The effect of new atypical antipsychotic drugs on the expression of transcription factors regulating cytochrome P450 enzymes in rat liver

Background Our recent studies showed that prolonged administration of novel atypical antipsychotics affected the expression and activity of cytochrome P450 (CYP), as demonstrated in vitro on human hepatocytes and in vivo on the rat liver. The aim of the present work was to study the effect of repeated treatment with asenapine, iloperidone, and lurasidone on the expression of transcription factors regulating CYP drug-metabolizing enzymes in rat liver. Methods The hepatic mRNA (qRT-PCR) and protein levels (Western blotting) of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPARγ) were measured in male Wistar rats after 2 week-treatment with asenapine, iloperidone or lurasidone. Results The 2-week treatment with asenapine significantly diminished the AhR and PXR expression (mRNA, protein level), and CAR mRNA level in rat liver. Iloperidone lowered the AhR and CAR expression and PXR protein level. Lurasidone did not affect the expression of AhR and CAR, but increased PXR expression. The antipsychotics did not affect PPARγ. Conclusions Prolonged treatment with asenapine, iloperidone, or lurasidone affects the expression of transcription factors regulating the CYP drug-metabolizing enzymes. The changes in the expression of AhR, CAR, and PXR mostly correlate with alterations in the expression and activity of respective CYP enzymes found in our previous studies. Since these transcription factors are also engaged in the expression of phase II drug metabolism and drug transporters, changes in their expression may affect the metabolism of endogenous substrates and pharmacokinetics of concomitantly used drugs. Supplementary Information The online version contains supplementary material available at 10.1007/s43440-024-00608-2.

After prolonged treatment, antipsychotics can affect cytochrome P450 enzymes' expression and activity, which has been shown in vitro on human hepatocytes and in vivo on the rat liver (Table 1).Specifically, when added to the cell culture for 5 days, levomepromazine and clozapine enhanced the mRNA level and activity of CYP3A4, not affecting CYP1A1/2, CYP2C9 and CYP2C19 [7].In contrast, iloperidone diminished the mRNA level and activity of CYP3A4, asenapine diminished those of CYP1A2, while lurasidone did not affect the investigated CYP enzymes [8].
When given to rats for two weeks, the atypical neuroleptics: asenapine and iloperidone reduced the expression and activity of CYP1A, CYP2B, CYP2C11, and CYP3A enzymes in the liver, not affecting the CYP2C6 enzyme [9,10].On the other hand, lurasidone did not affect human CYP enzymes in hepatocyte culture, but decreased the expression of CYP2B1/2 and CYP2C11, and enhanced that of CYP3A1/2 in rat liver after chronic treatment [11].
The above-presented data indicate similarities in the downregulation of CYP1A by asenapine and of CYP3A by iloperidone in humans and rats.However, the difference in the effects of lurasidone between in vitro experiments on human hepatocyte culture and in vivo experiments performed in rats has been noticed, namely, lurasidone enhanced the CYP3A expression in rat liver, but not in human hepatocyte culture.Although there are similarities in the expression regulation of CYP enzymes in humans and rats, some differences between the two species have also been noticed, e.g. in the induction of CYP3A subfamily enzymes by rifampicin and dexamethasone [12,13].Moreover, in vivo, additional mechanisms are involved in CYP enzyme regulation by neuroactive drugs, including neuroendocrine and neuroimmune pathways [14][15][16].
Taking into account the contribution of cytoplasmic aryl hydrocarbon receptor (AhR) to the regulation of CYP1A expression and the involvement of nuclear receptors: pregnane X receptor (PXR), constitutive androstane receptor (CAR) and retinoid X receptor (RXR) in the expression of CYP3A and other CYP genes in humans and rodents [17][18][19][20] it can be expected that asenapine interacts with human and rat AhR, iloperidone interplays with PXR and/or CAR, while a possibility of cooperation of lurasidone with the mentioned CYP transcription factors is difficult to assess at this stage of studies.Thus, the molecular mechanisms of CYP enzyme regulation by antipsychotics in the liver need further investigation.
Table 1 The effect of asenapine, iloperidone and lurasidone on the mRNA, protein level and activity of cytochrome P450 enzymes in the liver of male rats treated with one of the antipsychotics for two weeks (A), and in human hepatocyte culture after 5 day-exposure to one of the antipsychotics (B) ↓ a decrease, ↑ an increase, -no change, nt not tested The effect of new atypical antipsychotic drugs on the expression of transcription factors… The present study aimed to identify the effect of 2 weektreatment treatment with the new atypical antipsychotic drugs: asenapine, iloperidone, and lurasidone on the expression of transcription factors regulating the CYP drug-metabolizing enzymes in rat liver.

