Jurij Trontelj

Simultaneous measurement of imatinib, nilotinib and dasatinib in dried blood spot by ultra high performance liquid chromatography tandem mass spectrometry

Imatinib, dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). However, achieving maximum benefit with these drugs may require optimal dosing and adherence to therapy. In those cases, therapeutic drug monitoring (TDM) can be a useful tool in managing patients with CML. The paper presents simple and high throughput method for simultaneous determination of all three TKIs in dried blood spot (DBS) samples from CML patients. DBS samples were prepared by applying 10 μL of spiked whole blood onto an Agilent DBS cards. Whole blood spot was punched out of the card, transferred to a well in a 96-well Captiva ND Lipids filter plate. After the addition of isotopically labelled internal standard, the drug was extracted with 0.1% formic acid in methanol. The collected extract (1 μL) was injected onto a Phenomenex Kinetex 50 mm × 2.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESI-MS/MS Agilent 6460 operated in positive mode. The total run time was only 2.6 min. The method was validated in terms of linearity, selectivity, specificity, accuracy, precision, absolute and relative matrix effect and stability. The effect of haematocrit (Hct) on the accurate concentration determination was also examined. The method was linear in the range of 50-5000 μg/L for imatinib and nilotinib and in the range of 2.5-250 μg/L for dasatinib, with correlation coefficient values higher than 0.997. Lower limits of quantification (LLOQ) were 50 μg/L for imatinib and nilotinib and 2.5 μg/L for dasatinib. The method proved to be accurate (% bias < 13.2) and precise (CV < 10.3%) on intra- as well as on inter-day basis. Sample matrix (% ME=94.5-106.7) and different Hct values had no significant effect on the accuracy of measured concentrations. Samples proved to be stable whilst stored on DBS cards at room temperature or in the refrigerator; however, at 40 °C the stability of dasatinib was compromised. The method presented was successfully applied to clinical samples.

Bioactivation of bisphenol A and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes

Bisphenol A analogs are a class of chemicals known as diphenylmethanes, which contain two benzene rings separated by one central carbon atom, usually with a para-hydroxy group on both benzene rings. Bisphenol A (BPA) can induce an uterotrophic response in immature CD-1 mice and elicits estrogenic responses in many other experimental systems. Besides highlighting endocrine effects, a number of metabolic studies provide strong support for the idea that reactive species of BPA are formed in vitro and in vivo that can form covalent adducts with nucleophilic macromolecules and/or produce oxidative stress. We used a liquid chromatography with a triple quadrupole tandem mass spectrometry (LC-MS/MS) for the detection of metabolites and glutathione conjugates of BPA and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes (HLM) or with recombinant CYP isozymes in the presence of NADPH and GSH as a trapping agent. We have confirmed that BPA and its structural analogs form hydroxylated metabolites and electrophilic species during bioactivation in HLM and CYP isozymes. These results provided important mechanistic insight into the metabolic fate of BPA structural analogs in vitro.

A Self-Microemulsifying Drug Delivery System to Overcome Intestinal Resveratrol Toxicity and Presystemic Metabolism

A mixed lipid-mixed surfactant self-microemulsifying drug delivery system (SMEDDS) was developed to exploit the health benefits of resveratrol, a Biopharmaceutical Classification System Class 2 natural polyphenol, subject to extensive intestinal presystemic metabolism. SMEDDS with a mixed lipid phase (castor oil/Capmul MCM 1:1) and a mixed surfactant phase (Kolliphor EL/Kolliphor RH 40 1:1) was developed and evaluated for its self-emulsifying properties and in vitro dispersion. The impact of SMEDDS on the permeability properties of resveratrol and its metabolite fluxes through the rat intestine and Caco-2 cells was monitored. The inhibitory effect of selected SMEDDS components on the efflux transporters multidrug resistance-associated protein and P-gp as well as cytotoxicity was assessed on Caco-2 cells. The formulation allowed for high resveratrol loading (122.5 mg/g SMEDDS), excellent self-emulsifying properties, and very rapid release. When formulated in SMEDDS, resveratrol metabolite efflux significantly declined. The formulation (SMEDDS without incorporated resveratrol) and its individual components did not compromise in vitro cell vitality and integrity. Mixed lipid-mixed surfactant SMEDDS is a prospective formulation to improve resveratrol biopharmaceutical, pharmacokinetic, and toxicological properties, leading the way to resveratrol use not only as a supplement but also as a pharmacological drug.

