The Atypical Antipsychotic Lurasidone Affects Brain but Not Liver Cytochrome P450 2D (CYP2D) Activity. A Comparison with Other Novel Neuroleptics and Significance for Drug Treatment of Schizophrenia

The aim of this work was to study the effect of prolonged lurasidone administration on the cytochrome 2D (CYP2D) expression and activity in the rat liver and selected brain structures involved in the therapeutic or side effects of this neuroleptic. Male Wistar rats received lurasidone (1 mg/kg ip.) for two weeks. The activity of CYP2D was measured in brain and liver microsomes as the rate of bufuralol 1′-hydroxylation. The CYP2D protein level was determined in microsomes by Western blot analysis. The CYP2D gene expression was estimated in liver tissue by a qRT-PCR method. Lurasidone decreased the activity and protein level of CYP2D in the frontal cortex but increased them in the striatum, nucleus accumbens, brain stem, substantia nigra, and the remainder of the brain. The neuroleptic did not affect CYP2D in the hippocampus, hypothalamus, and cerebellum. In the liver, lurasidone did not affect the CYP2D activity and protein level, though it enhanced the mRNA of CYP2D1 without affecting that of CYP2D2, CYP2D3, CYP2D4, and CYP2D5. In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns the frontal cortex (down-regulation) and nigrostriatal pathway (up-regulation) and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which individual monoaminergic receptors/signaling pathways are involved in the regulation of the rat CYP2D4 and human CYP2D6 enzyme in particular brain structures.


Introduction
Cytochrome P450 (CYP) enzymes are a family of heme-containing monooxygenases responsible for metabolizing xenobiotic and endogenous compounds. CYP2D is an important enzyme, as it metabolizes approximately 20% of clinically used drugs, including CNS and cardiovascular system therapeutics, neurotoxins, and endogenous compounds [1,2].
CYP2D enzymes have been detected in various species. In humans, only one enzyme (CYP2D6) is expressed in various tissues, while rats have six enzymes (CYP2D1, 2, 3, 4, 5, and 18) with unique substrate specificity, expression, metabolism, and inhibition properties [3,4]. In the rat, the enzymes CYP2D1 and CYP2D2 are the most abundant CYP2D isoforms in the liver, while CYP2D4 is mostly expressed in the brain [5]. However, rat CYP2D enzymes collectively are a useful model of human CYP2D due to their relatively high amino acid sequence identity (>70%) and the ability to perform metabolic reactions in vitro similar to human CYP2D6 [6].
CYPs are expressed in the brain of numerous animals (mice, rats, monkeys, dogs, and humans), where they may contribute to the local metabolism of exogenous and endogenous compounds and to neuroprotection [7]. Moreover, brain CYP2Ds were shown to

Animal Treatment and Preparation of Brain and Liver Microsomes
Rats (n = 15 for each treatment group) received intraperitoneally a pharmacological dose of lurasidone (1 mg/kg ip.) or vehicle (0.5% methylcellulose and 0.2% Tween 80 in sterile water) once daily for two weeks [18][19][20]. Rats were sacrificed by decapitation 24 h after the last dose. Whole livers and brains were removed, and the selected brain structures (in accordance with the Paxinos and Watson atlas [21]) (the cerebellum, brain stem, substantia nigra, hippocampus, frontal cortex, hypothalamus, striatum, nucleus accumbens, and the remainder of the brain) were isolated. All those tissues were immediately frozen in dry ice and then stored at −80 • C until analysis. The differential centrifugation methods [22,23] were used to obtain liver and brain microsomes.

Measurement of the CYP2D Enzyme Activity in Brain and Liver Microsomes
The CYP2D enzyme activity was determined using the CYP2D specific metabolic reaction, i.e., 1 -hydroxylation of bufuralol. Briefly, the incubation system included: 2 mM potassium phosphate buffer (pH = 7.4) and NADPH generating system (1.6 mM NADP, 4 mM MgCl 2 , 5 mM glucose 6-phosphate, and 2.5 U glucose 6-phosphate-dehydrogenase). The protein concentration in microsomes derived from selected brain structures of 1-3 rats (mg of protein/mL: ca. 0.3 for the nucleus accumbens and substantia nigra; 2.8 for the frontal cortex and remainder of the brain; 1.8 for the hippocampus, hypothalamus and the cerebellum; 1.2 for the brain stem and the striatum) or liver microsomes (0.5 mg of protein/mL) was used, as described earlier [15,24]. The appropriate concentrations of bufuralol (brain microsomes-125 µM or liver microsomes-10 µM) were applied in the final volume of 0.4 mL. The incubation time for brain microsomes was 60 min and for liver microsomes was 10 min at a temperature of 37 • C.
In all experiments, the amount of specific metabolic product (1 -hydroxybufuralol formed from bufuralol) was measured by an HPLC method with fluorometric detection [25].