Animals
The experiments were conducted on male Wistar Han rats (3 months old and weighing 280-300 g) from Charles River Laboratories, Sulzfeld, Germany.The rats were housed under standard laboratory conditions (

Determination of total protein in the liver
The frozen livers (20 mg) were homogenized (TissueLyser, Qiagen, Germany) in ice-cold Tissue Extraction Reagent I (Thermo Fisher Scientific, Walthman, MA, USA) supplemented with 1 mM PMSF (Sigma, Sigma-Aldrich, St. Louis, MO, USA; product number P7626).Then, the homogenates were centrifuged for 15 min at 15,000 × g at 4 °C, and supernatants were collected.The total protein content in the sample was assessed by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Walthman, MA, USA; product number 23225).

Data analysis
The antipsychotic drugs asenapine, iloperidone, and lurasidone were investigated in separate experiments.Therefore, the results of each experiment were calculated and statistically analyzed separately.The Kolmogorov-Smirnov test was used to assess normality.Changes in the mRNA and protein expression levels of transcription factors were statistically assessed using an unpaired Student's t-test (GraphPad Prism 10 Software, Inc., La Jolla, CA).The obtained values were indicated as the mean ± SD and were recognized as significant when p ≤ 0.05.

Results and discussion
This is the first report showing that prolonged treatment with the new atypical antipsychotic drugs: asenapine, iloperidone, and lurasidone affects the expression of transcription factors regulating the cytochrome P450 drug-metabolizing enzymes.The observed changes in the expression of transcription factors AhR, CAR, and PXR mostly correlate with the alterations in the expression and activity of respective CYP enzymes found in our previous studies in rats [9][10][11] and humans [8], after prolonged exposure to the antipsychotics (Table 1).
The two-week treatment with asenapine significantly decreased the expression (mRNA and protein level) of AhR (Figs. 1, 2) in rat liver, which corresponded with the reduction in the CYP1A2 expression and activity after two-week treatment of rats with this antipsychotic drug, observed in previous experiments [9].Simultaneously, asenapine decreased PXR expression and CAR mRNA level, which, in turn, was consistent with the depletion of CYP2B1 and CYP3A1 expression and activity found earlier in those animals (Figs. 1, 2).Both transcription factors are engaged in the regulation of those two CYP subfamily enzymes to different degrees.CAR is the main regulator of CYP2Bs, while PXR governs the expression of CYP3As [19].The above results concerning the effect of asenapine on rat AhR and CYP1A2 enzyme in vivo remain in agreement with the results obtained for the corresponding CYP1A2 enzyme in humans in vitro.Just as in rats in vivo, asenapine decreased the CYP1A2 mRNA and activity in humans in vitro after 5 day-incubation with hepatocyte culture [8] (Table 1).
Prolonged administration of iloperidone exerted similar effects on the expression of the investigated transcription factors in rat liver.Iloperidone lowered the expression of AhR (mRNA and protein level) (Figs. 1, 2), which was Fig. 1 The effect of asenapine (0.3 mg/kg sc), iloperidone (1 mg/kg ip), and lurasidone (1 mg/kg ip) on the mRNA level of the transcription factors PXR, CAR, AhR, and PPARγ in rat liver, after 2 week-treatment with one of the antipsychotics.All values are shown as the mean ± SD (n = 8).The results were evaluated statistically using Student's t-test: *p < 0.05; ****p < 0.0001 versus control group (asenapine PXR t 14 = 2.164, p = 0.048; lurasidone PXR t 14 = 2.438, p = 0.0287; aseanpine CAR t 14 = 2.299, p = 0.0374; iloperidone CAR t 14 = 23.79,p < 0.0001; asenapine AHR t 14 = 2.171, p = 0.0476; iloperidone AhR t 14 = 2.261, p = 0.0402) consistent with the reduced CYP1A expression and activity observed after prolonged antipsychotic treatment in our previous experiment [10].However, iloperidone did not influence the CYP1A2 expression in human hepatocytes, which may suggest different amino acid sequence in the ligand binding domain of AhR receptor between species [8,21].At the same time, iloperidone diminished CAR expression and PXR protein level, but not PXR mRNA level (Figs. 1, 2).The observed discrepancy between PXR mRNA and protein level may result from the decreased stability of PXR protein.The revealed decreases in the level of CAR and PXR proteins evoked by iloperidone remain in agreement with the decrease in the expression and activity of CYP2B1/2 and CYP3A1/2 enzymes in the liver of chronically treated rats, detected in our earlier studies [10].In this case, the results obtained in rats correlated positively with those concerning changes in the CYP3A4 mRNA and activity produced by iloperidone in human hepatocyte culture [8] (Table 1).
