The Psychedelic N,N-Dipropyltryptamine Prevents Seizures in a Mouse Model of Fragile X Syndrome via a Mechanism that Appears Independent of Serotonin and Sigma1 Receptors

The serotonergic psychedelic psilocybin shows efficacy in treating neuropsychiatric disorders, though the mechanism(s) underlying its therapeutic effects remain unclear. We show that a similar psychedelic tryptamine, N,N-dipropyltryptamine (DPT), completely prevents audiogenic seizures (AGS) in an Fmr1 knockout mouse model of fragile X syndrome at a 10 mg/kg dose but not at lower doses (3 or 5.6 mg/kg). Despite showing in vitro that DPT is a serotonin 5-HT2A, 5-HT1B, and 5-HT1A receptor agonist (with that rank order of functional potency, determined with TRUPATH Gα/βγ biosensors), pretreatment with selective inhibitors of 5-HT2A/2C, 5-HT1B, or 5-HT1A receptors did not block DPT’s antiepileptic effects; a pan-serotonin receptor antagonist was also ineffective. Because 5-HT1A receptor activation blocks AGS in Fmr1 knockout mice, we performed a dose–response experiment to evaluate DPT’s engagement of 5-HT1A receptors in vivo. DPT elicited 5-HT1A-dependent effects only at doses greater than 10 mg/kg, further supporting that DPT’s antiepileptic effects were not 5-HT1A-mediated. We also observed that the selective sigma1 receptor antagonist, NE-100, did not impact DPT’s antiepileptic effects, suggesting DPT engagement of sigma1 receptors was not a crucial mechanism. Separately, we observed that DPT and NE-100 at high doses caused convulsions on their own that were qualitatively distinct from AGS. In conclusion, DPT dose-dependently blocked AGS in Fmr1 knockout mice, but neither serotonin nor sigma1 receptor antagonists prevented this action. Thus, DPT might have neurotherapeutic effects independent of its serotonergic psychedelic properties. However, DPT also caused seizures at high doses, showing that DPT has complex dose-dependent in vivo polypharmacology.

M any studies are investigating the therapeutic potential of serotonergic psychedelics, including psilocybin and related psychedelic tryptamines, for various psychiatric conditions.Indications under study include but are not limited to major depressive disorder (MDD) and substance-use disorders, whereas other indications under consideration include autism spectrum disorder (ASD) and fragile X syndrome (FXS). 1−9 Despite the proliferation of clinical studies, the specific pharmacodynamic properties that contribute to the therapeutic efficacies of psychedelics are not well understood.−18 This poses the question of whether targets in addition to 5-HT 2 Rs contribute to the pharmacotherapeutic effects of psychedelics. 19,20−23 FXS is a monogenic neurodevelopmental disorder that is the leading cause of intellectual disability and ASD. 24In addition to various other neurobehavioral issues, individuals with FXS present with auditory hypersensitivities and seizures. 25Seizures affect approximately 12% of FXS patients, 26−31 with three times higher risk in comorbid ASD patients. 32−35 Clinical trials are now underway assessing the efficacy of selective 5-HT 2C R agonists for treating seizures in Dravet syndrome, 36 based partly on historical studies showing susceptibility in 5-HT 2C R knockout (KO) mice to audiogenic seizures (AGS). 37−41 A classical study showed that the serotonergic psychedelic 5-methoxy-N,N-dimethyltryptamine inhibited myoclonic seizures caused by photic stimulation in lateral geniculate-kindled felines. 42−45 Still, others show that nonselective 5-HT 2 R antagonists block psychostimulant-induced convulsions, 46 and there are clinical reports of seizures induced by certain, full-efficacy 5-HT 2A agonist psychedelics. 47Thus, the functions of distinct 5-HT 2 R subtypes in modulating distinct types of epilepsy remain unsolved.
With few exceptions, e.g., in absence seizures, 48,49 in preclinical models, 5-HT 1A R activation is antiepileptic.−52 WAY-100635, a selective 5-HT 1A R antagonist, inhibits the anti-AGS effect in Fmr1 KO mice of the selective 5-HT 1A agonist, NLX-112. 50WAY-100635 also inhibits the anticonvulsant effects of the 5-HT 1A/1B R agonist RU24969 on pentylenetetrazol-induced seizures and inhibits the anticonvulsant effects of the 5-HT 1A R agonist 8-OH-DPAT on picrotoxin-induced seizures in mice, 45,50,53 demonstrating that activation of 5-HT 1A Rs is antiepileptic in various seizure models.−56 This provides ample evidence that targeting the central 5-HT system may be a fruitful approach for treating epilepsies.
Sigma1Rs are another target of tryptamines that can modulate epileptiform activity. 11,57−59 Also, fenfluramine was shown to act as a positive allosteric modulator at sigma1Rs in mice and zebrafish models, 60 and sigma1R modulation prevents seizures in models of Dravet syndrome, amphetamine-induced seizures, and epileptic encephalopathies. 59,61,62e tested the hypothesis that the serotonergic psychedelic N,N-dipropyltryptamine (DPT) would prevent AGS in juvenile Fmr1 KO mice and that it would be effective via a serotonergic or sigma1R mechanism.DPT is a short-acting psychedelic tryptamine, but there is limited knowledge about its pharmacology and behavioral effects; in vivo, it possesses agonist activity at 5-HT 1A and 5-HT 2A Rs, 63−66 and in vitro it has been shown to be a substrate of the 5-HT transporter. 14PT is not restricted as a Schedule 1 controlled substance and hence is accessible for laboratory research without possessing a Drug Enforcement Agency controlled substances license.Here, we report observations that DPT prevents AGS in Fmr1 KO mice, but our in vitro and in vivo pharmacology experiments did not provide evidence that its antiepileptic effects were 5-HTR-or sigma1R-mediated.