HIV protease inhibitors: garlic supplements and first-pass intestinal metabolism impact on the therapeutic efficacy

Background/Aims. The aim of this study was to elucidate the impact of first‐pass intestinal metabolism to therapeutic efficacy of antiretrovirals and to ascertain interaction mechanisms between garlic supplements (aged garlic extract) and HIV‐protease inhibitors.

Influence of cancer cachexia on drug liver metabolism and renal elimination in rats

Body wasting and cachexia change body composition and organ function, with effects on drug pharmacokinetics. The aim of this study was to investigate how cancer and cancer cachexia modify liver metabolism and renal drug elimination in rats.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.

Raloxifene Glucuronidation in Human Intestine, Kidney and Liver Microsomes and in Human Liver Microsomes Genotyped for the UGT1A1*28 Polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4′-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min−1 · kg−1, respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437–444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.

Liquid chromatography–tandem mass spectrometry method for simultaneous quantification of bisoprolol, ramiprilat, propranolol and midazolam in rat dried blood spots

Dried blood spot (DBS) sampling represents a suitable method for pharmacokinetic studies in rats, particularly if serial sampling is needed. To study the pharmacokinetics of drugs in a rat heart failure (HF) model, we developed and validated a method for the simultaneous determination of bisoprolol, ramiprilat, propranolol and midazolam in DBS samples. Bisoprolol and ramipril are widely used in the treatment of HF, and midazolam and propranolol are markers of hepatic metabolism, which can be altered in HF. A 20μL sample of rat blood was pipetted onto Whatman 903 Protein Saver Card and allowed to dry. The whole spot was excised and 300μL of solvent (methanol with 10% ultrapure water and 0.1% formic acid) was added. After mixing and incubating the sample in an ultrasonic bath, a mixture of isotopically labeled internal standards was added. After centrifugation, the extracts were cleaned on an Ostro™ plate and analyzed using liquid chromatography-tandem mass spectroscopy. The method was successfully validated. No significant interference was observed in the retention times of analytes or internal standards. The intraday and interday accuracy and precision were within a ±15% interval. The method was linear in the range 5-250μg/L and the lower limit of quantification was 5μg/L for all four analytes. The absolute matrix effect ranged from 98.7% for midazolam to 121% for ramiprilat. The recovery was lowest for ramiprilat and highest for propranolol. Samples were stable at all tested temperatures. The method has been used successfully in a real-time pharmacokinetic study in rats.

Bio-relevant media to assess drug permeability: Sodium taurocholate and lecithin combination or crude bile?

The assessment of in vivo drug absorption with in vitro permeability models demands the use of transport media with surface acting compounds. With the aim to establish their influence on in vitro permeability of 30 drugs through Caco-2 monolayers, cell vitality/integrity and micellar drug entrapment, taurocholate/lecithin (NaTC/Leci) and pig crude bile were applied. Drug permeabilities were correlated to fraction of drugs absorbed and appropriate NaTC/Leci and bile concentrations were proposed to simulate fasted/fed conditions in vitro (bile in the concentration range 1-5 v/v% or 0.2/0.05mM NaTC/Leci for fasted; 10 v/v% bile or 3/0.75mM NaTC/Leci for fed conditions) without detrimental effects on monolayer integrity/vitality (NaTC/Leci was more toxic than bile). Surfactants exerted different affinities for drugs; free drug concentration (c(free)) of some was significantly lowered only by bile, while for the others NaTC/Leci and bile significantly diminished c(free). For some substances NaTC/Leci and bile significantly increased their permeabilities (i.e. more than 3-times) in spite of profound c(free) decrease indicating the existence of an alternative absorption mechanism. Based on these data, the impact of bile on in vitro drug permeability and micellar drug entrapment cannot be adequately simulated by NaTC/Leci, because their effects on drug absorption differ.

The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity.

The enzymatic activity of butyrylcholinesterase (BChE) in the brain increases with the progression of Alzheimers disease, thus classifying BChE as a promising drug target in advanced Alzheimers disease. We used structure-based drug discovery approaches to develop potent, selective, and reversible human BChE inhibitors. The most potent, compound 3, had a picomolar inhibition constant versus BChE due to strong cation-π interactions, as revealed by the solved crystal structure of its complex with human BChE. Additionally, compound 3 inhibits BChE ex vivo and is noncytotoxic. In vitro pharmacokinetic experiments show that compound 3 is highly protein bound, highly permeable, and metabolically stable. Finally, compound 3 crosses the blood-brain barrier, and it improves memory, cognitive functions, and learning abilities of mice in a scopolamine model of dementia. Compound 3 is thus a promising advanced lead compound for the development of drugs for alleviating symptoms of cholinergic hypofunction in patients with advanced Alzheimers disease.