In contrast to asenapine and iloperidone, lurasidone did not affect the expression of AhR and CAR but increased the expression of PXR (mRNA and protein level) (Figs. 1, 2), which corresponded with the lack of lurasidone effect on CYP1A expression and activity, and enhanced expression and activity of CYP3A1/2 (but not CYP2B) enzymes found in our previous study in those animals [11].Since PXR is not the main regulator of CYP2B enzymes and the CAR expression was not changed by lurasidone, the observed reduction in the CYP2B expression in the rat may be ascribed to changes in hormone levels (decreased growth hormone and corticosterone, and increased T 3 level), as observed for other antipsychotics in rats in vivo [9,10].But the lack of effect of lurasidone on CYP3A4 noticed in human hepatocyte culture [7] compared to the increase in the CYP3A1/2 [11] and PXR expression observed in the rat liver in vivo may be explained by structural differences in the CYP3A genes' responsive elements or PXR protein, which leads to disparate regulation of CYP3A enzymes in humans and rodents [19,22].The above discrepancy in the regulation of CYP3A expression by lurasidone may also be related to structural and functional differences between human and rat glucocorticoid receptor (GR), which takes part in the regulation of PXR and CAR expression [18].Thus, rifampicin is a strong PXR and CYP3A4 inducer in humans, dexamethasone is a much more efficient inducer of those proteins in rats than in humans, while pregnenolone 16α-carbonitrile has been found to be a specific PXR ligand and CYP3A inducer in rats [12,23,24].On the other hand, other mechanisms may also be engaged in the regulation of cytochrome P450 by neuroactive drugs in vivo, which cannot be seen in hepatocyte culture, such as the presence of high concentrations  of drug metabolites, which may exert different effects on cytochrome P450 expression than their parent compounds.The investigated antipsychotics did not affect the expression of the transcription factor PPARγ, which is involved in the regulation of hepatic lipid metabolism and expression of CYP2D enzymes in the brain [25,26].However, it cannot be excluded that some of the tested drugs may act as direct ligands of cytoplasmic/nuclear receptors regulating CYP genes [25], which requires further molecular studies.Such a possibility was suggested for the typical antipsychotic chlorpromazine in relation to CAR [18,28].
In summary, the results obtained in rats indicate that the atypical antipsychotics: asenapine, iloperidone, and lurasidone influence the expression of the transcription factors AhR, PXR, and CAR engaged in the regulation of drugmetabolizing enzymes.Further studies are advisable to find out whether similar changes in the expression of transcription factors are evoked by the tested antipsychotics in humans.The issue seems to be of high importance, since these transcription factors are involved in the expression of enzymes catalyzing phase I drug metabolism (CYP1-3 enzymes) and phase II drug biotransformation (UDP-glucuronosyltransferase, sulfotransferase, glutathione S-transferase), and in the expression of drug transporters (P-glycoprotein, MRPs, OATP2) (reviewed in [29]).Thus, the investigated antipsychotics may affect the pharmacokinetics of concurrently administered drugs.Moreover, some endogenous, physiologically important substances are substrates for phase I and/or phase II drug-metabolizing enzymes (e.g.steroid hormones, cholesterol derivatives, bile acids, thyroid hormones) (discussed in [30]).Therefore, changes in the expression level of the investigated transcription factors may contribute to potential adverse effects of those antipsychotic drugs and drug-drug interactions.

Fig. 2
Fig.2The effect of asenapine (0.3 mg/kg sc), iloperidone (1 mg/kg ip), and lurasidone (1 mg/kg ip) on the protein level of the transcription factors PXR, CAR, AhR, and PPARγ in rat liver, after 2 weektreatment with one of the antipsychotics.All values are shown as the mean ± SD (n = 12).The results were evaluated statistically using the Local Ethics Commission for Experimentation on Animals at the Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków.