14 research outputs found
Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10
Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.Peer reviewe
Development of LC/MS techniques for plant and drug metabolism studies
Abstract
Liquid chromatography (LC) combined with mass spectrometry (MS) is a powerful tool for qualitative and quantitative analytics of organic molecules from various matrices, and the use of this hyphenated technique is very common in bioanalytical laboratories. In this study, LC/MS methods and the required sample preparation applications were developed for plant flavonoid and drug metabolism studies. The main focus was in developing methods to be used during cytochrome P450 (CYP) -specific drug interaction studies. Traditional high performance liquid chromatography (HPLC) and new, more efficient and faster ultra-performance liquid chromatography (UPLC) were utilized together with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry. In the flavonoid study, collision-induced radical cleavage of flavonoid glycosides was tested and observed to be a suitable tool for the structure elucidation of the 15 flavonol glycosides extracted from the medicinal plant Rhodiola rosea. Ten of these glycosides were previously unreported in the plant.
Several unreported in vivo bupropion metabolites were identified from human urine when developing the method for the new and more extensive in vitro and in vivo N-in-one interaction cocktail assays. The qualified analysis methods developed here enable faster analysis for the N-in-one cocktail assays, in turn enabling a more efficient screening of drugs that affect CYP-enzyme activities. In the case of the human in vitro cocktail assay, fourteen compounds were analyzed using a single LC/MS/MS run. The method has proven to be very reliable and has been used in several interaction studies utilizing different sample matrices. The in vivo cocktail assay that was developed enables totally non-invasive sample collection from the patients, the urine sample being sufficient for the UPLC/MS/MS analysis of all target compounds. The last part of the study consisted of developing a specific and very sensitive UPLC/MS/MS method for the analysis of one of the in vivo cocktail analytes, the antidiabetic drug repaglinide, from human placenta perfusates.Tiivistelmä
Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas työväline kvalitatiivisessa ja kvantitatiivisessa analytiikassa, ja tätä tekniikkaa käytetään erityisesti bioalan laboratorioissa. Tässä väitöskirjatyössä kehitettiin ja sovellettiin LC/MS- ja näytteenkäsittelymenetelmiä kasvien flavonoidimetabolian ja lääkeaineiden metaboliatuotteiden tutkimukseen keskittyen erityisesti sytokromi P450 (CYP) -entsyymispesifisten lääkeaineiden interaktiotutkimuksiin tarvittaviin menetelmiin. Työssä hyödynnettiin perinteistä korkean erotuskyvyn nestekromatografiaa (HPLC) ja uutta, suorituskyvyltään vielä tehokkaampaa ja nopeampaa nestekromatografiaa (UPLC) yhdessä lentoaika- (TOF) ja kolmoiskvadrupolimassaspektrometrian (QqQ) kanssa. Tutkimustyön flavonoidimetaboliaan keskittyneessä osuudessa havaittiin törmäyksen aiheuttaman (CID) radikaalipilkkoutumisen soveltuvan lääkinnällisenä kasvina käytetystä ruusujuuresta (Rhodiola rosea) uutettujen viidentoista flavonoliglykosidin rakennemääritykseen. Kymmentä näistä löydetyistä glykosideista ei oltu aiemmin raportoitu ruusujuuresta. Tutkimustyön keskeisimpänä tavoitteena kehitettiin kvalifioidut LC/MS -analyysimenetelmät käytettäväksi aikaisempaa kattavampien in vitro ja in vivo -olosuhteiden N-in-one -tyyppisten