Characterization of the Brain Penetrant Neuropeptide Y Y2 Receptor

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Characterization of the Brain Penetrant Neuropeptide Y Y2 Receptor Antagonist SF-11 Helena Domin,*,† Natalia Piergies,‡ Ewa Pięta,‡ Elżbieta Wyska,§ Bartłomiej Pochwat,† Piotr Wlaź,¶ Maria Śmiałowska,† Czesława Paluszkiewicz,‡ and Bernadeta Szewczyk†

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†

Maj Institute of Pharmacology, Polish Academy of Sciences, Department of Neurobiology, 31-343 Kraków, 12 Smętna Street, Poland ‡ Institute of Nuclear Physics Polish Academy of Sciences, PL-31342 Krakow, Poland § Department of Pharmacokinetics and Physical Pharmacy, Collegium Medicum, Jagiellonian University, Medyczna 9, 30-688 Kraków, Poland ¶ Department of Animal Physiology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, PL-20-033 Lublin, Poland S Supporting Information *

ABSTRACT: This paper discusses the biological and three-dimensional molecular structure of the novel, nonpeptide Y2R antagonist, SF-11 [N-(4ethoxyphenyl)-4-(hydroxydiphenylmethyl)-1-piperidinecarbothioamide]. Pharmacokinetic studies in a rat model indicated that, following intraperitoneal dosing, SF-11 crossed the blood−brain barrier and was able to penetrate the brain, making it a suitable tool for behavioral studies. We showed for the first time that SF-11 decreased the immobility time in the forced swim test (FST) after acute peripheral administration (10 and 20 mg/kg), indicating that it has antidepressant potential. Inhibitors of the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways blocked the anti-immobility effect of SF-11, suggesting that these pathways are involved in the antidepressant-like activity of SF-11 in the FST. The results of locomotor activity of rats indicate that the effects observed in the FST are specific and due to the antidepressant-like activity of SF-11. These findings provide further evidence for the antidepressant potential of Y2R antagonists. Also, the application of Fourier transform infrared absorption (FT−IR) and Raman spectroscopy (RS) methods combined with theoretical density functional theory (DFT) calculations allowed us to present the optimized spatial orientation of the investigated drug. Structural characterization of SF-11 based on vibrational spectroscopic data is of great importance and will aid in understanding its biological activity and pave the way for its development as a new antidepressant agent. KEYWORDS: Y2 receptor, Raman spectroscopy (RS), theoretical density functional theory (DFT), forced swim test, antidepressant-like activity, pharmacokinetics

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INTRODUCTION Neuropeptide Y (NPY), a 36-amino acid peptide, is widely distributed in the mammalian central nervous system (CNS) where it plays a vital role in various physiological functions by binding to different types of NPY receptors.1,2 NPY produces its effects through specific membrane-bound G-proteincoupled receptors (GPCRs) (Y1, Y2, Y3, Y4, Y5, and y6), which are negatively coupled to the Gαi signaling pathway. The activation of these receptors leads to the inhibition of adenylate cyclase and subsequently the inhibition of cyclic adenosine monophosphate (cAMP) formation.3 In some cells, NPY can also activate, through the G beta gamma subunit, a number of different kinase cascades, including protein kinase C (PKC), mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), and phosphatidylinositol 3kinase (PI3K).4,5 Recently, Y receptors (YRs) have attracted the attention of pharmacologists as potential targets for the © XXXX American Chemical Society

treatment of various neurological disorders (mood disorders, epilepsy).5,6 A large number of preclinical and clinical studies have shown that NPY plays an important role in the pathophysiology of mood disorders, such as depression.7−9 Several clinical studies have indicated reduced levels of NPY in the cerebrospinal fluid and plasma of depressed patients.10−13 Therefore, it has been postulated that increasing the NPY levels in the CNS may prove to be a useful mechanism in treating mood disorders. For example, in humans, it has been shown that treatment with a selective serotonin reuptake inhibitor (SSRI) results in an increase in the cerebrospinal fluid level of NPY.14 Received: February 5, 2019 Accepted: June 19, 2019 Published: June 19, 2019 A

