Przemysław Danek, MSc - 2022

DOCTORAL DISERTATION

Interakcje nowych atypowych neuroleptyków z cytochromem P450

Przemysław Danek

Department of Pharmacokinetics and Drug Metabolism Maj Institute of Pharmacology Polish Academy of Science

Cytochrome P450 enzymes are responsible for the metabolism of most drugs and other xenobiotics (promutagens, procarcinogens) and endogenous substances, such as cholesterol, steroids, monoaminergic neurotransmitters and vitamins. The highest concentrations of these enzymes are found in the liver, where detoxification processes take place in the body, but they have also been identified in other tissues, such as the brain. During treating schizophrenia, neuroleptics are administered for a long time and may be combined with other psychotropic drugs or drugs belonging to other pharmacological groups (e.g. with cardiological drugs or antibiotics), which patients are taking simultaneously. This can lead to pharmacokinetic interactions at the level of cytochrome P450 metabolism that are sometimes dangerous for patients. Due to the differences in the qualitative and quantitative composition of the cytochrome P450 enzymes in the brain and liver, as well as the differences in the regulation of enzymes in individual organs or tissues, it seems advisable to investigate the effect of long–term administration of new atypical neuroleptics on the expression of cytochrome P450 not only in the liver, but also in the brain, i.e. at the site of action of psychotropic drugs. The aim of this dissertation was to investigate the influence of atypical neuroleptics – asenapine, iloperidone and lurasidone on the activity and expression of cytochrome P450 in the brain and liver. For this purpose, several experimental models were used. Model I investigated the direct effect of drugs on the activity of CYP enzymes in the human liver. Human liver microsomes and supersomes containing recombinant human CYP enzymes with defined expression were used in this experimental model. The rate of specific metabolic reactions was measured in the absence and presence of an in vitro added antipsychotic. The obtained results indicate that asenapine strongly inhibited CYP1A2 and CYP2D6 and weakly decreased the activity of CYP3A4. Iloperidone strongly inhibited CYP3A4 and CYP2D6. In addition, it moderately decreased the activity of CYP2C19 and had little effect on CYP1A2. Strong inhibition of CYP2C9, moderate inhibition of CYP1A2 and CYP2C19, and CYP3A4 were observed with lurasidone. Model II investigated the indirect effect of drugs on the activity and expression of CYP enzymes in the human liver. For this purpose, cultures of human cryopreserved hepatocytes were used, which had been treated with drugs for 72 h. The rate of specific metabolic reactions was measured with the specific substrates added to the incubation medium, and the expression of CYP genes was measured by determining the mRNA level of CYP enzymes in liver cells. In human hepatocyte culture, asenapine and iloperidone at therapeutic concentrations reduce the expression and activity of the enzymes CYP1A2 and CYP3A4, respectively. Lurasidone in this study did not influence on the activity and expression of the tested CYP enzymes. Strong inhibition of CYP enzymes by the tested neuroleptics may slow down the metabolism of substrates of these enzymes in vivo and increase their concentration, which may result in a higher frequency and/or severity of side effects. Model III examined the direct effects of drugs on CYP2D enzyme activity in rat liver and brain. Rat liver microsomes control and whole brain brain microsomes were used in this experimental model. The rate of the specific metabolic reaction was measured in the absence and presence of an in vitro added antipsychotic. Atypical neuroleptics inhibit the activity of the enzyme CYP2D (by binding to the enzyme) in the brain microsomes. The Ki inhibition constants determined for the brain microsomes are significantly higher than those determined for the liver microsomes. This effect may be due to a lower affinity of test drugs for the CYP2D active site in the brain than for CYP2D in the liver. Model IV investigated the effect of chronic drug administration on the activity and expression of CYP enzymes in the rat liver. Liver microsomes from animals treated with the drug for two weeks were used in this experiment. Specific substrates were added to the in vitro incubation mixture, and the rate of specific reactions was tested in the absence of drugs which, when administered chronically in vivo, were washed out in vitro. In the obtained microsomes, the protein level of the respective CYP enzymes was measured, as well as the mRNA level of the respective CYP enzyme genes. Additionally, the levels of hormones regulationg the expression of CYP genes in the serum and in the pituitary glands were determined using ELISA kits. The obtained results indicate that chronic administration of asenapine and iloperidone to rats lowers the expression (mRNA and protein levels) and activity of cytochrome P450 and may slow down the metabolism of CYP1A, CYP2B, CYP2C11, CYP2D and CYP3A substrates (steroids and / or drugs). The observed effect of the studied drugs on cytochrome P450 may be related to their pharmacological action (in particular on D2, 5–HT1A, 5–HT2C and α2 receptors), which affects the endocrine regulation of hepatic CYP enzymes and signaling pathways mediating in the expression of enzymes in the liver. Model V investigated the effect of chronic drug administration on the activity and expression of the CYP2D enzyme in the rat brain. In this model, microsomes from the appropriate brain structures of rats that had been administered the drug for a period of two weeks were used. The specific substrate was added to the in vitro incubation mixture, and the rate of the specific reaction was tested in the absence of drugs which, when administered chronically in vivo, were washed out in vitro. The level of CYP2D4 protein and mRNA was also measured. The atypical neuroleptics under study act on the enzyme CYP2D in the brain in a manner specific for the structure of the brain. By modulating CYP enzymes in the brain, these drugs can affect the local metabolism of drugs and endogenous substances such as neurosteroids and monoaminergic messengers. Reduction of CYP2D activity by the studied drugs may slow the oxidative metabolism of neurosteroids (via 21–hydroxylation) and have a beneficial effect on symptoms of schizophrenia. On the other hand, an increase in CYP2D activity in selected brain structures (the nigrostriatal pathway) may increase the local synthesis of dopamine (through tyramine hydroxylation) and serotonin (through O–demethylation of 5– methoxytryptamine), and thus alleviate extrapyramidal symptoms. It therefore seems possible that psychotropic drugs may affect the cytochrome P450 in the liver through various mechanisms involving direct and indirect mechanisms. The obtained results indicate the need to test new neuroactive drugs in terms of their interaction with the cytochrome P450 not only in vitro, but also in vivo, which allows for the observation of the full spectrum of their mechanisms of action on the expression and activity of cytochrome P450 operating in the organism, including their action in the brain and the neuroendocrine regulation of the enzyme