Department of Pharmacokinetics and Drug Metabolism

Department of Pharmacokinetics and Drug Metabolism

OVERVIEW

Our research is focused on pharmacokinetics of psychotropic drugs (antidepressants and neuroleptics), an involvement of cytochrome P450 enzymes (CYPs) in the metabolism of these drugs in the liver and endogenous neuroactive substrates in the brain as well as an influence of the nervous system on the expression and activity of this enzyme.

Studies concerning pharmacokinetics and metabolism of psychotropics are carried out using animals. They include four stages of the drug's fates in the body: absorption from the site of administration, cytochrome P450-dependent metabolism, distribution (transport and penetration across the blood-brain barrier) and elimination from the body.

Studies on cytochrome P450-mediated metabolism of psychotropic drugs and endogenous substrates are carried out in human and animal enzyme preparations. Particular attention is paid to the involvement of this enzyme in alternative pathways of neurotransmitter synthesis (e.g. dopamine formation from tyramine, serotonin synthesis from 5-methoxytryptamine or melatonin), which may constitute new targets for psychotropic drug action.

Our research involves also the impact of psychotropic drugs on brain and liver cytochrome P450 expression and metabolic activity (drug-CYP enzyme or drug-CYP gene interaction). The results of these studies help to understand pharmacokinetic interactions between drugs and their effects on metabolism of endogenous substances (e.g. neurotransmitters, steroids, arachidonic acid).

We are also interested in the involvement of brain and peripheral neurotransmitters in the regulation of cytochrome P450 expression, including neuroendocrine mechanisms. These studies aim to understand the role of nervous system in the physiological and molecular regulation of hepatic and cerebral cytochrome P450 and to explain the influence of psychotropic drugs on this enzyme, which may be considered as a novel aspect of drug action.

The results of the above research allow to gain a better understanding of physiological and pharmacological role of cytochrome P450 in the brain and liver, and the influence of psychotropic drugs on this enzyme. Studies conducted in our Department are intended to assess the ability of psychotropic drugs to reach their site of action in the brain and their influence on metabolism of endogenous neuroactive substrates (looking for new mechanisms of drug action) and drugs (finding possible interactions between drugs).

Research methods

The Department of Pharmacokinetics and Drug Metabolism uses in vivo (systemic or intra-cerebral administration of substances, brain microdialysis), ex vivo (preparation of microsomal fractions) and in vitro (determination of drug and neurotransmitter concentration, enzymatic reactions and measurement of specific substrates and metabolites, measurement of enzyme expression, determination of hormone and cytokine levels) research methods.

Pharmacokinetic and biochemical methods:

- measurement of drugs and their metabolite concentration in biological material (plasma, brain, other tissues) using high-performance liquid chromatography (HPLC) with UV or fluorimetric detection and liquid chromatography coupled to LC/ESI-MS/MS tandem mass spectrometry;

- determination of monoaminergic neurotransmitters and their metabolite levels in plasma and brain structures, using high-performance liquid chromatography (HPLC) with electrochemical (coulometric) detection;

- in vitro measurement of cytochrome P450 enzyme activity, using recombinant cytochrome P450 enzymes, liver and brain microsomes, hepatocytes and neuronal cells. Determination of specific substrates related to cytochrome P450 enzymes and their metabolites formed during enzymatic reaction using high performance liquid chromatography (HPLC) with UV or fluorimetric detection and liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS);

- measurement of brain and liver expression of cytochrome P450 genes, nuclear receptors and transcription factors (Western blotting, qRT-PCR);

- determination of serum hormones and cytokines (ELISA).

- molecular biology: gene and recombinant DNA vector cloning, genomic PCR, RT-PCR, qPCR, DNA sequencing analysis, site-directed mutagenesis, southern blot, northern blot, DNA and RNA isolation and analysis, high titer AAV and lentivirus production (iodixanol and cesium

- cell biology: cell culture, DNA and RNA transfection, CRISPR/Cas9 knockout and direct mutagenesis, lentivirus target gene expression, flow cytometry, live cell imaging, cell lysis techniques, immunofluorescence, RNAi (with siRNA, shRNA, and lentivirus), multiwell plate-based cytotoxicity and proliferation assays (MTS, MTT), DNA damage assay (Comet Assay), protein level expression and genotyping, UV-visible absorption and fluorescence spectroscopy, bright field and fluorescence microscopy, fluorimeter and plate reading assays

- protein biochemistry: recombinant protein vector expression and purification, separation of proteins by FPLC (AKTA), surface plasmon resonance (SPR-Biacore), Microarray Printing Platform (Aushon 2470), antibody purification, Yeast Two-Hybrid, enzyme kinetics, protein quantization with Bradford methods, immunoprecipitation, and western blot analysis, dot blot analysis, ELISA, in vivo and in vitro labelling of proteins

Other:

- stereotactic implantation of cannula into the brain structures;

- intra-cerebral drug administration;

- brain microdialysis;

- brain dissection;

- preparation of brain and liver microsomes (differential centrifugation).