DOI: 10.1021/acschemneuro.9b00082 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience Over 300 million people worldwide suffer from depression, which can lead to suicide in severe cases.15 In more than 30% of these patients, antidepressant treatments are not effective; therefore, research aimed at developing new antidepressant agents with better efficacy and fewer side effects is imperative.16 Recent studies have demonstrated the potential antidepressant-like activity of NPY receptor ligands in animal models of depression; on the basis of these findings, the roles of Y1R, Y2R, and Y5R in depression-related disorders have been postulated.5,17−20 Our present study sought to unravel the role of Y2R in depression-like disorders. It is well-known that Y2Rs are mainly presynaptic and are involved in the inhibition of release of NPY as well as other neurotransmitters, such as GABA or glutamate that have been implicated in mood disorders.7,21,22 Considering the role of the Y2R in mood disorders, it is worth mentioning that mice lacking this receptor (Y2R knockout mice) exhibit anxiolytic and antidepressant-like behavioral effects.23−26 For many years, the lack of selective pharmacological tools has limited in vivo characterization of Y2R-mediated functions. Only a few studies have shown that Y2R antagonists, such as BIIE0246 and JNJ31020028, produced antidepressant-like effects in animal models of depression. 8,19,27 However, because of its pseudomimetic complex structure, high molecular weight (MW ≈ 900), poor capability to cross the blood−brain barrier, and off-target activity, BIIE0246 has found limited usefulness as an in vivo pharmacological tool.28,29 Also, the results obtained in behavioral tests using the small molecule, JNJ-31020028, are fragmentary because of its reduced antidepressant-like effects, which show up only after intracerebroventricular (i.c.v.) administration.19 Recently, the novel, nonpeptide Y2R antagonist, SF-11 [N(4-ethoxyphenyl)-4-(hydroxydiphenylmethyl)-1-piperidinecarbothioamide], has received attention. It is a low molecular weight (MW ≈ 450) compound with high brain penetrability, thus making it proper for use in animal models of mood disorders.28 However, this compound still has to be thoroughly investigated in experimental models of mood disorders. Consequently, we characterized the pharmacokinetic properties of SF-11 and examined its possible antidepressant potential in adult male Sprague−Dawley (SD) rats. Since cellular processes engaged in the mechanisms of the antidepressant action of Y2R antagonists have not been investigated, we also examined the role of cell signaling pathways in the antiimmobility effect of SF-11. To the best of our knowledge, this is the first report of the structural analysis of SF-11 (see Figure 1 for the molecular structure). Research confirming the different biological activities of compounds with chemical similarities but different 3D structures to Y2R abounds.30 Thus, we performed a structural characterization of the Y2R antagonist on the basis of Raman spectroscopy (RS) and infrared absorption (IR) spectroscopic data and theoretical density functional theory (DFT) calculations. RS and IR spectroscopies provide characteristic spectra of molecules often referred to as vibrational fingerprints. These methods also provide complementary information regarding molecular structure and composition of the compounds being investigated.31 Thus, RS and IR spectroscopies find broad application in qualitative and quantitative analysis of different types of samples.32−34 The experimental results are often supported by theoretical, computational quantum chemistry. This is associated with the fact that the interpretation of the

Figure 1. Optimized structure of [N-(4-ethoxyphenyl)-4-(hydroxydiphenylmethyl)-1-piperidinecarbothioamide] (SF11) (A) and the atom numbering scheme together with the 2D structural image (B).

recorded spectra can be ambiguous and may hinder structural elucidation.35 Spectroscopic analyses based on experimental and theoretical data provide more reliable evidence about the molecular structure of the compounds under investigation.36 Currently, hybrid density functional theory (DFT) calculations with the use of the B3LYP functional are often used for computing vibrational frequencies, molecular structures, and other properties of various chemical systems.37 B3LYP works well in predicting structures and experimental RS and IR spectra of many biologically active compounds, especially peptides, amino acids, and their analogues.32,38 This prompted us to apply DFT-B3LYP calculations to the Y2R antagonist to support the interpretation of the experimentally obtained spectra.

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RESULTS AND DISCUSSION Vibrational Spectroscopy Study. Geometric Structure. The analysis of the geometric structure of SF-11 was performed using the DFT B3LYP 6-311G(d,p) level of theory. The conformational study enabled us to predict the stable structure of the molecule (see Figure 1). The selected calculated bond lengths and angles of SF-11 are listed in Table S1, while the the atom-numbering scheme is presented in Figure 1. The optimized C−C and C−N bond lengths of the Pip ring are in agreement with those found in the DFT calculations39 and X-ray analysis40,41 performed for substituted piperidines. The X-ray analysis indicates that the C−C bond lengths are in the range of 1.549 to 1.599 Å, whereas the values resulting from the DFT calculations are in the range of 1.502 to 1.536 Å, and the C−N bond lengths are ∼1.469 and ∼1.464 Å, respectively. Also, a good correlation was observed in the case of 4-ethoxyphenyl bond lengths. Briefly, the DFT and Xray literature indicate that the C−C bond length is ∼1.400 Å and the C−O distance is ∼1.359 Å, while the C−O−C angle is equal to 118.7°.42,43 Moreover, the optimized bond distances and angles of the hydroxy(diphenyl)methyl group also correlate well with DFT44 and X-ray45 studies for hydroxydiphenyl-methyl and benzene derivatives, wherein, e.g., C−O = 1.435 Å, C20−C19−C26 = 109.1°, and C20−C19−O32 = 108.5°. As it was mentioned above, the detailed information about the remaining bond lengths, angles, and dihedral angles for SF-11 is displayed in Table S1. B


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ACS Chemical Neuroscience RS, FT-IR, and DFT Studies. As mentioned in the Introduction, the molecular structure of SF-11 may strongly affect its biological activity. Therefore, we decided to perform an in-depth structural characterization of the molecule on the basis of its vibrational spectroscopic data. Figure 2 shows the

Table 1. Wavenumber and Suggested Assignments for Selected Experimental and Calculated RS and FT-IR Bandsa wavenumber/cm−1 experimental

Figure 2. Experimental and calculated RS and IR spectra of the stable SF11 monomer in the spectral ranges of 3200−2750 and 1800−350 cm−1.

calculated

assignment

RS

FT-IR

RS

FT-IR

ν(CH)ring νs(CH2) νas(CH2) ν(CC)ring, ν8a, ν(NH) ν(CC)ring, ν8b, δ(NH) δ(NH), ν(CN) δ(CH2), δ(CH3), ν19b ρw(CH2), ρw(CH3) ρw(CH2) ν(CC), ν14 δ(CH2), δ(CH), ν3 ρt(CH2), ν(CN) ν(CO), δring, ν(CN), ρt(CH2) ν(CN) ρb(CH), ν9a ν(CC), ν(CO) ν(CC)ring, Phebreath, ν12 γ(Phe), γ(CH), ν17 γ(CH) ν(CC), δ(CH), ν(CS) γ(ring) ring breath ν(CS) δ(Phe), ν6b