CYP-entsyymi-interaktiotutkimusten analyyttisenä työkaluna. Näitä analyysimenetelmiä kehitettäessä löydettiin ja tunnistettiin ihmisen virtsasta aiemmin raportoimattomia metaboliitteja CYP2B6 -entsyymin malliaineena käytetyn bupropionin annostelun jälkeen. Kyseisten kehitettyjen analyysimenetelmien avulla CYP-entsyymien toimintaan vaikuttavien lääkeaineiden tutkiminen on aiempaa nopeampaa ja antaa yhdellä kertaa samasta tutkimuksesta entistä laaja-alaisempaa tietoa. In vitro -tutkimusta varten kehitetty LC/MS/MS -analyysimenetelmä on osoittautunut erittäin käyttökelpoiseksi lukuisissa interaktiotutkimuksissa, ja in vivo -tutkimusta varten kehitetty UPLC/MS/MS -analyysimenetelmä mahdollistaa täysin ei-invasiivisen näytteenoton potilaista. Tutkimustyön viimeisessä vaiheessa kehitettiin erittäin herkkä ja spesifinen UPLC/MS/MS -analyysimenetelmä CYP2C8-entsyymin toiminnan malliaineena käytetyn repaglinidin analysoimiseksi koejärjestelystä, jossa tutkitaan yhdisteiden kulkeutumista raskausaikana äidin ja sikiön verenkierron välillä istukan kautta
Blood-brain barrier permeability and brain uptake mechanism of kainic Acid and dihydrokainic Acid
A liquid chromatography-tandem mass spectrometry analysis of nine cytochrome P450 probe drugs and their corresponding metabolites in human serum and urine
Species-Specific Differences in the <i>in Vitro</i> Metabolism of Lasiocarpine
There are species-related differences
in the toxicity of pyrrolizidine
alkaloids (PAs) partly attributable to the hepatic metabolism of these
alkaloids. In this study, the metabolism of lasiocarpine, a potent
hepatotoxic and carcinogenic food contaminant, was examined <i>in vitro</i> with human, pig, rat, mouse, rabbit, and sheep
liver microsomes. A total of 12 metabolites (M1–M12) were detected
with the human liver microsomes, of which M1, M2, M4, and M6 were
unstable in the presence of reduced glutathione (GSH). With the exception
of M3 and M8, the formation of all metabolites of lasiocarpine was
catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected
several new metabolites, termed M4–M7; their toxicological
significance is unknown. M9 (<i>m</i>/<i>z</i> 398), identified as a demethylation product, was the main metabolite
in all species, although the relative dominance of this metabolite
was lower in humans. The level of the reactive metabolites, as measured
by M1 ((3<i>H</i>-pyrrolizin-7-yl)methanol) and the GSH
conjugate, was higher with the liver microsomes of susceptible species
(human, pig, rat, and mouse) than with the species (rabbit and sheep)
resistant to PA intoxication. In general, in addition to the new metabolites
(M4–M7) that could make humans more susceptible to lasiocarpine-induced
toxicity, the overall metabolite fingerprint detected with the human
liver microsomes differed from that of all other species, yielding
high levels of GSH-reactive metabolites
A liquid chromatography-tandem mass spectrometry analysis of nine cytochrome P450 probe drugs and their corresponding metabolites in human serum and urine
<i>In Silico</i> Prediction of the Site of Oxidation by Cytochrome P450 3A4 That Leads to the Formation of the Toxic Metabolites of Pyrrolizidine Alkaloids
In
humans, the metabolic bioactivation of pyrrolizidine alkaloids
(PAs) is mediated mainly by cytochrome P450 3A4 (CYP3A4) via the hydroxylation
of their necine bases at C3 or C8 of heliotridine- and retronecine-type
PAs or at the N atom of the methyl substituent of otonecine-type PAs.
However, no attempts have been made to identify which C atom is the
most favorable site for hydroxylation <i>in silico</i>.
