva per il paziente. Invece, la quantificazione di IDA e del suo metabolita attivo nel plasma umano era richiesta come obiettivo secondario di uno studio clinico di fase II (CRO-2017-42) in corso presso il CRO. Per tutti questi farmaci, i dati di esposizione-risposta e -tossicità sono ancora limitati o mancanti, evidenziando così la necessità di approfondire questo tipo di indagine. Per valutare la concentrazione plasmatica del farmaco nelle matrici biologiche sono necessari metodi analitici affidabili. In questo contesto, sono stati sviluppati e validati due metodi LC-MS/MS secondo linee guida EMA ed FDA per la quantificazione contemporanea di SORA, REGO e 3 metaboliti attivi (SORA-N-ossido, REGO-N-ossido e N-desmetil-REGO-N-ossido), sia in plasma umano sia in DBS. Questi metodi hanno lo stesso range analitico (50-8000 ng/mL) per SORA e REGO e (30-4000 ng/mL) per i metaboliti. Dopo le validazioni, questi metodi sono stati applicati per quantificare 66 campioni di plasma e 63 DBS ottenuti da 16 pazienti, trattati con SORA o REGO e arruolati nello studio CRO 2018 83. Tale analisi ha permesso di ottenere dati preliminari riguardanti le concentrazioni dei farmaci nei pazienti e la correlazione tra campioni accoppiati di plasma e DBS. L'applicazione di adeguate analisi statistiche per lo studio di cross-validazione ha mostrato l'assenza di una forte correlazione tra le concentrazioni plasmatiche e quelle in DBS ed ha suggerito di aumentare il numero di pazienti e di rivalutare l'effetto dell'ematocrito e del volume dello spot per migliorare tale conversione. Sono stati sviluppati altri due metodi LC-MS/MS per la quantificazione di LENVA sia nel plasma umano sia nel DBS utilizzando due diverse carte da filtro (Whatman 31 ET CHR e Whatman 903). Questi metodi sono stati validati secondo le linee guida di riferimento di EMA, FDA e per il DBS. Il metodo basato sul plasma ha un ampio range analitico (0,5-2000 ng/mL) ed è stato applicato per quantificare 24 campioni di plasma ottenuti da 6 pazienti trattati con LENVA e arruolati nello studio CRO-2018-83. Il corrispondente metodo basato su DBS ha un range analitico leggermente ridotto (5-2000 ng/mL) ed è stato applicato per l'analisi di 4 campioni di pazienti DBS. Non sono stati ancora condotti studi di correlazione a causa della scarsità di campioni dei pazienti raccolti fino ad ora. L'applicazione di questi metodi analitici agli studi in corso presso il CRO di Aviano consentirebbe di raccogliere dati utili per approfondire le conoscenze sulla possibile correlazione tra i livelli plasmatici e l'esito del trattamento o tossicità Therapeutic Drug Monitoring (TDM) is the clinical practice of measuring a specific drug at defined intervals of time to maintain plasma concentrations within a targeted therapeutic window, to maximize efficacy and minimize toxicity. Robust, sensitive, and reproducible analytical methods to quantify anticancer drugs in biological fluids are needed to make this approach feasible. This PhD project focused on four different oral anticancer drugs used to treat patients with advanced HCC: sorafenib (SORA), regorafenib (REGO), lenvatinib (LENVA), and idarubicin (IDA), whose safety and efficacy as a third-line therapy option is currently under investigation. SORA, REGO, and LENVA were also involved in a cross-validation study (CRO-2018-83) ongoing at Centro di Riferimento Oncologico di Aviano (CRO), where the primary aim was the cross-validation between the plasma-based LC-MS/MS method and the Dried Blood Spot (DBS)-based LC-MS/MS assay to demonstrate that DBS can be used alternatively to plasma sample to quantify these drugs. DBS may improve TDM applicability thanks to its more patient-friendly procedures for sample collection. Instead, the quantification of IDA and its active metabolite (idarubicinol) in human plasma was required as a secondary aim of a phase II clinical study (CRO-2017-42) ongoing at CRO. For all these drugs, exposure-response and -toxicity data are still limited or lacking, thus highlighting the necessity of deepening this type of investigation. To assess drug plasma concentration in biological matrices, reliable analytical methods are needed. In this contest, two LC-MS/MS methods were developed and validated according to international guidelines to simultaneously quantify SORA, REGO, and 3 active metabolites (SORA N oxide, REGO N oxide, and N