■ RESULTS
DPT Is an Antiepileptic in Fmr1 KO Mice.Compounds that target the central 5-HT system, such as fenfluramine, have antiepileptic effects in individuals with neurodevelopmental disorders. 33,34,41,67Hence, we evaluated the antiepileptic effects of DPT in juvenile Fmr1 KO mice using the AGS assay.As shown in Figure 1, vehicle-treated male and female Fmr1 KO mice showed a prevalence of AGS of 72%, and DPT completely prevented AGS at 10 mg/kg (p < 0.0001).Sixtyfive percent of mice treated with 10 mg/kg DPT showed normal behavior during the presentation of the seizure-eliciting alarm (akin to wild-type (WT) mice), whereas 35% showed a wild-running and jumping (WRJ) response.The duration of WRJ in these mice was significantly longer than WRJ in vehicle-treated mice (vehicle (mean ± SEM), 22 ± 6.26 s vs 10 mg/kg DPT (mean ± SEM), 78 ± 20.7 s; p < 0.01) which could indicate that DPT treatment prevented the transition to the tonic−clonic seizure (TCS) stage of AGS in these mice. 68,69Relative to vehicle, DPT did not significantly affect the prevalence of AGS at 3 (p = 0.69) or 5.6 mg/kg (p = 0.69).In addition, 3 and 5.6 mg/kg DPT did not impact latency to seizure onset, seizure duration, or lethality caused by AGS (p values ≥ 0.46, Supplemental Figure S1).
In Vitro, DPT Is a Modest Potency 5-HT 2A R Agonist, and In Vivo, DPT Elicits Peak 5-HT 2A R-Dependent Head-Twitch Responses at a Dose Equivalent to Its Antiepileptic Dose.We next tested DPT's in vitro and in vivo pharmacology at 5-HT 2A Rs to explore whether 5-HT 2A R activation could be mediating its antiepileptic effects.In vitro, DPT was a moderate potency full agonist at 5-HT 2A Rs with E max of 106% ± 1.13 (mean ± SEM), relative to 5-HT, the positive control.EC 50 and K i values are reported in Table 1.See Figure 2A,B for affinity and function nonlinear regression curves.
Antagonism of 5-HT 2A/2C Rs Does Not Block DPT's Antiepileptic Effects.As DPT engaged 5-HT 2A Rs in vivo at 10 mg/kg, which matched its effective dose to block AGS, we tested whether selective antagonism of 5-HT 2A/2C Rs blocks DPT's anti-AGS effects (Figure 2D).Pretreatment with 3 mg/ kg pimavanserin slightly reversed the antiepileptic effects of DPT 10 mg/kg (AGS prevalence 25% for pimavanserin 3 mg/ kg plus DPT 10 mg/kg vs AGS prevalence of 0% for DPT 10 mg/kg alone), but this increase in the seizure prevalence was not significant (p = 0.10).Pretreatment with pimavanserin 10 mg/kg did not impact DPT's anti-AGS effects (AGS prevalence 0 vs 0%; p > 0.99).Mice treated with pimavanserin 3 mg/kg behaved normally, i.e., like vehicle-treated mice.Mice treated with pimavanserin 10 mg/kg showed signs of mild sedation which included partial ptosis, low sensory responses, hypolocomotion, and immobility that took effect within 2 min of injection and which lasted ∼30 min.These observations suggest that the anti-AGS effects of DPT are not mediated by 5-HT 2A / 2C R activation.These data align with our previous observations that the 5-HT 2C R-preferring agonist, lorcaserin, does not prevent AGS in juvenile Fmr1 KO mice. 70n Vitro, DPT Is a Low-Potency 5-HT 1A R Agonist, and In Vivo, DPT Elicits 5-HT 1A R-Dependent Behavioral Effects at Doses Higher than Its Antiepileptic Dose.As we and others recently showed that 5-HT 1A R activation blocks AGS in Fmr1 KO mice, 50,51 we next tested whether DPT activation of 5-HT 1A Rs could be mediating its anti-AGS effects.We tested its pharmacology at 5-HT 1A Rs in vitro and in vivo.In vitro, we observed that DPT is a low potency partial agonist at 5-HT 1A Rs, with an E max of 53% ± 3.67 (mean ± SEM) relative to 5-HT, the positive control.These observations are similar to a previous study of DPT at 5-HT 1A Rs. 71 EC 50 and K i values are reported in Table 1.See Figure 3A and 3B for affinity and function curves.As shown in Figure 3C, relative to vehicle, juvenile and adult mice treated with DPT at 20 mg/kg, but not lower doses, showed distinct behavioral symptoms, which included flat body posture (0 vs 90%, p = 0.0001), hind limb abduction (0 vs 90%, p = 0.0001), tremors (0 vs 70%, p = 0.003), and Straub tail (0 vs 90%, p = 0.0001).These behaviors are 5-HT 1A R-dependent, 50,72 providing evidence that DPT does not sufficiently engage 5-HT 1A R at its antiepileptic dose of 10 mg/kg.