3064 2978 2931 1612 1583 1513 1441 1400 1367 1331 1305 1268

3055 2976 2928 1612 1596 1512 1446 1393

3064 2982 2929 1629 1597 1522 1458 1404 1380 1326 1299 1261

3058 2976 2928 1629 1600 1522 1456 1401 1380 1319 1299 1259 1240 1222 1172 1048 996 969 920 832 750 707 640 623

1231 1172 1002 969 922 820 749 707 635 619

1326 1298 1240 1220 1169 1045 1000 968 919 823 748 703 635

1223 1172 1049 1000 971 922 820 753 708 640 625

Abbreviations: ν, stretching; δ, deformation; γ, deformation out of plane; ρb, bending; ρw, wagging; ρt, twisting; as, asymmetric; s, symmetric; breath, breathing; Phe, phenyl. a

calculated and experimental RS and FT−IR spectra of the stable SF-11 monomer. As can be observed, the band positions and intensities observed in the vibrational spectra indicate a good correlation with those obtained from the DFT B3LYP 6311G(d,p) calculations. Additionally, the RS and FT-IR results of SF-11 show similarities to the spectral patterns of aniline,46,47 piperidine,48,49 derivatives of carbothioamide,50 thiazole,42 and anthraquinone,51 anilinium sulfate,52 benzene and monosubstituted benzenes,53,54 and thioamides.55 Table 1 contains the vibrational assignments for the most pronounced bands. Additionally, Table S2 provides the experimental and calculated wavenumbers with the acquired potential energy distribution (PED, in %) of the discussed RS and FT−IR spectra. Note that there are currently no reports regarding the vibrational spectroscopic characterization of SF-11. Aromatic Vibrations. The SF-11 molecule consists of phenyl and ethoxyphenyl rings, which strongly influence the RS spectra (Figure 2). The spectra are thus dominated by the bands associated with these aromatic moieties which appear at 1629−1602, 1597−1583, and 1172−1171 cm−1 and are attributed to the ν8a, ν8b, and ν9a modes, respectively (according to the Wilson nomenclature).56 Moreover, the spectral features observed at 640−630 cm−1 and 550−485 cm−1, 420 cm−1 are due to the δ(Phe)/δ(Eth-Phe) and γ(Phe)/γ(Eth-Phe) vibrations, accordingly (see Tables 1 and S2). Some of the previously mentioned bands also occur in the corresponding FT-IR spectral pattern (see Figure 2). On the other hand, in the RS and Fourier transform infrared absorption (FT−IR) spectra, there are bands which can be separately assigned to the phenyl and ethoxyphenyl rings. The bands are at 3064−3055, 1493−1492, 1331−1326, 1305−

1298, 1190−1187, 1159−1157, 1079−1064, 1033, 1002− 1000, 990−982, 971−968, and 625−619 cm−1 and 885−878, 772−748, 708−703, and 354−343 cm−1, and they are related to the ν2, ν19a, ν14, ν3, ν7a, ν9b, ν15, ν18a, ν12, ν5, ν17, ν6b, γ(Phe) vibrations of the phenyl rings, respectively (Tables 1 and S2). Consequently, the stretching C−C [ν(CC)] and out-of-plane deformation [γ(Eth-Phe)] modes of the ethoxyphenyl aromatic moiety appear at 1427−1425 cm−1 and 792−783, 732−725, 692−683, 368−366 cm−1, respectively. The aromatic functional groups also contribute to the bands observed at 1522−1512 cm−1 [ρr(CC(H)C)Eth‑Phe], 1109− 1107 cm−1 [ρb(CC(H)C)Phe], and 666−644 cm−1 [δ(Phe)]. Aliphatic Vibrations. In the vibrational spectra, the medium- and weak-intensity bands due to the piperidine, ethoxy, and carbothioamide moieties of SF-11 antagonist are visible (Figure 2). The presence of the piperidine ring is manifested by the bands observed at 3030−3025, 2978−2876, 2931−2928, 1369−1351, 1268−1261, 1231−1220, 1113− 1107, 856−852, 823−820, 666−644, and 579−568 cm−1. These spectral features are associated with the following vibrational modes: νas(CH2)Pip, νs(CH2)Pip, νas(CH2)Pip, ρw(CH2)Pip, ρt(CH2)Pip/ν(CNPip), ν(CNPip), ν(CC)Pip/ν(CNPip), δ(CC)Pip, Pipbreath, ρr(CH2)Pip, respectively. In addition, the rocking vibrations of the CH2 group of the piperidine ring [ρr(CH2)Pip] contributed to the bands at 708− 707 cm−1, and the deformation mode of the aliphatic ring [δ(Pip)] influenced the bands at 550−485 cm−1 (Tables 1 and S2). C


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ACS Chemical Neuroscience The ethoxy moiety occurs in the RS and FT-IR spectra as stretching, scissoring, rocking, and wagging vibrational modes of −CH2 and −CH3 fragments. These vibrations correspond to the bands at 2969−2962 cm−1 [νas(CH3)Eth, νas(CH2)Eth,], 1477−1476 cm−1 [ρs(CH2)Eth, ρs(CH3)Eth], 1458−1441 cm−1 [ρr(CH2)Eth, ρr(CH3)Eth], and 1404−1393 cm−1 [ρw(CH2)Eth, ρw(CH3)Eth], respectively (Tables 1 and S2). On the other hand, the stretching modes of C−C/C−O bonds [ν(CC)/ ν(CO)] appear at the spectral ranges 1047−1044 and 922− 919 cm−1. Additionally, the vibrational bands observed at 395−382 cm−1 are due to the bending vibrations of the −COC and −CCO fragments [ρb(COC)Eth/ρb(CCO)Eth]. The spectral features due to the carbothioamide moiety vibrations are observed at 1522−1512 and 446−434 cm−1 and correspond to the bending vibrations of the NH fragment [ρb(NH)]. These bands exhibit the strongest intensity in the experimental and theoretical IR spectra. Moreover, the stretching C−S modes [ν(CS)] contribute to the bands at 732−725, 692−683, and 640−635 cm−1. Pharmacokinetic Study. To determine the concentrations of SF-11 in serum, selected brain structures, and several peripheral organs, we performed a pharmacokinetic study in rats. The results of the noncompartmental analysis revealed that, following an intraperitoneal (i.p.) dose of 10 mg/kg, SF11 attained a peak serum concentration of approximately 3 μM at 15 min (Table 2 and Figures 3 and 4).

