Title Ispitivanje mogućnosti inhibicije tiopurin S-metiltransferaze pripravcima odabranih biljnih vrsta Title (english) Examination of the possibility of thiopurine S-methyltransferase inhibition by preparations of selected plant species Author Marko Mataija Mentor Ana Mornar Turk (mentor)Committee member Ana Mornar Turk (predsjednik povjerenstva)Committee member Ivana Perković (član povjerenstva)Committee member Daniela Amidžić Klarić (član povjerenstva)Granter University of Zagreb Defense date and country 2022-09-09, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Abstract Upalne bolesti crijeva bolesti su koje predstavljaju globalni zdravstveni problem, a u zadnjih nekoliko desetljeća bilježi se porast njihove incidencije. Kao dva najznačajnija predstavnika ističu se Crohnova bolest i ulcerozni kolitis koje karakterizira kronična recidivirajuća upala. Tiopurin S-metiltransferaza (TPMT, E.C. 2.1.1.67) citosolni je enzim koji metabolizira reakcije S-metilacije aromatskih i heterocikličnih sulfhidrilnih skupina kao što su tiopurinski i tiopirimidinski lijekovi. Među ksenobiotike koje TPMT metabolizira spadaju i azatioprin i 6-merkaptopurin koji se u reakciji posredovanom ovim enzimom inaktiviraju. Pored konvencionalne farmakoterapije, sve više pacijenata rješenje svojih zdravstvenih problema pokušava pronaći u komplementarnoj i alternativnoj medicini. Neki od biljnih pripravaka za koje postoje znanstvene informacije o protuupalnom i imunomodulatornom učinku koji bi pozitivno utjecao na ishode liječenja ovog tipa bolesti uključuju kurkumu (Curcuma longa), crni papar (Piper nigrum), indijski tamjanovac (Boswellia serrata) i kineski androfagis (Andrographis paniculata). Cilj ovog istraživanja bio je utvrditi dovode li navedeni biljni pripravci do inhibicije tiopurin S-metiltransferaze, enzima koji je ključan u metabolizmu azatioprina i merkaptopurina, ili istodobna primjena biljnih pripravaka i navedenih lijekova ne uzrokuje takvu vrstu interakcije.
Istraživanje je uključivalo razvoj i validaciju analitičke metode koja bi učinkovito pratila enzimsku rekaciju metilacije 6-MP i nastanak 6-MMP, ispitivanje enzimske kinetike TPMT i, u konačnici, ispitivanje njegove potencijalne inhibicije ekstraktima odabranih biljnih droga. Razvijena je HPLC metoda u trajanju od 22 min. Kao nepokretna faza korištena je Cortecs Phenyl kolona (150 × 4,6 mm, veličina čestica 2,7 um). Provedena je gradijentna eluacija s dvije sastavnice mobilne faze (0,1 % mravlje kiseline u vodi, odnosno metanolu). Metoda je zadovoljila sve ispitane parametre validacije. Ispitivanje enzimske kinetike dovelo je do zaključka da TPMT dobrovoljca pokazuje visoku enzimsku aktivnost te da se dobiveni rezultati mogu translatirati na najveći udio populacije. Na kraju, određeni su i inhibicijski potencijali ekstrakata biljnih droga te su dobivene vrijednosti za ekstrakt podanka kurkume (IC50 = 275 μg/mL), ekstrakt ploda crnog papra (IC50 = 490 μg/mL), ekstrakt lista kinsekog androfagisa (IC50 = 2300 μg/mL) i smole indijskog tamjanovca (IC50 = 1900 μg/mL). No, niti jedan od ovih biljnih ekstrakata nije inhibirao TMPT učinkovito kao furosemid (IC50 = 15 μg/mL) koji je poznati inhibitor ovog enzima.