Antagonism of 5-HT 1A Rs Does Not Block DPT's Antiepileptic Effects.To further evaluate whether DPT's antiepileptic effects in the AGS model were 5-HT 1A Rdependent, we tested whether selective antagonism of 5-HT 1A Rs blocks DPT's effects.Pretreatment with WAY-100635 0.1 and 1 mg/kg did not significantly reverse DPT's anti-AGS effects (13% vs 0% p = 0.33, and 0% vs 0% p > 0.99, respectively) (Figure 3D).Importantly, we previously showed that WAY-100635 at a 0.1 mg/kg dose blocks the antiepileptic effects of the highly selective 5-HT 1A R agonist, NLX-112, in the AGS assay in Fmr1 KO mice. 50Thus, these data support the conclusion that DPT's antiepileptic effects are not 5-HT 1A R-mediated.
In Vitro, DPT Is a Very Low-Potency 5-HT 1B R Agonist, and In Vivo, Antagonism of 5-HT 1B Receptors Does Not Block DPT's Antiepileptic Effects.We next tested DPT's in vitro pharmacology at 5-HT 1B Rs and observed that it is a very low-potency 5-HT 1B R agonist, relative to 5-HT, the positive control.DPT's E max was 83% ± 1.95 (mean ± SEM), relative to 5-HT.EC 50 and K i values are reported in Table 1.See Figure 4A,B for affinity and function curves.We tested whether selective 5-HT 1B R antagonism would block DPT's anti-AGS effects.As shown in Figure 4C, pretreatment with SB-224289 at 5 mg/kg increased seizure prevalence when compared to treatment with DPT 10 mg/kg alone (25 vs 0%).However, the difference was not statistically significant (p = 0.10).These observations suggest that activation of 5-HT 1B Rs does not mediate DPT's anti-AGS effects.
Pan-5-HTR Antagonism Does Not Block DPT's Antiepileptic Effects.Given DPT's structural similarity and shared targets with 5-HT, and our objective to investigate the potential involvement of multiple 5-HTRs in mediating the anti-AGS effects of DPT, we next tested the effects of the pan- 5-HTR antagonist methiothepin.Pretreatment with methiothepin at 4 mg/kg did not influence DPT's anti-AGS effects (seizure prevalence 0 vs 0%; p > 0.99) (Figure 5).During the observation period, mice pretreated with methiothepin appeared to be highly sedated (partial or complete ptosis) while lying in a prone or medial-lateral position being either slightly responsive or unresponsive to the AGS-eliciting alarm.These effects were more prominent in the methiothepintreated group as compared to the pimavanserin 10 mg/kg group (data not shown).We did a pilot test of pretreatment with methiothepin at 2 mg/kg; it also did not impact the antiepileptic effects of DPT (data not shown).Collectively, our observations suggest that the anti-AGS effects of DPT may be mediated by a mechanism(s) that is independent of 5-HTRs.
Antagonism of Sigma1Rs Does Not Block DPT's Antiepileptic Effects.Since no 5-HTR antagonist challenged DPT's antiepileptic efficacy in the AGS assay, we tested whether a nonserotonergic receptor could mediate DPT's antiepileptic effects.DPT and other tryptamines bind sigma1Rs, which studies have shown modulate epileptic activity. 14,59,61,62,73NE-100, a selective sigma1R antagonist, is structurally similar to DPT, sharing an N,N-dipropyl moiety 74 (see Figures 1 and 6).NE-100 potentiates seizures at a 25 mg/ kg dose and induces seizures at 50 mg/kg and above. 75To determine suitable doses of NE-100 for evaluation in the AGS assay�doses of NE-100 that straddle the threshold dose for NE-100 causing seizures on its own�we tested NE-100 at six different doses, 5, 10, 15, 20, 30, and 50 mg/kg, in juvenile WT Figure 2. In vitro and in vivo pharmacology of DPT at 5-HT 2A Rs and examination of the impact of inhibiting 5-HT 2A/2C Rs on the antiepileptic properties of DPT.(A) In vitro radioligand competition binding of DPT and 5-HT at human (h) 5-HT 2A Rs.The 100 μM data point was interpolated, so curves reached asymptote (no specific binding).Data were obtained from two separate experiments in which 5-HT was tested in duplicate and DPT was tested in sextuplicate per concentration.Data best fit to a "two-site, fit K i " model, which is shown.(B) In vitro, functional activity of 5-HT and DPT at h5-HT 2A Rs.Data were obtained from three experiments in which 5-HT and DPT were tested in quadruplicate per concentration.(C) DPT dose-dependently elicited the 5-HT 2A R-dependent HTR, with maximal effects at 10 mg/kg in juvenile and adult WT mice.(We previously found no difference in DOI-elicited HTRs between WT and Fmr1 KO mice. 22) Note that this dose was equivalent to the effective dose of DPT to prevent seizures, demonstrating that DPT engaged 5-HT 2A Rs while it prevented seizures.*represents p < 0.05 and ## , **represents p < 0.01 compared to vehicle.Despite this, as shown in (D), the selective 5-HT 2A/2C R antagonist, pimavanserin, did not significantly block the anti-AGS effects of DPT in juvenile Fmr1 KO mice.**** represents p < 0.0001 DPT 10 mg/kg compared to vehicle; data reproduced from Figure 1  and Fmr1 KO mice, and observed them for 30 min following administration.Like a previous report, 75 we observed that 50 mg/kg NE-100 induced generalized TCS (Table 2), whereas the mice exhibited normal behavior when administered the lower doses.Thus, we tested the impact of 30 mg/kg and 50 mg/kg NE-100 on DPT's anti-AGS effects.Neither 30 mg/kg nor 50 mg/kg NE-100 pretreatment reversed the anti-AGS effects of 10 mg/kg DPT (all mice showed no AGS; 0%, p > 0.99) (Figure 6).