Figure 3. Observed (circles) and pharmacokinetic model-predicted (line) serum concentrations of SF-11 (10 mg/kg) administered i.p. to rats. Symbols represent the mean ± SEM (n = 3 to 4).

Table 2. Pharmacokinetic Parameters of SF-11 Following i.p. Administration (10 mg/kg) to Rats Estimated Using Noncompartmental Analysisa tissue parameter tmax (min) Cmax (μM) λz (min−1) t0.5λz(min) AUClast (μM·min) AUC0−∞ (μM·min) MRT (min) Vz/F (L/kg) CL/F (L/min/kg)

serum

hippocampus

cortex

15 2.90 0.017 40.41

30 0.49 0.002 372.23

30 0.47 0.003 225.98

190.98 191.41 62.40 6.82 0.117

55.84 94.95 438.09

45.14 61.48 264.35

Figure 4. Concentration versus time profiles of SF-11 (10 mg/kg) in serum and brain structures after i.p. administration to rats. Values are presented as the mean ± SEM (n = 3 to 4).

the heart, where the value of terminal half-life was comparable to that in the hippocampus (Tables 2 and S5). When a one-compartment pharmacokinetic model was used to describe the serum concentration versus time profile of SF11 (Figure 3), the values of pharmacokinetic parameters were close to those obtained from the noncompartmental analysis. The estimated volume of distribution (V/F) was 5.87 L/kg and the elimination rate constant (ke) was 0.019 min−1. As a result, the half-life of SF-11 in the rat serum was 36.59 min. The absorption rate constant (ka) was relatively high (0.084 min−1), indicating a fast absorption of SF-11 from the peritoneal cavity. Characterization of the pharmacokinetic properties of a drug candidate is necessary before its preclinical pharmacological evaluation. In the present study, we have shown, for the first time, the pharmacokinetic profile of SF-11 in rats at different time points. A previous study performed by other authors28 in mice evaluated serum and brain concentrations of SF-11 but only at one time point (30 min after administration). Our present data confirm that SF-11 crosses the blood−brain barrier and can penetrate into the brain following intra-

a

n = 3 to 4 per time point.

In the hippocampus and cortex, maximum concentrations occurred later, that is 30 min postdosing (Table 2 and Figure 2). These concentrations were similar in both brain structures and constituted about 16% of the peak serum concentration. The elimination of this compound from serum was relatively fast with a terminal half-life of about 40 min and the clearance value of 0.12 L/min/kg. In turn, the values of t0.5λz in the hippocampus and cortex were 9.2 and 5.6 times longer than in serum. These observations were further confirmed by the much longer mean residence times in both brain structures in comparison to serum (Table 2). Concentrations of SF-11 in peripheral organs, such as the liver, kidneys, lungs, and heart were higher than those in serum (Tables S3 and S4). The maximum concentrations were attained 30 min postdose in all tissues (Table S5). The compound reached the highest concentrations in the liver and the lowest in the heart. SF-11 was eliminated faster from these organs in comparison to brain structures with the exception of D


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ACS Chemical Neuroscience

Figure 5. (A) FST was performed 60 min after SF-11 treatment. SF-11 (10 and 20 mg/kg) induces the antidepressant-like effect in the FST performed 60 min after administration. Values are presented as the mean ± SEM of two separate experiments (n = 10−16). (B) The effect of SF-11 in the FST was studied 30 min after the treatment. Values are presented as the mean ± SEM, n = 8. (C) The effect of SF-11 in the FST was studied 4 h after the treatment. Values are presented as the mean ± SEM, n = 8. Differences between SF-11-treated and control groups were analyzed by one-way ANOVA followed by the Dunnett’s multiple comparisons test (A) or Student’s t test (B, C). **p < 0.01 vs control group.

