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.2023 Sep 18;6(10):1492-1507.
doi: 10.1021/acsptsci.3c00142. eCollection 2023 Oct 13.

In Vitro andIn Vivo Evaluation of Pellotine: A HypnoticLophophora Alkaloid

Affiliations

In Vitro andIn Vivo Evaluation of Pellotine: A HypnoticLophophora Alkaloid

Christian B M Poulie et al. ACS Pharmacol Transl Sci..

Abstract

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), aLophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate variousin vitro andin vivo properties of1. We demonstrate that1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally,1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM,Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM,Emax = -98.6). Finally, we demonstrated that1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

© 2023 American Chemical Society.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Chemical structuresof a selection ofLophophora alkaloids. Pellotine(1) and anhalonidine (4) are representativesof the phenolic THIQ class, mescaline (2) is a representativeof the β-PEA class, and lophophorine(3) is a representative of the non-phenolic THIQ class.
Scheme 1
Scheme 1. Synthesis of Pellotine (1)
Reagents and conditions:(i)(a) 2,2-dimethoxyethylamine (2.2 equiv), MeOH, room temperature, overnight(b) NaBH4 (1.3 equiv), 0 °C, 2 h, quantitative; (ii)8 M HCl(aq), room temperature, 7 days, 62%; (iii) Et3SiH (2.2 equiv), TFA, Ar, room temperature, overnight, 27%;(iv) (a) 37% formaldehyde(aq) (1.6 equiv), MeOH, room temperature,30 min, (b) NaBH4 (1.3 equiv), 0°C, 30 min, 85%.
Figure 2
Figure 2
In vitro andin vivo metabolicdata. All data points are from the AUC obtained from MS scan in ESI-positivemode from 200 to 500 Da. Graphs are displayed as mean ± SEM.(a) Structures of pellotine (1) and metabolites observedin vitro andin vivo. (b) Relative metabolicstability of pellotine (1)in vitro:stability displayed as the percent of pellotine (1) remainingover time. Data from three independent experiments, each measuredin triplicate. The combined data for both analytical and experimentalreplicates is displayed. Microsomal stability in male mouse livermicrosomes (MLMs) and female human liver microsomes (HLMs) were monitoredover 240 min. Plasma stability in human plasma (HP) was monitoredover 180 min. (c) Relative relationship between pellotine (1) and the observed metabolites in mouse liver microsomes displayedas % of total peak area for each data point. (d) Metabolic stabilityof pellotine (1)in vivo: Distributionof metabolites observed in blood from three different animals at both30 min and 4 h after administration. Distribution displayed as %AUC.(e) Distribution displayed as total AUC. (f) Metabolic stability ofpellotine (1)in vivo: Distributionof metabolites observed in urine from 16 different animals dosed ingroups of four with either 10, 20, 30, or 40 mg/kg pellotine (1) at 2.5 h after administration. Distribution displayed as%AUC. (g) Distribution displayed as AUC.
Figure 3
Figure 3
Biodistribution of pellotine(1) (m/z 238.1438)analyzed with DESI-MSI in whole-bodymice and sagittal section of the rat brain. (a, c, o) Picture of whole-bodymice prior to cryo-sectioning. (b) DESI-MSI of whole-body mice dosedwith 40 mg/kg pellotine (1) and sacrificed 30 min (n = 1) and (d) 4 h (n = 1) after drug administration.(e, g, i, k, m) HE-stained rat brain slices. (f) Sagittal sectionof brain of a rat dosed with 10 mg/kg pellotine (1) andsacrificed 30 min (n = 1) and (h) 4 h (n = 1) after drug administration. (j) Sagittal section of brain ofa rat dosed with 40 mg/kg pellotine (1) and sacrificed30 min (n = 1) and (l) 4 h (n =1) after drug administration. (n) Sagittal section of control ratbrain (not dosed with pellotine (1)). (p) Whole-bodycontrol mouse brain (not dosed with pellotine (1)).