DPT and NE-100 Cause Seizures on Their Own that Are Qualitatively Distinct from AGS.During dose− response testing of DPT and NE-100, we observed that at high doses, i.e., 20 mg/kg DPT and 50 mg/kg NE-100, both compounds caused seizures on their own.Hence, we compared the behavioral signs of convulsions due to AGS, DPT, and NE-100; each caused a unique time-dependent repertoire of behavioral symptoms (Table 2).DPT 20 mg/kg induced convulsions in 90% of mice tested (N = 10 total WT mice tested).These began between 5 and 10 min after administration, and the duration was brief.Mice exhibited myoclonic tremors that transitioned to tonic convulsions with prominent opisthotonos, and 50% of the mice vocalized during the episode.The entire seizure episode lasted less than 20 s.All mice recovered, and then showed behavioral signs of 5-HT 1A R activation (see Table 2).One adult male mouse also showed excessive salivation after the seizure.
NE-100 50 mg/kg induced convulsions in 69% of mice tested (N = 13 total tested, including 10 Fmr1 KO and 3 WT mice).Behaviors that preceded seizures and behaviors indicative of seizures were similar to those reported by Vavers et al. 75 NE-100-induced seizures lasted 150−200 s, and mice recovered to normal behavior between 20 and 30 min after treatment.Like DPT, NE-100-induced seizures were not lethal.Importantly, we did not observe a treatment by genotype effect (Supplemental Figure S4).In the AGS experiments, cotreatment with NE-100 50 mg/kg and DPT 10 mg/kg (N = 8, the same subjects as those in Figure 6) caused seizures within ∼2 min of administration, i.e., prior to sounding the AGSeliciting alarm.These seizures presented the same symptomology as observed with NE-100 50 mg/kg alone.After sounding the alarm, the characteristics of the seizures observed were like NE-100-induced seizures.In other words, mice did not exhibit AGS (see Methods and Table 2).In conclusion, DPT and NE-100 caused seizures that were qualitatively distinct from AGS, suggesting different mechanisms.

■ DISCUSSION
We discovered that DPT prevents AGS in juvenile Fmr1 KO mice, a genetic model of FXS.We also report DPT's affinity and function at 5-HT 2A , 5-HT 1A , and 5-HT 1B receptors.DPT was effective at preventing AGS only at a 10 mg/kg dose, whereas at lower doses AGS persisted.In separate studies of in vivo receptor engagement, DPT activated 5-HT 2A receptors, i.e., elicited the HTR, 76,77 at its antiepileptic dose.It took a higher dose to engage 5-HT 1A Rs, e.g., to elicit hind-limb abduction and flat body posture, which are elicited by 5-HT 1A R activation. 72,78These results corroborated our in vitro data which showed DPT was a moderate-potency full agonist at 5-HT 2A Rs but a low-potency partial agonist at 5-HT 1A Rs.We also showed that DPT was a low-potency 5-HT 1B R agonist in vitro.The in vitro and in vivo receptor pharmacology studies suggested that DPT engaged 5-HT 2A Rs but not 5-HT 1A or 5-HT 1B Rs at its antiepileptic dose.As an additional approach to investigate if these receptors contributed to DPT's antiepileptic properties, we tested if co-administration of selective 5-HT 2A/2C , 5-HT 1A , or 5-HT 1B R antagonists could block DPT's antiepileptic effects.None did nor did a pan 5-HTR antagonist.Collectively, our results suggest that DPT blocks AGS in juvenile Fmr1 KO mice via a nonserotonergic mechanism.
Interestingly, we observed that at high doses, DPT switched from an antiepileptic (in the AGS assay) to a proconvulsant, eliciting tonic seizures when administered on its own.The proconvulsant effects of DPT that we observed at 20 mg/kg align with a study in rats that showed DPT was proconvulsant at 30 mg/kg. 64Such dose-dependent switches in effects were also described with the 5-HT 1 R agonist, sumatriptan.Sumatriptan increases pentylenetetrazol-induced seizure thresholds in mice at 1 mg/kg but reduces the threshold at 20 mg/kg, suggesting the engagement of different targets or neural circuits at different doses. 79Similarly, 5-methoxy-N,Ndimethyltryptamine, mentioned in the Introduction section as having antiseizure effects in lateral geniculate kindled felines, has also been reported anecdotally to induce convulsions� akin to seizures�in humans when administered at strong doses (see experience ID:39420 and 76059 at Erowid.org).Based on the available evidence, the effects of DPT are dosedependent, consistent with DPT having polypharmacology like other tryptamines.