Here, in order to determine the site of hydroxylation that eventually
leads to the formation of the toxic metabolites produced from lasiocarpine,
retrorsine, and senkirkin, we utilized the ligand-based electrophilic
Fukui function <i>f</i><sup>–</sup>(<b>r</b>) and hydrogen-bond dissociation energies (BDEs) as well as structure-based
molecular docking. The ligand-based computations revealed that the
C3 and C8 atoms of lasiocarpine and retrorsine and the C26 atom of
senkirkin were chemically the most susceptible locations for electrophilic
oxidizing reactions. Similarly, according to the predicted binding
orientation in the active site of the crystal structure of human CYP3A4
(PDB code: 4I4G), the alkaloids were positioned in such a way that the C3 atom of
lasiocarpine and retrorsine and the C26 of senkirkin were closest
to the catalytic heme Fe. Thus, it is concluded that the C3 atom of
lasiocarpine and retrorsine and C26 of senkirkin are the most favored
sites of hydroxylation that lead to the production of their toxic
metabolites
Identification of a New Reactive Metabolite of Pyrrolizidine Alkaloid Retrorsine: (3<i>H</i>‑Pyrrolizin-7-yl)methanol
Pyrrolizidine alkaloids (PAs) such
as retrorsine are common food
contaminants that are known to be bioactivated by cytochrome P450
enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites
known as dehydropyrrolizidine alkaloids (DHPs). We compared how both
electrochemical (EC) and human liver microsomal (HLM) oxidation of
retrorsine could produce short-lived intermediate metabolites; we
also characterized a toxicologically important metabolite, (3<i>H</i>-pyrrolizin-7-yl)methanol. The EC cell was coupled online
or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas
the HLM oxidation was performed in 100 mM potassium phosphate (pH
7.4) in the presence of NADPH at 37 °C. The EC cell oxidation
of retrorsine produced 12 metabolites, including dehydroretrorsine
(<i>m</i>/<i>z</i> 350, [M + H<sup>+</sup>]),
which was degraded to a new reactive metabolite at <i>m</i>/<i>z</i> 136 ([M + H<sup>+</sup>]). The molecular structure
of this small metabolite was determined using high-resolution mass
spectrometry and NMR spectroscopy followed by chemical synthesis.
In addition, we also identified another minor but reactive metabolite
at <i>m</i>/<i>z</i> 136, an isomer of (3<i>H</i>-pyrrolizin-7-yl)methanol. Both (3<i>H</i>-pyrrolizin-7-yl)methanol
and its minor isomer were also observed after HLM oxidation of retrorsine
and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the
presence of reduced glutathione (GSH), each isomer formed identical
GSH conjugates at <i>m</i>/<i>z</i> 441 and <i>m</i>/<i>z</i> 730 in the negative ESI-MS. Because
(3<i>H</i>-pyrrolizine-7-yl)methanol) and its minor isomer
subsequently reacted with GSH, it is concluded that (3<i>H</i>-pyrrolizin-7-yl)methanol may be a common toxic metabolite arising
from PAs
Quantitative ADME Proteomics - CYP and UGT Enzymes in the Beagle Dog Liver and Intestine
International audiencePURPOSE:Beagle dogs are used to study oral pharmacokinetics and guide development of drug formulations for human use. Since mechanistic insight into species differences is needed to translate findings in this species to human, abundances of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) drug metabolizing enzymes have been quantified in dog liver and intestine.METHODS:Abundances of enzymes were measured in Beagle dog intestine and liver using selected reaction monitoring mass spectrometry.RESULTS:Seven and two CYPs were present in the liver and intestine, respectively. CYP3A12 was the most abundant CYP in both tissues. Seven UGT enzymes were quantified in the liver and seven in the intestine although UGT1A11 and UGT1A9 were present only in the intestine and UGT1A7 and UGT2B31 were found only in the liver. UGT1A11 and UGT1A2 were the most abundant UGTs in the intestine and UGT2B31 was the most abundant UGT in the liver. Summed abundance of UGT enzymes was similar to the sum of CYP enzymes in the liver whereas intestinal UGTs were up to four times more abundant than CYPs. The estimated coefficients of variation of abundance estimates in the livers of 14 donors were separated into biological and technical components which ranged from 14 to 49% and 20 to 39%, respectively.CONCLUSIONS:Abundances of canine CYP enzymes in liver and intestine have been confirmed in a larger number of dogs and UGT abundances have been quantified for the first time. The biological variability in hepatic CYPs and UGTs has also been estimated