Evaluation of CYP2D Protein in Brain and Liver Microsomes
The CYP2D protein levels in the brain and liver microsomes of control and lurasidonetreated rats were estimated by Western immunoblot analysis, as described previously [15]. The total protein concentration in samples was determined in the microsomes using the Lowry methods [26]. All samples were heated in a Laemmli sample buffer for 5 min at 100 • C. Next, the microsomal proteins (10 µg) were separated using an SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The blots were probed with the following primary antibodies: rabbit antirat CYP2D4 (1:1000) or rabbit antihuman CYP2D6 (1:2000). In order to dilute the primary and secondary antibodies, the specified solutions from the SignalBoost™ Immunoreaction Enhancer Kit (Merck Millipore, Burlington, MA, USA) were used. As a positive control, we used rat cDNA-expressed CYP2D4 (2.5 µg) or human CYP2D6 (1 µg). The blots were visualized using the Luminescent Image Analyzer LAS-1000 and Image Gauge 3.11 programs (Fuji Film, Tokyo, Japan). The results were normalized to β-actin protein level.

Examination of the Expression of Genes Coding for CYP2D Enzymes in the Liver
The total RNA was extracted from the liver samples using Total RNA Mini kit. RNA quality was determined by chip-based capillary electrophoresis using an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). The quantity of the isolated RNA was verified using a Synergy/HTX multimode reader (BioTek, Winoosk, VT, USA). RNA (1 µg) was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit. RT-Cells 2022, 11, 3513 4 of 11 PCR was performed using the Bio-Rad CFX96 PCR system (Bio-Rad, Hercules, CA, USA). TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) were used, including Rn01775090_mH (CYP2D1), Rn00562419_m1 (CYP2D2), Rn00597330_m1 (CYP2D3), Rn00593393_m1 (CYP2D4), Rn01790051_s1 (CYP2D5), and Rn00667869_m1 (ACTB), which was used for normalization. Gene expression was determined using 2-delta Ct method, as reported previously [27].

Data Analysis
The statistical significance of alterations in enzyme activity, protein level, or gene expression was calculated using Student's t-test compared to the control value (GraphPad Prism 8.0; GraphPad Prism Software Inc., CA, USA). The results of chronic lurasidone treatment are reported as the mean ± S.E.M. The changes were considered as statically significant when p < 0.05.

The Effect of Chronic Lurasidone Treatment on the CYP2D Activity in the Brain and Liver Microsomes of Rats
The activity of CYP2D measured as the rate of bufuralol 1 -hydroxylation was significantly changed in different brain regions after prolonged administration of lurasidone ( Figure 1). A two-week treatment with lurasidone diminished the activity of CYP2D in the frontal cortex (to 77% of control) but increased the enzyme activity in the striatum (to 131% of the control), nucleus accumbens (to 182% of control), brain stem (to 133% of control), substantia nigra (to 156% of control), and the remainder of the brain (to 115% of control). In contrast, the examined neuroleptic did not affect significantly the CYP2D activity in the hippocampus, hypothalamus, cerebellum, and in the liver (Table 1).

Examination of the Expression of Genes Coding for CYP2D Enzymes in the Liver
The total RNA was extracted from the liver samples using Total RNA Mini kit. RNA quality was determined by chip-based capillary electrophoresis using an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). The quantity of the isolated RNA was verified using a Synergy/HTX multimode reader (BioTek, Winoosk, VT, USA). RNA (1 μg) was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit. RT-PCR was performed using the Bio-Rad CFX96 PCR system (Bio-Rad, Hercules, CA, USA). TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) were used, including Rn01775090_mH (CYP2D1), Rn00562419_m1 (CYP2D2), Rn00597330_m1 (CYP2D3), Rn00593393_m1 (CYP2D4), Rn01790051_s1 (CYP2D5), and Rn00667869_m1 (ACTB), which was used for normalization. Gene expression was determined using 2delta Ct method, as reported previously [27].

Data Analysis
The statistical significance of alterations in enzyme activity, protein level, or gene expression was calculated using Student's t-test compared to the control value (GraphPad Prism 8.0; GraphPad Prism Software Inc., CA, USA). The results of chronic lurasidone treatment are reported as the mean ± S.E.M. The changes were considered as statically significant when p < 0.05.

The Effect of Chronic Lurasidone Treatment on the CYP2D Activity in the Brain and Liver Microsomes of Rats
The activity of CYP2D measured as the rate of bufuralol 1′-hydroxylation was significantly changed in different brain regions after prolonged administration of lurasidone ( Figure 1). A two-week treatment with lurasidone diminished the activity of CYP2D in the frontal cortex (to 77% of control) but increased the enzyme activity in the striatum (to 131% of the control), nucleus accumbens (to 182% of control), brain stem (to 133% of control), substantia nigra (to 156% of control), and the remainder of the brain (to 115% of control). In contrast, the examined neuroleptic did not affect significantly the CYP2D activity in the hippocampus, hypothalamus, cerebellum, and in the liver (Table 1). Figure 1. The influence of the two-week treatment with lurasidone on the CYP2D activity measured in microsomes derived from the selected brain structures or liver. The presented values are the means ± S.E.M. of 15 samples (from 15 animals) for the cerebellum, remainder of brain, and the liver; of 7 samples (each sample contained 2 pooled brain structures from 2 animals) for the frontal cortex; brain stem, striatum, and hippocampus; and of 5 samples (each sample contained 3 pooled brain structures from 3 animals) for the hypothalamus, nucleus accumbens, and substantia nigra. Student's t-test: * p < 0.05; ** p < 0.01 vs. control group. FCx-the frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, Bs-the brain stem, SN-the substantia nigra, CB-the cerebellum, and RM-the remainder.