Sigma1Rs are targets of several tryptamines 11 and modulate epileptiform activity, 59 which provided us the rationale to investigate them as antiepileptic targets of DPT.We used the sigma1R antagonist, NE-100, based on its structural similarity to DPT.Two observations lead us to conclude that DPT's anti-AGS effects were not caused by activation or inactivation of sigma1Rs.NE-100 failed to reverse DPT's effects, and DPT failed to impact (either suppress or potentiate) NE-100elicited seizures.Also, NE-100 caused characteristically distinct convulsions at a 50 mg/kg dose.These seizures differed from AGS and DPT-induced seizures in terms of the behavioral sequelae and duration.Furthermore, drug-elicited seizures differ in underlying anatomical loci than AGS.Drug-elicited seizures affect various neural systems, 80 whereas in Fmr1 KO mice, AGS have a localized origin, being dependent on altered activity in the inferior colliculus, an auditory structure in the midbrain. 81ne possible mechanism for why DPT was antiepileptic in the AGS assay is that it directly modulates auditory processing, reducing auditory hypersensitivity in Fmr1 KO mice.In humans, a closely related tryptamine N,N-diisopropyltryptamine reduces sound pitch and causes harmonic distortion while keeping the relationship between tones intact; subjects report that sounds from music are an octave lower than usual, i.e., as if they are listening to music underwater. 63The possibility that DPT or related tryptamines can target auditory processing is worth exploring in the future, as it may help improve understanding of auditory hypersensitivity in FXS and other neurodevelopmental disorders.
A limitation of the in vivo pharmacological antagonism studies is the side-effect of sedation (no locomotion, flaccid bodies, and eyes closed or partially closed) caused by pimavanserin and methiothepin.The sedation caused by brain-wide inhibition of 5-HT 2A/2C Rs in the case of pimavanserin and brain-wide inhibition of 5-HT 1 , 5-HT 2 , 5-HT 3 , 5-HT 5 , 5-HT 6 , 5-HT 7 Rs (and other receptors) in the case of methiothepin may have been sufficient to block auditory signals from reaching the inferior colliculus to cause AGS.For example, 5-HT 2A R blockade in the frontal cortex may have diminished auditory processing, and potentiated the anti-AGS effects of DPT, i.e., could have had anti-AGS effects independent of DPT.However, we previously showed that the selective 5-HT 2A R antagonist/inverse agonist, M100907, which causes sedation in mice, does not block AGS in juvenile Fmr1 KO mice. 70Another possibility for the inefficacy of pimavanserin to block the anti-AGS effects of DPT is that DPT's effects were due to precise, localized modulation of 5-HT 2A Rs. 5-HT 2A R modulation�5-HT 2A R biased signaling, antagonism or agonism of distinct 5-HT 2A R signal transduction pathways�in auditory neural pathways might have underlied the antiepileptic effects of DPT, and we were unable to detect this because of brain-wide inhibition of 5-HT 2A Rs that obfuscated this effect.It is yet to be determined whether local blockade or inactivation of 5-HT 2A Rs in auditory neural pathways would block DPT's anti-AGS effects, and conversely, whether local activation of 5-HT 2A Rs by DPT would be sufficient to block AGS.
DPT has not been studied extensively.A PubMed search of articles with "dipropyltryptamine" in their abstracts produced only 23 results.Little is known about DPT's pharmacodynamics.We investigated DPT's functional effects at 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs, using new TRUPATH technology, which probes the activity of ligands to stimulate individual Gα subunits coupled to GPCRs, and often, ligands have unique potencies to activate different Gα subunits. 82,83Future studies might find, for example, that DPT has different potencies at 5-HT 1A and 5-HT 1B coupled to Gα i/o family subunits other than Gα i3 , which we examined.One study assessed DPT's affinity and function at 5-HT 1A receptors, using [ 3 H]8-OH-DPAT and GTPγS incorporation, respectively; 71 DPT's 5-HT 1A R affinity was substantially higher than the affinity we measured with [ 3 H]5-CT competition binding, but it was a 5-HT 1A R partial agonist, like we observed.Another study reported that DPT was inactive at 5-HT 1A Rs up to 10 μM but used calcium mobilization as the functional readout. 12The higher affinity of DPT at 5-HT 1A compared to 5-HT 1B that we observed is similar to other psychedelic tryptamines. 11DPT's functional activity at 5-HT 2A Rs was also measured by Blough et al., 12 and its potency to stimulate canonical 5-HT 2A -Gα q signaling was higher than what we observed with TRUPATH; still, its full agonist efficacy was consistent with our results.Finally, there is no information to our knowledge about DPT's pharmacokinetics in any species.Thus, we are parsimonious in our conclusion about DPT's in vivo mechanism(s).We conjecture that our observations gel with recent research that concludes that some effects of psychedelics are mediated by nonserotonergic mechanisms. 19,20Our observations of an apparent nonserotonergic mechanism underlying the antiepileptic effects of DPT add to the growing literature about the pharmacological mechanisms underlying the potential therapeutic effects of serotonergic psychedelics.

Animals.
All experimental protocols involving FVB.129P2-Pde6b + Tyr c-ch Fmr1 tm1Cgr /J (Fmr1 KO mice, stock #004624, Jackson Laboratory) and FVB.129P2-Pde6b + Tyr c-ch /AntJ (sighted FVB or WT mice, stock #004828) were approved by the Mercer University Institutional Animal Care and Use Committee and were performed following the Guide for the Care and Use of Laboratory Animals, 8th edition.We used Fmr1 KO juvenile mice (P23−P25), male and female, for tests of AGS.The mice were bred and raised in the vivarium at Mercer University College of Pharmacy as previously described. 22All tests were performed during the light cycle (7:00−19:00).