peritoneal dosing, making it a suitable tool for behavioral studies. In addition, the detectable level of this compound persists longer in the brain than blood. The micromolar concentrations of SF-11 in the brain tissues obtained after i.p. injection of a dose of 10 mg/kg (0.47 μM 30 min after treatment and 0.20 μM 60 min after treatment; see Table S3) were able to inhibit Y2Rs (IC50 = 199 nM28). Finally, the pharmacokinetic profile obtained for SF-11 allowed us to properly design the behavioral experiments. Behavioral Study. Effect of SF-11 Administration in the Forced Swim Test in Rats. To ascertain if SF-11 exhibits antidepressant-like activity, we examined its effect in the forced swim test (FST). As shown in Figure 5A, SF-11 administered at doses of 10 and 20 mg/kg 60 min before the FST significantly (p < 0.01) decreased the immobility time in rats [one-way ANOVA, F(3,47) = 6117, p = 0.0013]. However, at a dose of 3 mg/kg, no significant influence on the behavior of rats was observed in the FST (p > 0.05) compared to the control group. As demonstrated by pharmacokinetic studies, the maximum penetration of the compound into the brain is achieved at 30 min. The high level of the compound in the brain also persists until 60 min, after which it begins to gradually decrease. Very low values are observed at 4 h after administration. Initially, we planned to check the SF-11 antidepressant effect in the FST in rats at 60 min after administration (Figure 5A). However, on the basis of the results described above, we decided to check whether the observed antidepressant effects of SF-11 will be correlated with the time of penetration of this compound. We did not observe the SF-11 antidepressant-like effect either at 30 min (Figure 5B) or at 4 h (Figure 5C) after administration. The FST was chosen for the study because it is a commonly used and accepted behavioral screening test for the selection of potential antidepressant drugs; it is easy to perform and has excellent reproducibility. The primary indication of antidepressant activity of any drug in the FST is a decrease in the immobility time.57,58 We show for the first time that the selective Y2 receptor antagonist (SF-11) can reduce the immobility time in the FST in rats (p < 0.01) at the doses of both 10 and 20 mg/kg, indicating that it does possess antidepressant potential. The observed pharmacological effect was not dose dependent, despite the fact that SF-11 serum concentrations measured 60 min postdose increased proportionally with the dose administered, and they were 0.44 ± 0.10, 1.23 ± 0.23, and 2.84 ± 0.48 μM for doses 3, 10, and 20 mg/

kg, respectively. The effect was found to reach saturation between 10 and 20 mg/kg. Our present findings are congruent with those of other authors who reported that acute treatment with another Y2 antagonist BIIE0246 induced antidepressant effects in the FST in naive control mice.27 However, BIIE0246 is a big molecular weight compound with poor brain penetrability, and this limits its usefulness in clinical applications. It is worth emphasizing that, in our present study, SF-11 showed antidepressant-like effects after acute peripheral administration. Our findings are thus interesting concerning therapeutics because of the inefficiency of current antidepressant therapies, which often require long treatment times. Other studies on the antidepressant action of Y2 receptor antagonists have largely focused on multiple dosing schemes and found that chronic i.c.v. administration of BIIE0246, as well as a brain-penetrant JNJ-31020028, induced a decrease in the immobility time in the FST in the olfactory bulbectomized (OBX) rat.8,19 These findings suggest that Y2 receptor antagonists could be therapeutically useful for treating depression-like symptoms. In this study, SF-11 showed antidepressant-like activity under normal conditions; in contrast to other findings, its beneficial effects were seen after acute peripheral administration. However, further studies are needed especially under challenging conditions in behavioral models of mood disorders to elucidate its usefulness as a therapeutic agent. The antidepressant potential of the Y2R antagonist, SF-11, is congruent with the findings of other studies, which reported that NPY and its associated receptor ligands exhibited antidepressant-like effects in animal models of depression.5,7,8 Our study also confirmed that Y2 receptors are involved in the modulation of depression-related behavior. The modulation of depression-related behavior by Y2 receptors may be linked to the presynaptic location of the receptors in neurons that contain NPY where they negatively regulate NPY release.21 Therefore, any action that would reverse the activity of Y2R would likely increase NPY levels in the CNS, making this mechanism useful in treating mood disorders. Effect of the MAPK/ERK and PI3K Signaling Pathway Inhibitors on the Antidepressant-Like Activity of SF-11 in the FST in Rats. To determine the roles of the MAPK/ERK and PI3K signaling pathways in the antidepressant-like activity of SF-11, U0126, and LYS294002, inhibitors of both intracellular pathways were used. As shown in Figure 6, SF11(10 mg/kg) alone significantly decreased the immobility E


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activity. Nevertheless, further studies are required to fully elucidate the role of other signaling pathways in this effect. It should be noted that the PI3K/Akt signaling pathway is also associated with the activation of the mammalian target of rapamycin (mTOR), a protein kinase that has been implicated in rapid antidepressant responses.62−64 Taking into account that in our present study SF-11 showed an antidepressant-like effect after acute treatment, it is likely that the mTOR signaling pathway is implicated in its antidepressant effect, and this hypothesis should be verified in future studies. Effect of SF-11 and the MAPK/ERK and PI3K Signaling Pathway Inhibitors on the Locomotor Activity of Rats. To determine whether the tested compounds produce nonspecific responses in the FST, the spontaneous locomotor activity of the rats was measured. As shown in Table 3A−C, SF-11

Figure 6. Effects of pretreatment with U0126 (5 μg/2 μL/rat, i.c.v.; MAPK/ERK inhibitor) and LY294002 (10 nmol/2 μL/rat, i.c.v.; PI3K inhibitor) on the antidepressant-like effect of SF-11 (10 mg/kg, i.p.) in the FST in rats. MAPK/ERK and PI3K inhibitors (75 min before the test) reversed the anti-immobility effect of SF-11 (60 min before the test) in the FST in rats. The values are expressed as means ± SEM (n = 12). Data were analyzed by two-way ANOVA, followed by the Tukey’s multiple comparisons test. **p < 0.01 vs control group; #p < 0.05 vs SF-11 treated group.