The Effect of Chronic Lurasidone Treatment on the CYP2D Protein Level in Microsomes from the Brain and Liver
The CYP2D protein level was measured by Western blot analysis in the microsomes from selected brain structures of control and lurasidone-treated rats. Lurasidone exerted a significant effect on the CYP2D protein level in the brain. The observed changes in CYP2D protein level in most cases corresponded well with the changes in CYP2D activities. Lurasidone diminished the protein level in the frontal cortex (down to 84.5% of control) and in the hypothalamus (down to 66% of control) but enhanced the enzyme protein level in the striatum (up to 157% of control), nucleus accumbens (up to 131% of control, not significant), brain stem (up to 128% of control), substantia nigra (up to 131% of control), and in the remainder of the brain (up to 122% of control) ( Figure 2). Lurasidone did not significantly affect the CYP2D4 protein level in the hippocampus, cerebellum, and in the liver (Table 1).

The Effect of Chronic Lurasidone Treatment on the CYP2D Gene Expression in the Rat Liver
The mRNA levels of CYP2D enzymes, i.e., CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D5 were measured in the liver. Lurasidone produced a significant increase in the mRNA level of CYP2D1 gene, up to 151% of the control value ( Figure 3). However, the neuroleptic did not significantly influence the expression of other examined CYP2D genes in the liver.
Considering the small size of brain structures (in particular, in the case of the nucleus accumbens and substantia nigra, we pooled three structures per one sample to measure the activity) and the fact that brain CYP2D may be induced at the posttranscriptional level, the effects of lurasidone on the CYP2D mRNA levels were not investigated in the brain in this study. The effect of two-week treatment with lurasidone on the CYP2D protein levels measured in microsomes derived from the selected brain structures or liver. The presented values are the means ± S.E.M. of 15 samples (from 15 animals) for the cerebellum, remainder of brain, and the liver; of 7 samples (each sample contained 2 pooled brain structures from 2 animals) for the frontal cortex, brain stem, striatum, and hippocampus; or of 5 samples (each sample contained 3 pooled brain structured from 3 animals) for the hypothalamus, nucleus accumbens, and substantia nigra. The representative CYP2D protein bands of the Western blot analysis are shown. Brain or liver microsomal protein (10 µ g) was subjected to Western blot analysis. cDNA-expressed CYP2D4 protein (Bactosomes) and cDNA-expressed CYP2D6 protein (Supersomes) was used as a positive control. Student's t-test: * p < 0.05 vs. control group. FCx-the frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, CB-the cerebellum, and RM-the remainder.

The Effect of Chronic Lurasidone Treatment on the CYP2D Gene Expression in the Rat Liver
The mRNA levels of CYP2D enzymes, i.e., CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D5 were measured in the liver. Lurasidone produced a significant increase in the mRNA level of CYP2D1 gene, up to 151% of the control value ( Figure 3). However, the neuroleptic did not significantly influence the expression of other examined CYP2D genes in the liver. The effect of two-week treatment with lurasidone on the CYP2D protein levels measured in microsomes derived from the selected brain structures or liver. The presented values are the means ± S.E.M. of 15 samples (from 15 animals) for the cerebellum, remainder of brain, and the liver; of 7 samples (each sample contained 2 pooled brain structures from 2 animals) for the frontal cortex, brain stem, striatum, and hippocampus; or of 5 samples (each sample contained 3 pooled brain structured from 3 animals) for the hypothalamus, nucleus accumbens, and substantia nigra. The representative CYP2D protein bands of the Western blot analysis are shown. Brain or liver microsomal protein (10 µg) was subjected to Western blot analysis. cDNA-expressed CYP2D4 protein (Bactosomes) and cDNA-expressed CYP2D6 protein (Supersomes) was used as a positive control. Student's t-test: * p < 0.05 vs. control group. FCx-the frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, CB-the cerebellum, and RM-the remainder.  Considering the small size of brain structures (in particular, in the case of the nucleus accumbens and substantia nigra, we pooled three structures per one sample to measure the activity) and the fact that brain CYP2D may be induced at the posttranscriptional level, the effects of lurasidone on the CYP2D mRNA levels were not investigated in the brain in this study.