Compounds.DPT hydrochloride, NE-100 hydrochloride, DOI hydrochloride, and methiothepin maleate were purchased from Cayman Chemical.WAY-100635 maleate was purchased from Tocris.Pimavanserin was obtained from Selleckchem, and SB-224289 hydrochloride was purchased from R&D Systems.5-HT hydrochloride and mianserin hydrochloride were obtained from Alfa Aesar.For in vivo pharmacology tests, all compounds were dissolved in Milli-Q (Millipore Sigma) water, which served as the vehicle, except for SB-224289, which was dissolved in 2% DMSO, 4% Tween-80, and 4% PEG-20 and subsequently q.s. with Milli-Q.The solutions were made fresh on the day of the experiments.Vehicle and all compounds for in vivo studies were administered intraperitoneally (i.p.) to Fmr1 KO and WT mice at a volume of 1 mL/100 g.Doses of compounds were selected based on studies showing their in vivo efficacy.For in vitro pharmacology studies, 10 mM stocks of test ligands were prepared in DMSO.[ 3 H]Lysergic acid diethylamide (LSD) and [ 3 H]5-Carboxamidotryptamine (5-CT) were purchased from PerkinElmer and were diluted in assay buffer.
Cell Growth, Maintenance, and Transfection.Plasmids encoding human 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs were obtained from the cDNA Resource Center.Dulbecco's modified Eagle's medium (DMEM) and OptiMEM were obtained from Gibco.Fetal bovine serum (FBS) and dialyzed FBS (dFBS) were purchased from Corning Life Sciences and Gibco.HEK 293 T cells (CRL-3216, ATCC) were used for in vitro binding and functional assays.Cells were cultured in 10 cm dishes with DMEM medium containing 10% FBS and were maintained in an incubator at 37 °C, 5% CO 2 , and 95% humidity.
Radioligand Competition Binding.Cell membranes expressing 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs were collected after 48 h of transfection.Cells were collected and homogenized in ice-cold 50 mM Tris HCl buffer.Homogenate was spun thrice at 12,000 × g for 10 min at 4 °C using an Avanti JXN-26 centrifuge (Beckman Coulter).The supernatant was discarded after each spin and the final pellet was stored at −80 °C for later testing.Competition binding assays with DPT and control compounds were performed in 96 well plates, using ∼0.7 nM [ 3 H]LSD to radiolabel 5-HT 2A Rs, and ∼0.2 and ∼0.3 nM [ 3 H]5-CT to radiolabel 5-HT 1A and 5HT 1B Rs, respectively.Nonspecific binding was determined in the presence of 10 μM mianserin for 5-HT 2A Rs, 10 μM serotonin for 5-HT 1A Rs, and 10 μM SB-224289 for 5-HT 1B Rs.After the addition of assay buffer (50 mM tris−HCl, 10 mM MgCl 2 , and 0.1 mM EDTA, pH = 7.4 at room temperature), test ligands, radioligand, and cell membranes expressing 5-HT 2A , 5-HT 1A , or 5-HT 1B Rs, the plates were covered and incubated on a shaker for 90 min at room temperature.Plate contents were rapidly filtered through Whatman GF/B filter mats using a 96-well cell harvester (PerkinElmer) and then washed with ∼800 mL ice-cold 50 mM Tris•HCl to remove unbound radioligand.Filter mats were dried and saturated with a scintillation cocktail (ScintiVerse Cocktail, Fisher Scientific), and scintillations were counted using a PerkinElmer Microbeta 2 instrument.
Bioluminescence Resonance Energy Transfer 2 Assay (TRUPATH).Cells were plated in white opaque 96-well microplates (Perkin Elmer) 48 h after transfection in bioluminescence resonance energy transfer (BRET) buffer at a density of 50,000 cells/well.After 2 h in an incubator, cells were treated with freshly prepared luminescent enzyme substrate coelenterazine (5 μM).After 5 min of the equilibration period, 5-HT (positive control) and DPT were added to the wells.After another 5 min, plates were then read in an LB940 Mithras plate reader (Berthold Technologies, Oak Ridge, TN) with 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters.G-protein activation was measured as BRET2 ratios (the ratio of the GFP2 emission to RLuc8 emission). 83udiogenic Seizures.Experiments testing the induction of AGS in Fmr1 KO were conducted as previously described. 22uvenile mice were acclimated to the test room for 30−60 min in their home cages.Mice were then administered vehicle or DPT at 3, 5.6, or 10 mg/kg.To determine the potential contribution of 5-HT 2A , 5-HT 1A, 5-HT 1B , other 5-HTRs, and sigma1Rs to the antiepileptic effects of DPT, separate groups of mice were pretreated with the selective 5-HT 2A R antagonist/inverse agonist pimavanserin (3 and 10 mg/kg), 84 the selective 5-HT 1A R antagonist WAY100635 (0.1 and 1 mg/ kg), 85,86 the selective 5-HT 1B R antagonist SB-224289 (5 mg/ kg), 87 the pan-5-HTR antagonist methiothepin (2 and 4 mg/ kg), 88,89 and the sigma1R antagonist NE-100 (30 and 50 mg/ kg) 75 10 min before treatment with DPT (10 mg/kg).All mice were placed back in their cages and were tested 5 min after injection with DPT.−93 Also, we previously showed that 0.1 mg/kg WAY100635 at this pretreatment interval is effective at preventing the anticonvulsant effects of the selective 5-HT 1A R agonist, NLX-112. 50Mice were placed in a clear, polycarbonate box (46 cm × 20 cm × 20 cm) covered with a perforated, clear, polycarbonate lid 1 min before being exposed to an alarm (RadioShack Kit #49-1010, doorstop alarm).The alarm was held by hand ∼10 cm away from the test box and the duration of exposure was 5 min.A sound-level meter/data logger (REED Model SD-4023) was placed ∼20 cm from the alarm and read during testing to ensure a uniform level of sound pressure in each experiment.Tests were video-recorded using a high-definition camcorder (Vixia HF R800, Canon).A maximum of 4 mice (2 per box) were observed simultaneously by two experimenters. 70The average (±standard deviation (SD)) baseline sound pressure in the testing room was 55 ± 9 dB, and the average alarm sound pressure was 105 ± 4 dB.
