Razvoj in optimizacija metode za določanje kromatografskega hidrofobnega indeksa

Kovačič, Maja (author), Ilaš, Janez (mentor)

Določitev lipofilnosti zdravilnih učinkovin je pomemben korak v farmacevtskem razvoju, saj lipofilnost vpliva na absorpcijo, distribucijo, metabolizem, izločanje in toksičnost zdravilnih učinkovin (ADMET lastnosti). Ko omenimo lipofilnost, pomislimo na kvantitativno opredelitev z logaritmom porazdelitvenega koeficienta – logP. Čeprav poznamo več računalniških modelov za izračun logP na podlagi strukture spojine, imajo rezultati v večini primerov veliko napako; prav tako se modeli razlikujejo in s tem tudi rezultati logP. Najbolj tradicionalna metoda za eksperimentalno določanje logP je metoda stresanja (angl. shake-flask method), ki je kljub njeni zamudnosti in potratnosti še vedno zlati standard za določanje logP. Pri njej gre za direktno določanje lipofilnosti, poznamo pa tudi indirektno določanje lipofilnosti. V tej magistrski nalogi smo se osredotočili na indirektno določanje lipofilnosti z metodo gradientne reverznofazne tekočinske kromatografije visoke ločljivosti (HPLC), kjer smo določili kromatografski hidrofobni indeks (CHI). CHI vrednost je ponavadi med 0 in 100 – višja, kot je vrednost CHI, bolj je analit hidrofoben. V primerjavi z metodo stresanja je metoda HPLC hitrejša, bolj občutljiva in selektivna ter potrebuje manjši volumen vzorca. V člankih smo zasledili tudi metodo ultravisokotlačne kromatografije (UHPLC), ki je bila predstavljena kot hitrejša in bolj optimizirana metoda kot HPLC. Ker pa je bilo podatkov o razvoju UHPLC metode za določevanje lipofilnosti zelo malo, smo se odločili da se optimizacije lotimo sami. Določili smo logD – logaritem distribucijskega koeficienta, ki upošteva tudi ionizacijo spojin pri določenem pH. Odločili smo se za eksperimentalno določitev logD pri fiziološkem pH – logD7,4, da bi dobili realen vpogled v lipofilne lastnosti zdravilnih učinkovin. Preučevali smo predvsem vpliv ionizacije na retencijo in logD7,4/logP vrednosti in kako se ti lahko v nekaterih primerih razlikujeta ravno na račun ionizacije. Po uspešno postavljeni metodi smo eksperimentalno določili CHI vrednost več kot dvesto unikatnim spojinam in dobljene rezultate primerjali z računsko pridobljenimi podatki. Ugotovili smo dobro korelacijo med izračunanimi in eksperimentalnimi vrednostmi logD7,4. V primerjavi z učinkovinami v razvoju smo z našo optimizirano metodo dosegli primerljive rezultate glede na korelacijo rezultatov in tudi povprečno razliko med vrednostmi. Naša metoda se je izkazala za bolj natančno za nevtralne/bazične spojine in manj natančno za kisline.

• Language: Slovenian
• Keywords: Lipofilnost, gradientna reverznofazna tekočinska kromatografija visoke ločljivosti, porazdelitveni koeficient, distribucijski koeficient, metoda stresanja, kromatografski hidrofobni indeks, fiziološki pH.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-148427
• Publication date in RUL: 23.08.2023

Secondary language

• Language: English
• Title: Development and optimization of a chromatographic hydrophobic index determination method

Abstract

Lipophilicity determination of active pharmaceutical ingredients is an important step in drug discovery since lipophilicity affects the absorption, distribution, metabolism, excretion, and toxicity of drugs (ADMET properties). When we mention lipophilicity, quantitative determination with the logarithm of partition coefficient – logP comes to mind. Even though we now know many computer-based models for calculating logP based on the chemical structure, these results come with a calculation error in many cases. On top of that, we know many different models with different logP results for the same compound. The most traditional method for experimental determination of logP is the shake flask method which is still the gold standard for logP determination even though it is not time or cost-effective. In this case, it involves a direct determination of lipophilicity. However, we also know indirect determination. In this master’s thesis, we focused on the indirect determination of lipophilicity with the gradient reversed-phase high-pressure liquid chromatography (HPLC) method where we determined the chromatographic hydrophobic index (CHI). CHI value is usually between 0 and 100. The higher the CHI value, the more hydrophobic the analyte is. In comparison to the shake flask method, the HPLC method is faster, more sensitive, and more selective, and it also requires less sample volume. In some articles, we also came across a UHPLC method which was presented as a faster and more optimized method in comparison to HPLC. However, the data for UHPLC determining lipophilicity was scarce. Therefore, we decided to optimize the method ourselves. We determined the logD – logarithm of the distribution coefficient which takes into account the ionization of the compound at the specific pH. We decided to determine logD at physiological pH – logD7,4 experimentally to get a realistic insight into the lipophilic properties of the drug. We studied the effect of ionization on retention and logD7,4/logP values and how they differ based on ionization. Following a successfully established method, we determined the CHI value of more than two hundred unique compounds experimentally and compared the obtained results with computationally obtained data. We observed a strong correlation between the calculated and experimental values of logD7.4. In comparison to compounds in development, our optimized method achieved comparable results in terms of correlation and the average difference between the values. Our method has proven to be more accurate for neutral/basic compounds and less accurate for acids.

• Keywords: Lipophilicity, gradient reversed-phase high-pressure liquid chromatography, partition coefficient, distribution coefficient, shake flask method, chromatographic hydrophobic index, physiological pH.