Discussion
The results of the present study show the impact of chronic administration of

Discussion
The results of the present study show the impact of chronic administration of lurasidone on cytochrome P450 2D (CYP2D) activity and expression in the brain and liver. They indicate that lurasidone exerts organ-and brain structure-depended effects on the CYP2D enzyme.
The effect of the tested neuroleptic on brain CYP2D is different than that observed in the liver. In the liver, no changes in the activity and protein level of the total hepatic CYP2D enzyme were observed after prolonged administration of lurasidone, though the mRNA of CYP2D1, i.e., one of the six CYP2Ds expressed in the liver (CYP2D1, 2, 3, 4, 5, 18) was moderately increased. However, in the brain, where CYP2D4 is mainly expressed, the activity and protein level of CYP2D was altered in a cerebral structure-dependent way.
Our previous in vitro studies carried out on human liver microsomes and cDNAexpressed human CYP2D6 Supersomes showed a weak potency of lurasidone to inhibit the enzyme activity [17]. Thus, no direct interaction of lurasidone with human CYP2D6 protein (observed earlier) and lack of changes in the liver CYP2D activity after chronic neuroleptic treatment (observed in the present study) indicate that lurasidone has no potential for metabolic drug-drug interactions with CYP2D substrates during their biotransformation in the liver.
However, 14-day treatment with lurasidone influenced brain CYP2D, and the effect varied between cerebral regions. Lurasidone diminished the CYP2D activity and protein level in the frontal cortex but significantly enhanced the enzyme activity and protein level in the striatum, nucleus accumbens, brain stem, substantia nigra, and the remainder of the brain, which may modify its pharmacological effect. Reducing the CYP2D activity in the frontal cortex lurasidone may slow down the oxidative metabolism of neurosteroids (via 21-hydroxylation), thereby exerting beneficial effects on the symptoms of schizophrenia. It was shown that neurosteroids have a wide range of potential clinical applications for the treatment of schizophrenia [28,29] due to their ability to modulate GABA A and NMDA receptors and neuroprotective properties [30,31].
In contrast, lurasidone increased the CYP2D activity and protein level in the striatum, substantia nigra, nucleus accumbens, brain stem, and remainder of the brain. Thus, lurasidone may increase the local synthesis of monoamonergic neurotransmitters catalyzed by CYP2D, i.e., tyramine hydroxylation to dopamine and 5-methoxytryptamine O-demethylation to serotonin [25,32]. The stimulation of CYP2D-mediated dopamine formation in the nigrostriatal pathway and serotonin formation in the brainstem may alleviate extrapyramidal symptoms of the neuroleptic [33,34].
The observation that lurasidone did not produce any changes in the expression and activity of liver CYP2D (in particular CYP2D4), while it did in the region-dependent manner in the brain (where CYP2D4 is the main CYP2D enzyme), implies that the neuroleptic exerted its effect on the brain enzyme via acting at monoaminergic receptors, and all the more so as similar effects on brain CYP2D in the frontal cortex and nigrostriatal pathway were observed for the atypical neuroleptics previously studied by us, namely, iloperidone and asenapine, which share with lurasidone similar receptor mechanisms in the brain [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D 2 , 5-HT 1A ) or positively (e.g., D 1 , 5-HT 2A , 5-HT 6 , 5-HT 7 ) via G-protein to adenylate cyclase or Cphospholipase and A 2 -phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D 1 receptor dominates over D 2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, Cells 2022, 11, 3513 8 of 11 the mentioned atypical neuroleptics display different affinities for dopaminergic D 1 , D 3 , D 4 , 5-HT 6 , α 1 , and histaminergic H 1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Table 2. The effects of novel atypical neuroleptic drugs on the CYP2D activity and protein level in the liver and brain.

Drug
Liver α2) Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Table 2. The effects of novel atypical neuroleptic drugs on the CYP2D activity and protein level in the liver and brain.

Drug
Liver α2) Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Table 2. The effects of novel atypical neuroleptic drugs on the CYP2D activity and protein level in the liver and brain.

Drug
Liver α2) Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Table 2. The effects of novel atypical neuroleptic drugs on the CYP2D activity and protein level in the liver and brain.

Drug
Liver  [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level.  [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, [15,16]. These three atypical neuroleptics act at different type serotonergic, or noradrenergic receptors (Table 2), which are throughout the brain in different structures and on differen cells. They usually function through metabotropic receptors 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-p C-phospholipase and A2-phospholipase to evoke second-m ing (reviewed by [35,36]). Because of differential regional di inergic receptors and transcription factors in the brain, the r logically active proteins including cytochrome P450 enzym (discussed by [37,38]). For example, in the prefrontal and fro inates over D2 receptor compared to other brain areas, which regulation of CYP2D4 in this brain area compared to the stri In contrast, the mentioned atypical neuroleptics display diff ergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Ta ence their effect on brain CYP2D4 activity/protein level.  [15,16]. These three atypical neuroleptics act at dif serotonergic, or noradrenergic receptors (Table 2), throughout the brain in different structures and o cells. They usually function through metabotropic 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT C-phospholipase and A2-phospholipase to evoke ing (reviewed by [35,36]). Because of differential r inergic receptors and transcription factors in the b logically active proteins including cytochrome P4 (discussed by [37,38]). For example, in the prefron inates over D2 receptor compared to other brain are regulation of CYP2D4 in this brain area compared In contrast, the mentioned atypical neuroleptics d ergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 rec ence their effect on brain CYP2D4 activity/protein Table 2. The effects of novel atypical neuroleptic drugs the liver and brain.