Dobiveni podaci doveli do zaključka da se koncentracije koje su u ovom istraživanju dobivene kao inhibicijske ne mogu ostvariti pri koncentracijama u kojima se odrabrani pripravci nalaze na tržištu i pokazuju svoje farmakološke učinke. Sve navedeno ukazuje da istodobna primjena ovih pripravaka i liječenja azatioprinom i 6-merkaptopurinom ne dovodi do značajnijih farmakokinetičkih interakcija zbog kojih bi istodobna primjena ovih tvari bila kontraindicirana te da, barem prema ispitivanim parametrima, ne postoji ograničenje u njihovom zajedničkom korištenju.
Abstract (english) Inflammatory bowel disease is disease that represents a global health problem, and an increase in their incidence has been recorded in the last few decades. The two most significant representatives are Crohn’s disease and ulcerative colitis, which are characterized by relapsing chronic inflammation. Thiopurine S-methyltransferase (TPMT, E.C. 2.1.1.67) is a cytosolic enzyme that metabolizes S-methylation reactions of aromatic and heterocyclic sulfhydryl groups such as thiopurine and thiopyrimidine drugs. The xenobiotics metabolized by TPMT include azathioprine and 6-mercaptopurine, which are inactivated in a reaction mediated by this enzyme. In addition to conventional pharmacotherapy, more and more patients are trying to find a solution to their health problems in complementary and alternative medicine. Some of the herbal preparations for which there is scientific information on the anti-inflammatory and immunomodulatory effect that would positively influence the treatment outcomes of this type of disease include turmeric (Curcuma longa), black pepper (Piper nigrum), Indian frankincense (Boswellia serrata) and Chinese Andrographis (Andrographis paniculata). The aim of this research was to determine whether the mentioned herbal preparations lead to the inhibition of thiopurine S-methyltransferase, an enzyme that is key in the azathioprine and mercaptopurine metabolism, or whether the simultaneous use of herbal preparations and the mentioned drugs does not cause this type of interaction.
The research included the development and validation of an analytical method that would effectively monitor the enzymatic reaction of methylation of 6MP and the formation of 6MMP, the investigation of the enzymatic kinetics of TPMT and, finally, the investigation of its potential inhibition by extracts of selected herbal drugs. An HPLC method lasting 22 min was developed. A Cortecs Phenyl column (150 × 4.6 mm, particle size 2.7 μm) was used as the stationary phase. A gradient elution with two components of the mobile phase (0.1% formic acid in water or methanol) was performed. The method met all the tested validation parameters. Examination of enzyme kinetics led to the conclusion that the volunteer’s TPMT shows high enzyme activity and that the obtained results can be translated to the largest part of the population. Finally, the inhibitory potentials of herbal drug extracts were determined, and values were obtained for turmeric root extract (IC50 = 275 μg/mL), black pepper fruit extract (IC50 = 490 μg/mL), Chinese Andrographis leaf extract (IC50 = 2300 μg /mL) and Indian frankincense resin (IC50 = 1900 μg/mL). However, none of these plant extracts inhibited TMPT as efficiently as furosemide (IC50 = 15 μg/mL), which is a known inhibitor of this enzyme.
The obtained data led to the conclusion that the concentrations obtained in this research as inhibitory cannot be achieved at the concentrations in which the selected preparations are on the market and show their pharmacological effects. All of the above indicates that the simultaneous use of these preparations and treatment with azathioprine and 6-mercaptopurine does not lead to significant pharmacokinetic interactions due to which the simultaneous use of these substances would be contraindicated and that, at least according to the tested parameters, there is no limitation in their joint use.
Keywords
crijeva
upalne bolesti crijeva
tiopurin S-metiltransferaza
6-merkaptopurin
kurkuma
crni papar
indijski tamjanovac
kineski androfagis
Keywords (english)
bowels
inflammatory bowel diseases
thiopurine S-methyltransferase
6-mercaptopurine
turmeric
black pepper
Indian frankincense
Chinese andrographis
Language croatian URN:NBN urn:nbn:hr:163:576138 Study programme Title: Pharmacy Type of resource Text File origin Born digital Access conditions Embargoed access Terms of use Created on 2022-09-09 12:48:19