Table 3. Effect of SF-11 and Inhibitors of Signaling Pathways on the Locomotor Activity of Ratsa treatment

activity counts [5 min]

A. 30 min after the Treatment 100.00 ± 15.63 132.40 ± 17.89 B. 60 min after the Treatment control 100.00 ± 20.5 SF-11 3 mg/kg 57.19 ± 16.57 SF-11 10 mg/kg 88.96 ± 9.32 SF-11 20 mg/kg 94.98 ± 15.68 C. 4 h after the Treatment control 100.00 ± 14.41 SF-11 10 mg/kg 81.30 ± 14.77 D. 75 min after U0126 or LY294002 and 60 min after SF-11 Administration control 100.00 ± 15.57 SF-11 10 mg/kg 69.00 ± 23.75 U0126 86.00 ± 12.94 U0126 + SF-11 10 mg/kg 58.6 ± 4.42 LY294002 78.8 ± 18.55 LY294002 + SF-11 10 mg/kg 108.5 ± 26.00 control SF-11 10 mg/kg

time in the FST in rats (p < 0.01). Pretreatment with U0126 (5 μg/2 μL) reversed the anti-immobility effect of SF-11 (p < 0.05) but did not affect the behavior of rats given only SF-11 (p > 0.05). Furthermore, we found that LY294002 (10 nmol/2 μL) did not evoke any changes in the behavior of rats in comparison to the control group (p > 0.05) but significantly reversed the anti-immobility effect of SF-11 in the FST (p < 0.05) (Figure 6). Values of the immobility time of the control group of rats subjected to surgery did not differ from those obtained for the control group of naive rats. Data were analyzed by two-way ANOVA, followed by the Tukey’s multiple comparisons test. A two-way ANOVA analysis showed a significant effect of inhibitors [F(2,66) = 3.346, p = 0.0413], a significant effect of SF-11 [F(1,66) = 7.804, p = 0.0068], and significant interaction [F(2.66) = 5.05, p = 0.0091] (n = 12). The MAPK/ERK and PI3K signaling pathways are thought to be involved in the pathophysiology of depression and the antidepressant-like effects of different compounds.59−61 We demonstrated for the first time that the anti-immobility effect of SF-11 was prevented by inhibitors of the MAPK/ERK and PI3K signaling pathways, suggesting that these pathways are involved in the antidepressant-like activity of SF-11 in the FST in rats. It has been shown that NPY can activate a number of different kinase cascades, e.g., MAPK/ERK and PI3K.4,5 On the basis of the above-mentioned data as well as the fact that antagonism of Y2R would be expected to increase NPY levels in the CNS, we suggest that a possible mechanism underlying the antidepressant-like action of SF-11 has been associated with the activation of MAPK/ERK and PI3K intracellular signaling pathways. Interestingly, we showed recently that both signaling pathways are involved in the antidepressant-like activity of another NPY receptor ligand (Y5R antagonist, Lu AA33810).20 Therefore, our previous and present studies show that the antagonism of Y2R and/or Y5R can result in antidepressant-like properties of compounds through the modulation of the MAPK/ERK and PI3K signaling pathways. It is worth emphasizing that ours is the first study to show the possible mechanism of Y2R-mediated antidepressant-like

a

Data were presented as a percentage of the control value and as the mean ± SEM (n = 4−9 rats per group).

treatment (regardless of the time of administration and the dose administered) did not change the locomotor activity of rats. As shown in Table 3D, SF-11 administered alone (10 mg/ kg) or in combination with MAPK/ERK and PI3K inhibitors did not significantly alter the locomotor activity of rats. Both U0126 (5 μg/2 μL) and LY294002 (10 nmol/2 μL) when given alone also did not evoke any significant changes in the locomotor activity of rats compared to the control group. The findings indicate that the effect observed in the FST is specific and due to the antidepressant-like activity of SF-11. Our findings are in agreement with results obtained with other Y2R antagonists such as BIIE0246 and JNJ-31020028.8,19

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CONCLUSION In this study, we performed the first comprehensive characterization of SF-11 as an antidepressant agent. Vibrational spectroscopic methods, namely, RS and FT−IR, supported by theoretical DFT calculation allowed us to present the optimized molecular structure of the compound. Such an approach is extremely important and timely since the spatial molecular orientation strongly affects the biological activity of drugs. Given the fact that current antidepressant therapies are F