Drug
Liver FCx St NA Lurasidone (D2, 5-HT2A, 5-HT7, 5-HT1A, α2C) Iloperidone (D2, D3, 5-HT2A, α1, α2) Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, [15,16]. Th serotonerg throughou cells. They 5-HT1A) or C-phospho ing (review inergic rec logically a (discussed inates ove regulation In contras ergic D1, D ence their  exerted its effect on the brain enzyme via acting at monoaminergic receptors, and all the more so as similar effects on brain CYP2D in the frontal cortex and nigrostriatal pathway were observed for the atypical neuroleptics previously studied by us, namely, iloperidone and asenapine, which share with lurasidone similar receptor mechanisms in the brain [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Asenapine 1, D2, D3, D4, 5-T1A, 5-HT1B, 5-, 5-HT2B, 5-HT2C, exerted its effect on the brain enzyme via acting at monoaminergic receptors, and all the more so as similar effects on brain CYP2D in the frontal cortex and nigrostriatal pathway were observed for the atypical neuroleptics previously studied by us, namely, iloperidone and asenapine, which share with lurasidone similar receptor mechanisms in the brain [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, exerted its effect on the brain enzyme via acting at monoaminergic receptors, and all the more so as similar effects on brain CYP2D in the frontal cortex and nigrostriatal pathway were observed for the atypical neuroleptics previously studied by us, namely, iloperidone and asenapine, which share with lurasidone similar receptor mechanisms in the brain [15,16]. These three atypical neuroleptics act at different types/subtypes of dopaminergic, serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously distributed throughout the brain in different structures and on different types of neuronal and glial cells. They usually function through metabotropic receptors coupled negatively (e.g., D2, 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate cyclase or C-phospholipase and A2-phospholipase to evoke second-messenger intracellular signaling (reviewed by [35,36]). Because of differential regional distribution of those monoaminergic receptors and transcription factors in the brain, the regulation of a variety of biologically active proteins including cytochrome P450 enzymes is also region-dependent (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 receptor dominates over D2 receptor compared to other brain areas, which may be a reason for different regulation of CYP2D4 in this brain area compared to the striatum or nucleus accumbens. In contrast, the mentioned atypical neuroleptics display different affinities for dopaminergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also may influence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, exerted its effect on the brain enzyme via acting at monoaminergic receptors, an more so as similar effects on brain CYP2D in the frontal cortex and nigrostriatal were observed for the atypical neuroleptics previously studied by us, namely, ilo and asenapine, which share with lurasidone similar receptor mechanisms in [15,16]. These three atypical neuroleptics act at different types/subtypes of dopam serotonergic, or noradrenergic receptors (Table 2), which are heterogeneously di throughout the brain in different structures and on different types of neuronal cells. They usually function through metabotropic receptors coupled negatively 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to adenylate c C-phospholipase and A2-phospholipase to evoke second-messenger intracellula ing (reviewed by [35,36]). Because of differential regional distribution of those m inergic receptors and transcription factors in the brain, the regulation of a varie logically active proteins including cytochrome P450 enzymes is also region-d (discussed by [37,38]). For example, in the prefrontal and frontal cortex, D1 recep inates over D2 receptor compared to other brain areas, which may be a reason for regulation of CYP2D4 in this brain area compared to the striatum or nucleus acc In contrast, the mentioned atypical neuroleptics display different affinities for d ergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), which also m ence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, exerted its effect on the brain enzyme via acting at monoaminergic re more so as similar effects on brain CYP2D in the frontal cortex and nig were observed for the atypical neuroleptics previously studied by us, n and asenapine, which share with lurasidone similar receptor mecha [15,16]. These three atypical neuroleptics act at different types/subtype serotonergic, or noradrenergic receptors (Table 2), which are heterogen throughout the brain in different structures and on different types of cells. They usually function through metabotropic receptors coupled n 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT7) via G-protein to a C-phospholipase and A2-phospholipase to evoke second-messenger i ing (reviewed by [35,36]). Because of differential regional distribution inergic receptors and transcription factors in the brain, the regulation logically active proteins including cytochrome P450 enzymes is also (discussed by [37,38]). For example, in the prefrontal and frontal cortex inates over D2 receptor compared to other brain areas, which may be a regulation of CYP2D4 in this brain area compared to the striatum or n In contrast, the mentioned atypical neuroleptics display different affin ergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 receptors (Table 2), wh ence their effect on brain CYP2D4 activity/protein level. Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, exerted its effect on the brain enzyme via acting a more so as similar effects on brain CYP2D in the fr were observed for the atypical neuroleptics previo and asenapine, which share with lurasidone sim [15,16]. These three atypical neuroleptics act at dif serotonergic, or noradrenergic receptors (Table 2), throughout the brain in different structures and o cells. They usually function through metabotropic 5-HT1A) or positively (e.g., D1, 5-HT2A, 5-HT6, 5-HT C-phospholipase and A2-phospholipase to evoke ing (reviewed by [35,36]). Because of differential r inergic receptors and transcription factors in the b logically active proteins including cytochrome P4 (discussed by [37,38]). For example, in the prefron inates over D2 receptor compared to other brain are regulation of CYP2D4 in this brain area compared In contrast, the mentioned atypical neuroleptics d ergic D1, D3, D4, 5-HT6, α1, and histaminergic H1 rec ence their effect on brain CYP2D4 activity/protein Table 2. The effects of novel atypical neuroleptic drugs the liver and brain.