desmethyl REGO N oxide), both in human plasma and DBS. These methods have the same analytical range (50-8000 ng/mL) for SORA and REGO and (30-4000 ng/mL) for the metabolites. After the validations, these methods were applied to quantify 66 plasma samples and 63 DBS obtained from 16 patients, treated with SORA or REGO and enrolled in CRO 2018 83. This analysis allowed to obtain preliminary data regarding the concentrations of the drugs in patients and the correlation between plasma and DBS paired samples the application of proper statistical analyses for the cross-validation study showed the absence of a strong correlation between plasma and DBS concentrations and it suggested to enlarge patients number and to re-evaluate Hct and spot-volume effect to enhance the DBS-to-plasma conversion performance. Two additional LC-MS/MS methods were developed for the quantification of LENVA both in human plasma and DBS using two different filter papers (Whatman 31 ET CHR and Whatman 903). These methods were validated according to EMA, FDA, and DBS references guidelines. The plasma-based method has a wide analytical range (0.5-2000 ng/mL) and it was applied to quantify 24 plasma samples obtained from 6 patients treated with LENVA and enrolled in CRO-2018-83. The corresponding DBS-based method has a slightly reduced analytical range (5-2000 ng/mL) and it was applied for the analysis of 4 DBS patients’ samples. No correlation studies have yet been performed due to the paucity of patients’ samples collected till now. The application of these analytical methods to the ongoing studies at the C.R.O. di Aviano should allow to collect useful data to deepen the knowledge about the possibility of establishing a correlation between drug exposure levels and patients’ outcome or toxicity development. The aim is to implement the application of TDM for these anticancer drugs in clinical practice.

The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50–7500 ng/mL for imatinib and 10–1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 μL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 μm, 2x50 mm) while the trapping column was a POROS R1/20 (20 μm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients’ finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.

Sunitinib is approved for advanced renal cell cancer, imatinib-resistant or -intolerant gastrointestinal stromal tumors and pancreatic neuroendocrine cancers. It is prescribed at a fixed dose but its plasma exposure shows large inter-individual variations. Taking into account the narrow therapeutic window and the positive exposure-efficacy relationship, there is a robust rationale for its therapeutic drug monitoring. In fact, a target plasma concentration of sunitinib plus its active metabolite, N-desethyl sunitinib, ≥50 ng/mL was suggested. In order to quantify sunitinib and N-desethyl sunitinib in patients’ plasma, we developed and validated a new LC–MS/MS method applicable to clinical routine. In solution, sunitinib and N-desethyl sunitinib undergo to photo-isomerization and many published methods overcome this problem by conducting the entire procedures of samples collection and handling under strictly light-protection. Our method is based on a simple and fast procedure that quantitatively reconverts the E-isomer of both analytes, obtained during sample draw and processing without light-protection, into their Z-forms. Moreover, our method uses a small plasma volume (30 μL) and the analytes are extracted by a rapid protein precipitation. It was validated according to EMA-FDA guidelines. The calibration curves resulted linear (R2 always >0.993) over the concentration ranges (0.1–500 ng/mL for sunitinib, 0.1–250 ng/mL for N-desethyl sunitinib) with a good precision (within 7.7 % for sunitinib and 10.8% for N- desethyl sunitinib) and accuracy (range 95.8–102.9% for sunitinib and 92.3–106.2% for N-desethyl sunitinib). This method was applied to a pharmacokinetic study in one patient treated with sunitinib. Moreover, as incurred samples reanalysis is an established part of the bioanalytical process to support clinical studies, its assessment was performed early in order to assure that any reproducibility issues was detected as soon as possible. The percentage difference between the two runs resulted within ±20% in all the re-analysed samples for both sunitinib and N- desethyl sunitinib.