Figure 4
Figure 4
Concentration–responsecurves of pellotine (1) at the 5-HT6 and 5-HT7 receptors. (a) Concentration–responsecurve of pellotine (1) as agonist at 5-HT6 in the HTRF (homogeneous time-resolved fluorescence) cAMP assay.(b) Concentration–response curve of pellotine (1) as an agonist at 5-HT6 in the β-arrestin assay.(c) Concentration–response curve of pellotine (1) as agonist at 5-HT7 in the HTRF (homogeneous time-resolvedfluorescence) cAMP assay. (d) Concentration–inhibition curveof pellotine (1) as an inverse agonist at 5-HT7 in the GloSensor cAMP assay. The data in panels a–c representthe mean ± SEM (standard error of the mean) from three independentexperiments, and normalization of the response is relative to themaximal response mediated by 5-HT. Data in panel d represent the mean± SEM (standard error of the mean) from three independent experiments,each performed in duplicate, and normalization of the response isrelative to the maximal inhibition exerted by risperidone.
Figure 5
Figure 5
(a) In the majority ofcells, pellotine (1) inducedchanges in calcium, as exemplified by these representative recordingsfrom individual Fura-2AM-loaded cells within the DR in which increasesin fluorescence were induced by pellotine (1). (b) Inthe majority of responding cells in all three pontine nuclei, increasesin fluorescence were induced, indicative of increases in calcium,as indicated by the green boxes from the population data. However,in a minority of cells, decreases in fluorescence were noted as indicatedby the blue boxes, or no response was seen as indicated by the pinkboxes. Dorsal raphe (DR) (n = 64), laterodorsal tegmentum(LDT) (n = 67), and locus coeruleus (LC) (n = 89).
Figure 6
Figure 6
Locomotor activity inmice. (a) Total distance (cm) moved from10 to 70 min (n = 16). Pellotine (1)was administered as 40 mg/kg and zolpidem as 10 mg/kg. [One-way rANOVA:F(2,45) = 11.19,p = 0.0001;Tukey multiple comparison: (**)p < 0.01, (***)p < 0.001.] (b) Dose–response for pellotine (1) as total distance (cm) moved (n = 4 foreach dose). [One-way rANOVA:F(4,15) =9.714,p < 0.001; Tukey multiple comparison: (**)p < 0.01, (***)p < 0.001.] Dataare shown as mean ± SEM.
Figure 7
Figure 7
Total time spent in each sleep state after pellotine (1) treatment. Baseline (n = 5), vehicle (n = 5), 10 mg/kg (n = 4), and 50 mg/kg(n = 3). (a) Total length of NREM. (b) Total lengthof REM. [Two-way rANOVA; main effect (treatment):F(3,13) = 15.09, (###)p <0.001; interaction (treatment × time):F(3,13) = 1.06,p < 0.4.] (c) Total lengthof wake. (*)p < 0.05, (**)p < 0.01, (****)p < 0.0001 by Dunnettpost hoc analysis of two-way ANOVA. Data are shown as mean± SEM.
Figure 8
Figure 8
Bout analysiswas done according to a sleep state. Baseline (n =5), vehicle (n = 5), 10 mg/kg (n = 4), and 50 mg/kg (n = 3). (a) Numberof bouts of NREM. [Two-way rANOVA; main effect (treatment):F(3,13) = 9.43, (##)p < 0.01; interaction (treatment × time):F(9,39) = 2.87,p < 0.1.] (b) Averagelength of NREM. [Two-way rANOVA; main effect (treatment):F(3,13) = 5.87, (##)p < 0.01; interaction (treatment × time):F(9,39) = 2.92,p < 0.01.] (c) Numberof bouts of wake. [Two-way rANOVA; main effect (treatment):F(3,13) = 11.23, (###)p < 0.001; interaction (treatment × time):F(9,39) = 3.49,p < 0.01.] (d) Averagelength of wake. (*)p < 0.05, (**)p < 0.01, by Dunnett post hoc analysis of two-way ANOVA. Data areshown as mean ± SEM.
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