Drug
Liver FCx St NA Lurasidone (D2, 5-HT2A, 5-HT7, 5-HT1A, α2C) Iloperidone (D2, D3, 5-HT2A, α1, α2) Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, exerted its effect on the brain more so as similar effects on b were observed for the atypical and asenapine, which share w [15,16]. These three atypical ne serotonergic, or noradrenergic throughout the brain in differ cells. They usually function th 5-HT1A) or positively (e.g., D1, C-phospholipase and A2-phos ing (reviewed by [35,36]). Bec inergic receptors and transcrip logically active proteins inclu (discussed by [37,38]). For exa inates over D2 receptor compa regulation of CYP2D4 in this b In contrast, the mentioned aty ergic D1, D3, D4, 5-HT6, α1, and ence their effect on brain CYP2 Asenapine (D1, D2, D3, D4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, exerted its more so as were obse and asena [15,16]. Th serotonerg throughou cells. They 5-HT1A) or C-phospho ing (review inergic rec logically a (discussed inates ove regulation In contras ergic D1, D ence their  , -increase or decrease in activity, respectively; , -increase or decrease in protein level, respectively; -no effect. Receptors placed in the brackets display Ki below 20 nM [11]. FCxthe frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, Cb-the cerebellum, RM-the rest of brain. Lurasidone is an atypical antipsychotic with a unique receptor-binding profile including antagonism at the dopamine D2 receptor (but not other dopaminergic receptors), serotonin 5-HT2A and 5-HT7 receptor, and partial agonism at the 5-HT1A receptor [39]. Recent studies by Fukuyama et al. [40] showed that subchronic lurasidone administration attenuated adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling in cultured astrocytes due to its action at 5-HT7 receptors. In other studies, the suppression of AMPK increased CCAAT/enhancer-binding protein (C/EBPβ) and pCREB expression in hepatoma cells [41], while overexpression of C/EBPβ enhanced mRNA levels of CYP2D in HepG2 cells [42]. It is also worth noting that chronic administration of lurasidone attenuated ERK signaling, which was related to the combination of long-term inhibition of D2, 5-HT2A, and 5-HT7 receptors and downregulation of 5-HT1A and 5-HT7 receptors [40]. The inhibition of ERK may contribute to an increase in the PPARγ activity and, in turn, to the elevation of CYP2D expression. Since PPARγ contains a mitogen-activated protein kinase (MAPK) site, the phosphorylation by ERK leads to the inhibition of PPARγ activity [43,44]. In addition, the CYP2D6 gene is positively regulated by PPARγ, as shown in vitro in neuroblastoma SH-SY5Y cells and in vivo in the cerebellum and liver of mice [45]. Thus, the observed increases in the CYP2D activity/protein in specific brain structures in our experiment may be due to the receptor action and intracellular molecular effects of lurasidone. Further molecular studies will show which receptor-transduction pathway is involved in the regulation of CYP2D in particular brain regions and whether the enzyme regulation proceeds at a transcriptional or posttranscriptional level.

Conclusions
In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns frontal cortex (down-regulation) and nigrostriatal pathways (up-regulation), and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which indi- , -increase or decrease in activity, respectively; , -increase or decrease in protein level, respectively; -no effect. Receptors placed in the brackets display Ki below 20 nM [11]. FCxthe frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, Cb-the cerebellum, RM-the rest of brain.
Lurasidone is an atypical antipsychotic with a unique receptor-binding profile including antagonism at the dopamine D2 receptor (but not other dopaminergic receptors), serotonin 5-HT2A and 5-HT7 receptor, and partial agonism at the 5-HT1A receptor [39]. Recent studies by Fukuyama et al. [40] showed that subchronic lurasidone administration attenuated adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling in cultured astrocytes due to its action at 5-HT7 receptors. In other studies, the suppression of AMPK increased CCAAT/enhancer-binding protein (C/EBPβ) and pCREB expression in hepatoma cells [41], while overexpression of C/EBPβ enhanced mRNA levels of CYP2D in HepG2 cells [42]. It is also worth noting that chronic administration of lurasidone attenuated ERK signaling, which was related to the combination of long-term inhibition of D2, 5-HT2A, and 5-HT7 receptors and downregulation of 5-HT1A and 5-HT7 receptors [40]. The inhibition of ERK may contribute to an increase in the PPARγ activity and, in turn, to the elevation of CYP2D expression. Since PPARγ contains a mitogen-activated protein kinase (MAPK) site, the phosphorylation by ERK leads to the inhibition of PPARγ activity [43,44]. In addition, the CYP2D6 gene is positively regulated by PPARγ, as shown in vitro in neuroblastoma SH-SY5Y cells and in vivo in the cerebellum and liver of mice [45]. Thus, the observed increases in the CYP2D activity/protein in specific brain structures in our experiment may be due to the receptor action and intracellular molecular effects of lurasidone. Further molecular studies will show which receptor-transduction pathway is involved in the regulation of CYP2D in particular brain regions and whether the enzyme regulation proceeds at a transcriptional or posttranscriptional level.