Behavioral responses, including normal behavior, WRJ, TCS, and death, were documented during AGS testing.Normal behavior was defined as coordinated locomotion, alertness, exploring, sniffing, sitting, rearing, grooming, socializing, and squinting of eyes.The beginning of AGS was marked by a startle response, squinting of eyes, followed by WRJ phase(s), brief opisthotonos, a clonic phase with the mouse lying on either side of its body with head, neck, trunk, and limbs ventroflexed (muscle jerking and twitching with rigidity), a short (∼5 s) tonic seizure phase with full extension of extremities (muscle stiffening), and finally, respiratory arrest.Seizure was defined by TCS.In the case of recovery from the TCS phase, mice exhibited a second round of WRJ, Straub tail, a full body vibrating shudder, and tremors which finally ended with either freezing or a transition to normal behavior.The frequencies of AGS were documented by visual observations of video recordings. 70PT 5-HT 2A R and 5-HT 1A R In Vivo Pharmacology.WT mice were acclimated to a procedure room for ≥30 min before administering test compounds.Juvenile (P23-P25) and adult (>P60) mice were injected (i.p.) with Milli-Q water (vehicle) or DPT (3, 5.6, 10, 15, or 20 mg/kg) and were immediately placed in a clear open-field plexiglass chamber (43 × 43 cm; Med Associates).5-HT 2A R-dependent HTRs were counted using a hand-held tally counter for 15 min postinjection.Locomotor activity (distance traveled in cm) was video recorded and calculated by Ethovision software (Noldus Information Technology).Observations of 5-HT 1A R-dependent effects (see Figure 3C) were also documented.
Statistical Analysis.Statistical tests were performed using GraphPad Prism, version 9. AGS and other behaviors were analyzed using Fisher's exact test (two-sided, α = 0.05).To evaluate the efficacy of various doses of DPT to elicit the HTR compared to vehicle, a one-way ANOVA with Holm-S ̌i ́daḱ's multiple comparisons test was used.Student's t test was used for HTR comparison between DOI and vehicle treatment.Nonlinear regression was used for analyzing in vitro pharmacology results.Of note, for the 5-HT 2A R binding, data fit best to a two-site model.For 5-HT and DPT binding at 5-HT 1A R and 5-HT 1B R, we used a one-site model. 94,95 The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00137.
Figure2.In vitro and in vivo pharmacology of DPT at 5-HT 2A Rs and examination of the impact of inhibiting 5-HT 2A/2C Rs on the antiepileptic properties of DPT.(A) In vitro radioligand competition binding of DPT and 5-HT at human (h) 5-HT 2A Rs.The 100 μM data point was interpolated, so curves reached asymptote (no specific binding).Data were obtained from two separate experiments in which 5-HT was tested in duplicate and DPT was tested in sextuplicate per concentration.Data best fit to a "two-site, fit K i " model, which is shown.(B) In vitro, functional activity of 5-HT and DPT at h5-HT 2A Rs.Data were obtained from three experiments in which 5-HT and DPT were tested in quadruplicate per concentration.(C) DPT dose-dependently elicited the 5-HT 2A R-dependent HTR, with maximal effects at 10 mg/kg in juvenile and adult WT mice.(We previously found no difference in DOI-elicited HTRs between WT and Fmr1 KO mice.22 ) Note that this dose was equivalent to the effective dose of DPT to prevent seizures, demonstrating that DPT engaged 5-HT 2A Rs while it prevented seizures.*represents p < 0.05 and ## , **represents p < 0.01 compared to vehicle.Despite this, as shown in (D), the selective 5-HT 2A/2C R antagonist, pimavanserin, did not significantly block the anti-AGS effects of DPT in juvenile Fmr1 KO mice.**** represents p < 0.0001 DPT 10 mg/kg compared to vehicle; data reproduced from Figure1to show the comparison to the pimavanserin-treated groups.Veh: Vehicle; D3, 5.6, 10, 15 and 20: DPT 3, 5.6, 10, 15 and 20 mg/kg; P3 and 10: pimavanserin 3 and 10 mg/kg.N = number of mice tested.All data with error bars are means and SEMs.

Figure 3 .
