Razvoj bioanalitičke metode za određivanje inhibitora o ciklinu D ovisnih kinaza 4 i 6 i drugih lijekova u terapijskim kombinacijama za liječenje HR+/HER2− raka dojke

doctoral thesis

• Title (english): Development of a bioanalytical method for the determination of cyclin D dependent kinase 4 and 6 inhibitors and other drugs in therapeutic combinations for HR+/HER2− breast cancer treatment
• Author: Lu Turković
• Mentor: Miranda Sertić (mentor)
• Committee member: Biljana Nigović (predsjednik povjerenstva)
• Committee member: Anita Hafner (član povjerenstva)
• Committee member: Željko Debeljak (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical analysis); Zagreb
• Defense date and country: 2025-03-18, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

Rak dojke, kao jedan od najčešće dijagnosticiranih tumora u svijetu, velik je javnozdravstveni problem. Liječenje se nastoji unaprijediti razvojem novih lijekova, ali i personaliziranim pristupom postojećoj terapiji s obzirom na specifične karakteristike pacijentica kroz postupak terapijskog praćenja lijekova (engl. therapeutic drug monitoring, TDM). Temeljni preduvjeti TDM-u su provedena istraživanja farmakokinetičkih parametara u ciljanoj populaciji te povezanosti doze lijeka, njegove koncentracije u plazmi ili drugom tkivu i učinka ili toksičnosti. Za sve navedeno potrebna je prikladna, selektivna i osjetljiva bioanalitička metoda. U ovom doktorskom radu proveden je razvoj uvjeta pripreme uzorka ljudske plazme, kromatografskog razdvajanja i detekcije šest lijekova – kandidata za TDM: inhibitora o ciklinu D ovisnih kinaza 4 i 6 (CDK4/6) palbocikliba, ribocikliba, abemacikliba, inhibitora aromataze anastrozola i letrozola te selektivnog degradatora estrogenskih receptora fulvestranta. Ispitana su i optimizirana četiri različita pristupa pripremi složenog biološkog uzorka za analizu: taloženje proteina, disperzivna tekućinska mikroekstrakcija, ekstrakcija na čvrstoj fazi i pročišćavanje uklanjanjem fosfolipida. Razvijene su i validirane četiri nove kromatografske metode za razdvajanje lijekova od interesa primjenom fenilne i bifenilne stacionarne faze na različitim kromatografskim sustavima. Evaluacija pročišćavanja uzorka plazme od apsorbirajućih interferencija provedena je robusnim detektorom niza dioda i detektorom fluorescencije, dok su klinički relevantni linearni rasponi i praćenje zaostalih plazmatskih fosfolipida postignuti osjetljivim spektrometrima masa s analizatorom kvadrupola i vremena leta ili trostrukog kvadrupola. Provedena je validacija optimiziranih metoda u skladu s međunarodno prihvaćenim smjernicama, kao i unakrsna validacija naprednijih metoda pročišćavanja na istom kromatografskom sustavu i setu realnih uzoraka pacijentica. Ekološka i ekonomska svojstva razvijenih metoda vrednovana su primjenom Red-Green-Blue-12 (RGB-12), Analytical Greenness Metric (AGREE, AGREEprep) i Blue Applicability Grade Index (BAGI) alata. Naposlijetku, sve validirane metode su uspješno primijenjene za određivanje koncentracija te procjenu farmakokinetičkih parametara lijekova od interesa u stvarnim uzorcima pacijentica. Time je kao rezultat ovog doktorskog istraživanja stvorena potrebna bioanalitička podloga, kao i demonstrirana njena praktična valjanost za implementaciju u kliničkoj praksi u svrhu provođenja daljnjih ispitivanja TDM-a šest lijekova od interesa radi poboljšanja skrbi i terapijskih ishoda za pacijentice s rakom dojke.

Abstract (english)

Breast cancer, as one of the most diagnosed tumours in the world, presents a significant public health issue. Treatment options are being expanded with the development of new drugs and the personalisation of existing therapies based on patient-specific characteristics through therapeutic drug monitoring (TDM). The main prerequisites for TDM include gaining insight into pharmacokinetic parameters in the target population and establishing correlations between drug dose, its plasma or tissue concentration, and therapeutic or toxic effects. A selective and sensitive bioanalytical method is key to achieving these objectives. In this doctoral thesis the development of human plasma sample preparation conditions, chromatographic separation, and detection were performed for six drugs – candidates for TDM: cyclin D dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib, aromatase inhibitors anastrozole and letrozole, and the selective estrogen receptor degrader fulvestrant. Four different approaches for the preanalytical preparation of complex biological samples were tested and optimised: protein precipitation, dispersive liquid-liquid microextraction, solid-phase extraction and phospholipid removal. Additionally, four chromatographic methods for analyte separation on phenyl and biphenyl stationary phases were developed and validated using different chromatographic systems. Plasma sample cleanup from absorbing interferences was evaluated using robust diode-array and fluorescence detectors, while clinically relevant linear ranges and monitoring of leftover plasma phospholipids were achieved using sensitive mass spectrometers with quadrupole-time-of-flight or triple-quadrupole mass analysers. Method validation was conducted in accordance with internationally accepted guidelines, and cross-validation of advanced sample preparation methods was performed on the same chromatographic system with a set of real patient samples. The ecological and economic properties of the methods were assessed using Red-Green-Blue-12 (RGB-12), Analytical Greenness Metric (AGREE, AGREEprep) and Blue Applicability Grade Index (BAGI) tools. Finally, all validated methods were successfully applied to quantify the drugs of interest and estimate their pharmacokinetic parameters in real patient samples. As the main result of this doctoral research, the necessary bioanalytical foundation has been established and its practical feasibility for implementation in the clinical practice has been demonstrated, with the aim of facilitating further research on TDM for the drugs of interest, ultimately improving patient care and treatment outcomes for breast cancer patients.

• Keywords: terapijsko praćenje lijekova; LC-MS/MS; bioanalitička metoda; palbociklib; ribociklib; abemaciklib; antihormonska terapija; rak dojke; priprema uzorka
• Keywords (english): therapeutic drug monitoring; LC-MS/MS; bioanalytical method; palbociclib; ribociclib; abemaciclib; antihormonal therapy; breast cancer; sample preparation
• Language: croatian
• URN:NBN: urn:nbn:hr:163:758277
• Promotion: 2025
• Study programme: Title: Pharmceutical-biochemical sciences - doctoral study - university study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti u području biomedicine i zdravstva (doktor/doktorica znanosti u području biomedicine i zdravstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2025-04-09 13:36:47