The most important recent discoveries

(1) Studies performed using brain microdialysis technique revealed that cytochrome P450 2D (CYP2D) is involved in the synthesis of dopamine and serotonin in the brain via alternative pathway.

 (2) In vivo studies performed in animal model of depression showed that chronic mild stress (CMS) stimulates the CYP2D activity in the hippocampus and triggers the stimulatory effect of antidepressants on CYP2D in other brain structures.

(3) In vivo studies, performed after injury or activation of the brain monoaminergic systems (dopaminergic, noradrenergic or serotonergic) have demonstrated that those systems are involved in the neuroendocrine regulation of cytochrome P450 expression and activity in the liver.

(4) In vitro and in vivo studies showed that novel atypical neuroleptics influence liver and brain cytochrome P450 expression and activity via different direct and indirect mechanisms including neuroendocrine regulation.

(5)  Studies with brain deficiency of serotonin (TPH-2 knockout rats) revealed sex-differences in the regulation of brain cytochrome P450 2D (CYP2D).

Recent publications (selected)

The mechanisms of interactions of psychotropic drugs with liver and brain cytochrome P450 and their significance for drug effect and drug-drug interactions. Daniel WA, Bromek E, Danek PJ, Haduch A. Biochem Pharmacol. 2022, doi: 10.1016/j.bcp.2022.115006. 

The regulation of liver cytochrome P450 expression and activity by the brain serotonergic system in different experimental models. Bromek E, Daniel WA. Expert Opin Drug Metab Toxicol. 2021, doi: 10.1080/17425255.2021.1872543.

Cytochrome P450 2D (CYP2D) enzyme dysfunction associated with aging and serotonin deficiency in the brain and liver of female Dark Agouti rats. Haduch A, Danek PJ, Kuban W, Pukło R, Alenina N, Gołębiowska J, Popik P, Bader M, Daniel WA. Neurochem Int. 2022, doi: 10.1016/j.neuint.2021.105223.

Long-Term Treatment with Atypical Antipsychotic Iloperidone Modulates Cytochrome P450 2D (CYP2D) Expression and Activity in the Liver and Brain via Different Mechanisms. Danek PJ, Daniel WA. Cells, 2021, doi: 10.3390/cells10123472.

The Effect of Chronic Iloperidone Treatment on Cytochrome P450 Expression and Activity in the Rat Liver: Involvement of Neuroendocrine Mechanisms. Danek PJ, Kuban W, Daniel WA. Int J Mol Sci. 2021, doi: 10.3390/ijms22168447.

The Influence of Long-Term Treatment with Asenapine on Liver Cytochrome P450 Expression and Activity in the Rat. The Involvement of Different Mechanisms. Danek PJ, Bromek E, Daniel WA. Pharmaceuticals (Basel) 2021, doi: 10.3390/ph14070629.

Cytochrome P450 expression and regulation in the brain. Kuban W, Daniel WA. Drug Metab Rev. 2021, doi: 10.1080/03602532.2020.1858856.

The effect of ageing and cerebral serotonin deficit on the activity of cytochrome P450 2D (CYP2D) in the brain and liver of male rats. Haduch A, Pukło R, Alenina N, Nikiforuk A, Popik P, Bader M, Daniel WA. Neurochem Int. 2020, doi: 10.1016/j.neuint.2020.104884.

The activity of brain and liver cytochrome P450 2D (CYP2D) is differently affected by antidepressants in the chronic mild stress (CMS) model of depression in the rat. Haduch A, Rysz M, Papp M, Daniel WA. Biochem Pharmacol. 2018, doi: 10.1016/j.bcp.2018.09.005.

The engagement of brain cytochrome P450 in the metabolism of endogenous neuroactive substrates: a possible role in mental disorders. Haduch A, Daniel WA. Drug Metab Rev. 2018, doi: 10.1080/03602532.2018.1554674.