Figure 3.In vitro and in vivo pharmacology of DPT at 5-HT 1A Rs and examination of the impact of inhibiting 5-HT 1A Rs on the antiepileptic effects of DPT.(A) In vitro radioligand competition binding of DPT and 5-HT at human (h) 5-HT 1A Rs.The 100 μM data point was interpolated, so curves reached asymptote (no specific binding).Data were obtained from two separate experiments in which 5-HT was tested in duplicate and DPT was tested in sextuplicate per concentration.(B) In vitro functional activity of 5-HT and DPT at h5-HT 1A Rs.Data were obtained from four experiments in which 5-HT and DPT were tested in quadruplicate per concentration.(C) DPT at 20 mg/kg elicited behavioral signs of 5-HT 1A R engagement in juvenile and adult WT mice including flat body posture (FBP), hind limb abduction (HLA), tremors, and Straub tail; DPT did not elicit these behaviors at its antiepileptic dose of 10 mg/kg.**, *** represents p < 0.01 and p < 0.001, respectively, compared to vehicle.(D) The selective 5-HT 1A R antagonist, WAY-100635, did not block the antiepileptic effects of DPT in juvenile Fmr1 KO mice.**** represents p < 0.0001 DPT 10 mg/kg compared to vehicle; data reproduced from Figure 1 to show the comparison to the WAY-100635-treated groups.Veh: Vehicle; D3, 5.6, 10, 15 and 20: DPT (3, 5.6, 10, 15 and 20 mg/kg); W0.1 and W1: WAY-100635 (0.1 and 1 mg/kg).N = Number of mice tested.All data with error bars are means and SEMs.

Figure 4 .
Figure 4.In vitro and in vivo pharmacology of DPT at 5-HT 1B Rs and examination of the impact of inhibiting 5-HT 1B Rs on the antiepileptic properties of DPT.(A) In vitro, radioligand competition binding of DPT and 5-HT at human (h) 5-HT 1B Rs.The 100 μM data point was interpolated, so curves reached asymptote (no specific binding).Data were obtained from four separate experiments in which 5-HT was tested in duplicate and DPT was tested in sextuplicate per concentration.(B) In vitro functional activity of 5-HT and DPT at h5-HT 1B Rs.Data were obtained from four individual experiments per assay in which 5-HT and DPT were tested in quadruplicate per concentration.(C) The selective 5-HT 1B R antagonist, SB-224289, did not block the antiepileptic effects of DPT in juvenile Fmr1 KO mice.**** represents p < 0.0001 DPT 10 mg/ kg compared to vehicle; data reproduced from Figure 1 to show the comparison to the SB-224289-treated group.Veh: Vehicle; D10: DPT 10 mg/ kg; SB5: SB-224289 5 mg/kg.N = number of mice tested.All data with error bars are means and SEMs.

Figure 6 .
Figure 6.Examination of the impact of sigma1R blockade on the antiepileptic effects of DPT in juvenile Fmr1 KO mice.NE-100 (structure shown to illustrate the shared N,N-dipropyl moiety in NE-100 and DPT) at a subconvulsive dose (30 mg/kg) and at a convulsive dose (50 mg/kg) did not influence the anti-AGS effect of DPT.Note that this figure reports AGS and not NE-100-elicited seizures; see Table 2 for descriptive differences in the types of seizures.**** represents p < 0.0001 DPT 10 mg/kg compared to the vehicle; data reproduced from Figure 1 to show the comparison to the NE-100-treated groups.Veh: Vehicle; D10: DPT 10 mg/kg; NE30 and NE50: NE-100 30 and 50 mg/kg, respectively.N = number of mice tested.
K d values were set to 0.78 nM for [ 3 H]LSD at 5-HT 2A Rs, and 0.2 and 0.3 nM for [ 3 H]5-CT at 5-HT 1A and 5-HT 1B Rs, respectively, and were based on values reported in the literature.■ASSOCIATED CONTENT * sı Supporting Information Figure S1, latency to and duration of WRJ and TCS in Fmr1 KO mice in the AGS assay; Figure S2, effect of 1 mg/kg DOI on the HTR in juvenile and adult WT mice; Figure S3, effect of 3, 5.6, 10, 15, and 20 mg/kg DPT on locomotor activity in WT mice; Figure S4, latency to and duration of seizures induced by 50 mg/kg NE-100 in juvenile WT and Fmr1 KO mice (PDF) ■ AUTHOR INFORMATION Corresponding Author Clinton E. Canal − Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, Atlanta, Georgia 30341, United States; orcid.org/0000-0002-7940-933X;Phone: +1 (678) 547-6252; Email: canal_ce@mercer.edu

Table 1 .
DPT and 5-HT Affinities (K i ) and EC50Values, Determined Using TRUPATH Gαβγ Biosensors, at Human 5-HT 2A , 5-HT 1A , and 5-HT 1B Rs Expressed in HEK293T Cells a a[ 3 H]LSD was used to label 5-HT 2A Rs; [ 3 H]5-CT was used to label 5-HT 1A and 5-HT 1B Rs.For 5-HT 2A Rs, the affinity data fit best to a "two−site−fit K i " model.K i s and pK i s values are means.CI = confidence interval.

Table 2 .
AGS, 20 mg/kg DPT and 50 mg/kg NE-100 Elicited Seizures Are Qualitatively Distinct a Descriptions of AGS were gathered from experiments with vehicle-treated juvenile Fmr1 KO mice.Descriptions of seizures elicited by 20 mg/kg DPT were gathered from the dose−effect HTR study of DPT using juvenile and adult WT mice.Descriptions of seizures elicited by 50 mg/kg NE-100 were gathered from a dose-determining study of NE-100 using juvenile WT and Fmr1 KO mice and tests of NE-100 in the AGS study using juvenile Fmr1 KO mice. a