Research Article


mL) were spiked with 10 μL of an internal standard (IS) solution containing 5 μg/mL of diazepam and mixed by vortexing (Reax top, Heidolph, Germany) for 10 s. Proteins present in serum or tissue homogenate were precipitated using a concentrated solution of NaCl (10 g/100 mL; at a ratio of 1:1) after which samples were shaken on a vortex mixer for 15 s. Following this, samples were extracted with 3 mL of a mixture of ethyl acetate/hexane (30:70, v/v) for 20 min on a shaker (VXR Vibrax, IKA, Staufen, Germany) and centrifuged at 1000g for 15 min (Universal 32, Hettich, Germany). The organic layers were then transferred to fresh tubes and evaporated to dryness at 37 °C under a gentle stream of nitrogen. The residue in each tube was then dissolved in 100 μL of methanol with 50 μL aliquots injected into the HPLC system. The HPLC analysis was performed on a 250 × 4.6 mm LiChrospher100 RP-18 column (5 μm particle size) protected with a guard column (4 × 4 mm) containing the same packing material (Merck, Darmstadt, Germany). The mobile phase pumped at a flow rate of 1 mL/min was composed of deionized water and acetonitrile at a ratio of 46:54 (v/v). The column temperature was maintained at 21 °C. The HPLC system (Merck-Hitachi, Darmstadt, Germany) consisted of an L-7100 isocratic pump, an L7200 autosampler, and a UV/vis K-2600 detector (Knauer, Berlin, Germany) operating at 251 nm. For data acquisition and processing, the D-7000 HSM software was used. Under these conditions, the retention times of IS and SF-11 were approximately 8 and 13.5 min. The calibration curve constructed by plotting the ratio of the peak height of SF-11 to IS versus SF-11 concentrations was linear in the tested concentration ranges. The limit of quantification of the analytical method was 10 ng/mL (20 ng/g tissue). There were no interfering peaks observed in the chromatograms. The assay was reproducible as indicated by coefficients of variation of less than 10% for both intra- and interday assessments. The extraction efficiencies of SF-11 and IS were higher than 80%. SF-11 serum and tissue concentrations were expressed in μM (μM/kg of wet tissue). Pharmacokinetic Analysis. Pharmacokinetic parameters were calculated on the basis of the concentration vs time profile of SF-11 using noncompartmental and compartmental analysis with the aid of Phoenix WinNonlin v. 7.0 (Pharsight Corp, Certara, St. Louis, MO, USA) software. The peak concentration (Cmax) and the time to reach the maximum concentration (tmax) were both obtained directly from the serum or tissue concentration vs time graph. The linear trapezoidal rule was applied to calculate the areas under the concentration−time curve (AUC) from the time of dosing to the last measured data point (AUClast) or infinity (AUC0−∞). The terminal slope (λz) was estimated by linear regression. The terminal half-life (t0.5λz) was obtained by dividing ln 2 by λz. The volume of distribution based on the terminal phase (Vz/F) was calculated using the following equation: dose/(λz·AUC0−∞), where F is the fraction of the dose absorbed. The oral clearance (CL/F) was estimated from the formula: dose/AUC0−∞; the mean residence time (MRT) had the following equation: AUMC0−∞/AUC0−∞, where AUMC is the area under the first moment curve. In addition, SF-11 serum concentration−time data were fitted to a one-compartment pharmacokinetic model. Behavioral Study. Animals. Behavioral study experiments were performed as previously described.20 Experimental procedures were carried out according to the guidelines of the European Directive 2010/63/EU and approved by the Ethical Committee of the Institute of Pharmacology, Polish Academy of Sciences in Krakow. Male Sprague−Dawley rats (Charles River, Germany) weighing about 250−300 g were kept under standard laboratory conditions: room temperature, 22 ± 2 °C; humidity, 50 ± 5%; 12 h light/dark cycle (light on 6:00) with ad libitum access to water and food. Administration of Compounds. The following compounds were used in this study: [N-(4-ethoxyphenyl)-4-(hydroxydiphenylmethyl)1-piperidinecarbothioamide] (SF-11); 1,4-diamino-2,3-dicyano-1,4bis[2-aminophenylthio] butadiene (U0126), an inhibitor of MAPK/ ERK; 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002), an inhibitor of PI3K (Tocris Bioscience, UK). SF-11 (3, 10, or 20 mg/kg) was suspended in 0.5% methylcellulose and

not very effective, often requiring long-term use, it is worth noting that SF-11 showed an antidepressant-like effect after acute administration. Y2Rs may thus represent very promising targets in the treatment of depression. However, more research is necessary especially in other robust animal models of depression to fully elucidate the possible mechanism of a Y2Rmediated antidepressant-like effect.

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METHODS

Vibrational Spectroscopy Study. Sample Preparation. [N-(4Ethoxyphenyl)-4-(hydroxydiphenylmethyl)-1-piperidinecarbothioamide] (SF-11) was purchased from Tocris Bioscence (UK) and used without further purification. RS Measurements. The Raman spectra of SF-11, in the solid state, were measured using a Renishaw InVia Raman spectrometer combined with a CCD camera thermoelectrically cooled to 203 K and a confocal microscope (Leica with a 100× magnification objective). The excitation source was a 785 nm diode laser, which provides a 70 mW power output. The spectra were recorded with 1 cm−1 resolution in the spectral range of 350−3200 cm−1. Typically, 4 scans with 30 s of exposure time were collected. FT-IR Measurements. For FT-IR experiments, thin pellets containing 200 mg of KBr and about 1 mg of SF-11 were prepared The spectra were recorded in a transmission mode by means of Vertex70v (Bruker, Germany) vacuum spectrometer equipped with a KBr beamsplitter and a wide range DLaTGS detector in the spectral range of 350−3200 cm−1 with 4 cm−1 resolution. 256 scans were enough to acquire a good signal-to-noise ratio. Theoretical Calculations. The Gaussian 03 software package65 with density functional theory (DFT) at the B3LYP level was used to optimize the SF-11 molecular structure to generate the theoretical RS and IR spectra. 6-311G(d,p), the split-valence triple-ζ basis set with polarization functions, one set on heavy atoms and one set on hydrogens, was used as the basis set.66 No imaginary wavenumbers were observed proving that the optimized structure of SF-11 matched the energy minima on the potential energy surface for nuclear motion. Calculations were performed at the Academic Computer Centre “Cyfronet” in Krakow. The output data from the Gaussian was entered into the Raint program that converts Raman activities to Raman intensities.67 The 785 nm line was chosen as an excitation source. Theoretical RS and IR spectra were produced by the freeware GaussSum 0.8 software package.68 For a better comparison with experimental results, the 0.982 scaling factor, 11 cm−1 full width at half-maximum (fwhm), and 50%/50% Gaussian/Lorentzian band shape were applied. The calculated and experimental wavenumbers together with potential energy distribution (PED) for the RS and FTIR spectra of SF-11 are presented in Table S2. Pharmacokinetic Study. Sample Collection. Male Sprague− Dawley rats (Charles River, Germany) weighing between 250 and 300 g were used for the pharmacokinetic study of SF-11. The compound was dispersed in 0.5% methylcellulose and administered i.p. as a single dose of 10 mg/kg. Rats were decapitated at seven different time points (5, 15, 30, 60, 120, 240, and 360 min after administration of SF-11) to obtain blood and brain samples (n = 3 to 4 per time point). Blood was collected in Eppendorf tubes, allowed to clot at room temperature, and centrifuged at 3023g for 15 min, and the resulting serum was frozen at −20 °C until required for analysis. Also, immediately after decapitation, brain structures were dissected (frontal cortex with elements of the prefrontal cortex and the hippocampus), rinsed with 0.9% NaCl, and frozen at −20 °C until required for analysis. Moreover, several peripheral tissues, such as the liver, kidneys, lungs, and heart, were harvested. All samples were kept frozen at −20 °C until required for analysis. Determination of SF-11 in Serum and Tissue Samples Using High-Performance Liquid Chromatography (HPLC). For the isolation of SF-11 from tissues, the dissected brain structures or peripheral organs were homogenized in distilled water (1:4, w/v) using a tissue homogenizer TH220 (Omni International, Inc., Warranton, VA, USA). Aliquots of serum or tissue homogenate (0.5 G