Conclusions
In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns frontal cortex (down-regulation) and nigrostriatal pathways (up-regulation), and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which indi--increase or decrease in activity, respectively; Cells 2022, 11, x FOR PEER REVIEW 9 of 11 5-HT5A, 5-HT6, 5-HT7, α1, α2, H1) , -increase or decrease in activity, respectively; , -increase or decrease in protein level, respectively; -no effect. Receptors placed in the brackets display Ki below 20 nM [11]. FCxthe frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, Cb-the cerebellum, RM-the rest of brain.
Lurasidone is an atypical antipsychotic with a unique receptor-binding profile including antagonism at the dopamine D2 receptor (but not other dopaminergic receptors), serotonin 5-HT2A and 5-HT7 receptor, and partial agonism at the 5-HT1A receptor [39]. Recent studies by Fukuyama et al. [40] showed that subchronic lurasidone administration attenuated adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling in cultured astrocytes due to its action at 5-HT7 receptors. In other studies, the suppression of AMPK increased CCAAT/enhancer-binding protein (C/EBPβ) and pCREB expression in hepatoma cells [41], while overexpression of C/EBPβ enhanced mRNA levels of CYP2D in HepG2 cells [42]. It is also worth noting that chronic administration of lurasidone attenuated ERK signaling, which was related to the combination of long-term inhibition of D2, 5-HT2A, and 5-HT7 receptors and downregulation of 5-HT1A and 5-HT7 receptors [40]. The inhibition of ERK may contribute to an increase in the PPARγ activity and, in turn, to the elevation of CYP2D expression. Since PPARγ contains a mitogen-activated protein kinase (MAPK) site, the phosphorylation by ERK leads to the inhibition of PPARγ activity [43,44]. In addition, the CYP2D6 gene is positively regulated by PPARγ, as shown in vitro in neuroblastoma SH-SY5Y cells and in vivo in the cerebellum and liver of mice [45]. Thus, the observed increases in the CYP2D activity/protein in specific brain structures in our experiment may be due to the receptor action and intracellular molecular effects of lurasidone. Further molecular studies will show which receptor-transduction pathway is involved in the regulation of CYP2D in particular brain regions and whether the enzyme regulation proceeds at a transcriptional or posttranscriptional level.

Conclusions
In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns frontal cortex (down-regulation) and nigrostriatal pathways (up-regulation), and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which indi- , -increase or decrease in activity, respectively; , -increase or decrease in protein level, respectively; -no effect. Receptors placed in the brackets display Ki below 20 nM [11]. FCxthe frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, Cb-the cerebellum, RM-the rest of brain.
Lurasidone is an atypical antipsychotic with a unique receptor-binding profile including antagonism at the dopamine D2 receptor (but not other dopaminergic receptors), serotonin 5-HT2A and 5-HT7 receptor, and partial agonism at the 5-HT1A receptor [39]. Recent studies by Fukuyama et al. [40] showed that subchronic lurasidone administration attenuated adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling in cultured astrocytes due to its action at 5-HT7 receptors. In other studies, the suppression of AMPK increased CCAAT/enhancer-binding protein (C/EBPβ) and pCREB expression in hepatoma cells [41], while overexpression of C/EBPβ enhanced mRNA levels of CYP2D in HepG2 cells [42]. It is also worth noting that chronic administration of lurasidone attenuated ERK signaling, which was related to the combination of long-term inhibition of D2, 5-HT2A, and 5-HT7 receptors and downregulation of 5-HT1A and 5-HT7 receptors [40]. The inhibition of ERK may contribute to an increase in the PPARγ activity and, in turn, to the elevation of CYP2D expression. Since PPARγ contains a mitogen-activated protein kinase (MAPK) site, the phosphorylation by ERK leads to the inhibition of PPARγ activity [43,44]. In addition, the CYP2D6 gene is positively regulated by PPARγ, as shown in vitro in neuroblastoma SH-SY5Y cells and in vivo in the cerebellum and liver of mice [45]. Thus, the observed increases in the CYP2D activity/protein in specific brain structures in our experiment may be due to the receptor action and intracellular molecular effects of lurasidone. Further molecular studies will show which receptor-transduction pathway is involved in the regulation of CYP2D in particular brain regions and whether the enzyme regulation proceeds at a transcriptional or posttranscriptional level.