Research Article

ACS Chemical Neuroscience given i.p. in a volume of 1 mL/kg 60 min before the FST. Additionally, SF-11 at a dose of 10 mg/kg was also administered 30 min or 4 h before the FST. U0126 (5 μg/2 μL) and LY294002 (10 nmol/2 μL) were dissolved in 0.1 M phosphate buffer (pH = 7.4) and administered by the i.c.v. route (1 μL/site) 15 min before SF-11 (10 mg/kg) treatment. Control rats were administered with the appropriate vehicle. The doses of SF-11 used were based on results from our preliminary experiments and also on the study of Brothers et al.28 The doses of the kinase inhibitors and the schedule of drug administration were selected on the basis of our previous study.20 Intracerebroventricular Cannulae Implantation. Surgical implantation (i.c.v.) of cannulae was carried out as previously described.20 Briefly, the rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) administered intramuscular (i.m.), and guide cannulae were stereotaxically and bilaterally implanted into the brain ventricles as per the following coordinates: AP = −0.4 mm, L = +1.5 mm from the Bregma, and H = −4.6 mm from the skull.69 Seven days after i.c.v. implantation, solutions were infused bilaterally for 60 s using a Hamilton microsyringe. Following bilateral infusion, rats were subjected to behavioral testing. Forced Swim Test (FST). The FST was carried out as described previously.20 Briefly, each rat was placed in a glass cylinder containing 15 cm of water maintained at 25 °C for 15 min (pretest and habituation). After habituation, the rats were returned to their home cages. The FST was carried out after 24 h during which time rats were placed again in the cylinder, and the total duration of immobility was measured during a 5 min test. Separate control groups were used for each experiment. The FST has been done by the same experimenters. Locomotor Activity. Spontaneous locomotor activity of the rats was measured after the FST, following previously described methods.20 Locomotor activity was recorded for 5 min for each animal in Opto-Varimex cages (Columbus Instruments, San Diego, CA, USA) linked online to an IBM compatible PC. Rat behavior was analyzed using Auto-Track software (Columbus Instruments, USA); data is presented as the distance traveled in cm. Statistical Analysis. Results are presented as mean ± SEM. Behavioral data were analyzed using a Student’s t test or one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparisons post hoc test and two-way ANOVA followed by the Tukey’s multiple comparisons post hoc test (GraphPad Prism 7 Software). When appropriate, a value of p < 0.05 was considered statistically significant.

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Natalia Piergies: 0000-0003-4899-3534 Ewa Pięta: 0000-0001-7071-0284 Author Contributions

H.D. conceived the idea, contributed to the design of the research, participated in the pharmacokinetic and behavioral studies, and created the first and final versions of the manuscript. N.P. and E.P. contributed to the design of the vibrational spectroscopy study, performed vibrational spectroscopic measurements and density functional theory calculations, and analyzed the data. E.W. and P.W. contributed to the design of the pharmacokinetic study. E.W. developed and validated the analytical method and performed the pharmacokinetic analysis. N.P., E.P., and E.W. cowrote the paper. B.P. participated in the behavioral study. B.S. contributed to the design of the behavioral study, participated in the pharmacokinetic and behavioral studies, and analyzed the behavioral data. N.P., E.P., E.W., P.W., M.S., C.P., and B.S. contributed to revisions of the last version of the manuscript. All authors have approved the final version of the manuscript. Funding

The vibrational spectroscopy study was performed using equipment purchased under a project cofunded by the Małopolska Regional Operational Program Measure 5.1 Krakow Metropolitan Area as an important hub of the European Research Area for 2007−2013, project no. MRPO.05.01.00-12-013/15. Pharmacokinetic and behavioral studies were supported by statutory funds of the Maj Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland and partly by the grant “Depression-mechanisms-therapy” POIG.01.01.01-12-004/09. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the PL-Grid Infrastructure (Academic Computer Center “Cyfronet” in Krakow) for the use of their computational facilities.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.9b00082. Selected calculated bond lengths and angles of SF-11, the calculated and experimental wavenumbers and potential energy distribution (PED) for the RS and FT-IR spectra of SF-11, serum and brain concentrations of SF-11 following i.p. administration of this compound at a dose of 10 mg/kg to rats, SF-11 concentrations in peripheral organs following i.p. administration of this compound at a dose of 10 mg/kg to rats, and pharmacokinetic parameters of SF-11 in peripheral tissues following i.p. administration (10 mg/kg) to rats estimated using noncompartmental analysis (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +48 12 6623315. Fax: +48 12 6374500 (H.D.). ORCID

Helena Domin: 0000-0001-5831-7105 H


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