Conclusions
In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns frontal cortex (down-regulation) and nigrostriatal pathways (up-regulation), and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which indi--increase or decrease in protein level, respectively; 2022, 11, x FOR PEER REVIEW 9 of 11 HT5A, 5-HT6, 5-T7, α1, α2, H1) , -increase or decrease in activity, respectively; , -increase or decrease in protein level, respectively; -no effect. Receptors placed in the brackets display Ki below 20 nM [11]. FCxthe frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, Cb-the cerebellum, RM-the rest of brain.
Lurasidone is an atypical antipsychotic with a unique receptor-binding profile including antagonism at the dopamine D2 receptor (but not other dopaminergic receptors), serotonin 5-HT2A and 5-HT7 receptor, and partial agonism at the 5-HT1A receptor [39]. Recent studies by Fukuyama et al. [40] showed that subchronic lurasidone administration attenuated adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling in cultured astrocytes due to its action at 5-HT7 receptors. In other studies, the suppression of AMPK increased CCAAT/enhancer-binding protein (C/EBPβ) and pCREB expression in hepatoma cells [41], while overexpression of C/EBPβ enhanced mRNA levels of CYP2D in HepG2 cells [42]. It is also worth noting that chronic administration of lurasidone attenuated ERK signaling, which was related to the combination of long-term inhibition of D2, 5-HT2A, and 5-HT7 receptors and downregulation of 5-HT1A and 5-HT7 receptors [40]. The inhibition of ERK may contribute to an increase in the PPARγ activity and, in turn, to the elevation of CYP2D expression. Since PPARγ contains a mitogen-activated protein kinase (MAPK) site, the phosphorylation by ERK leads to the inhibition of PPARγ activity [43,44]. In addition, the CYP2D6 gene is positively regulated by PPARγ, as shown in vitro in neuroblastoma SH-SY5Y cells and in vivo in the cerebellum and liver of mice [45]. Thus, the observed increases in the CYP2D activity/protein in specific brain structures in our experiment may be due to the receptor action and intracellular molecular effects of lurasidone. Further molecular studies will show which receptor-transduction pathway is involved in the regulation of CYP2D in particular brain regions and whether the enzyme regulation proceeds at a transcriptional or posttranscriptional level.

Conclusions
In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns frontal cortex (down-regulation) and nigrostriatal pathways (up-regulation), and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which indi--no effect. Receptors placed in the brackets display K i below 20 nM [11]. FCx-the frontal cortex, St-the striatum, NA-the nucleus accumbens, Hp-the hippocampus, Ht-the hypothalamus, BS-the brain stem, SN-the substantia nigra, Cb-the cerebellum, RM-the rest of brain.
Lurasidone is an atypical antipsychotic with a unique receptor-binding profile including antagonism at the dopamine D 2 receptor (but not other dopaminergic receptors), serotonin 5-HT 2A and 5-HT 7 receptor, and partial agonism at the 5-HT 1A receptor [39]. Recent studies by Fukuyama et al. [40] showed that subchronic lurasidone administration attenuated adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) signaling in cultured astrocytes due to its action at 5-HT 7 receptors. In other studies, the suppression of AMPK increased CCAAT/enhancer-binding protein (C/EBPβ) and pCREB expression in hepatoma cells [41], while overexpression of C/EBPβ enhanced mRNA levels of CYP2D in HepG2 cells [42]. It is also worth noting that chronic administration of lurasidone attenuated ERK signaling, which was related to the combination of long-term inhibition of D 2 , 5-HT 2A , and 5-HT 7 receptors and downregulation of 5-HT 1A and 5-HT 7 receptors [40]. The inhibition of ERK may contribute to an increase in the PPARγ activity and, in turn, to the elevation of CYP2D expression. Since PPARγ contains a mitogen-activated protein kinase (MAPK) site, the phosphorylation by ERK leads to the inhibition of PPARγ activity [43,44]. In addition, the CYP2D6 gene is positively regulated by PPARγ, as shown in vitro in neuroblastoma SH-SY5Y cells and in vivo in the cerebellum and liver of mice [45]. Thus, the observed increases in the CYP2D activity/protein in specific brain structures in our experiment may be due to the receptor action and intracellular molecular effects of lurasidone. Further molecular studies will show which receptor-transduction pathway is involved in the regulation of CYP2D in particular brain regions and whether the enzyme regulation proceeds at a transcriptional or posttranscriptional level.

Conclusions
In conclusion, lurasidone regulates brain (but not liver) CYP2D activity/protein level in a region-dependent manner, which is similar to that of other atypical neuroleptics (iloperidone and asenapine) as concerns frontal cortex (down-regulation) and nigrostriatal pathways (up-regulation), and may be of pharmacological significance. However, further molecular studies with selective receptor agonists are necessary to find out which individual monoaminergic receptors/signaling pathways and transcription factors are involved in the rat CYP2D4 and human CYP2D6 enzyme regulation in particular brain structures.