Activity of Serotonin 5-HT1A Receptor Biased Agonists in Rat

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Activity of serotonin 5-HT receptor biased agonists in rat: anxiolytic and antidepressant-like properties Magdalena Jastrz#bska-Wi#sek, Anna Partyka, Joanna Rychtyk, Joanna #niecikowska, Marcin Ko#aczkowski, Anna Weso#owska, Mark Andrew Varney, and Adrian Newman-Tancredi ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00443 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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ACS Chemical Neuroscience

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Selective 5-HT1A agonists and behavior

Activity of serotonin 5-HT1A receptor biased agonists in rat: anxiolytic and antidepressant-like properties Regular Research article

Magdalena Jastrzębska-Więsek a, Anna Partyka a, Joanna Rychtyk a, Joanna Śniecikowska a, Marcin Kołaczkowski a, Anna Wesołowska a, Mark A. Varney b, Adrian Newman-Tancredi b

a

Jagiellonian University, Medical College, Medyczna 9 St., 30-688 Krakow, Poland

b

Neurolixis Inc. 34145 Pacific Coast Highway #504, Dana Point, CA 92629 USA

Corresponding author: Adrian Newman-Tancredi, PhD, DSc

[email protected]

ACS Paragon Plus Environment

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Abstract Although serotonin 5-HT1A receptors constitute attractive therapeutic targets, there is a lack of potential clinical candidates that have a high degree of selectivity and full agonist efficacy. Recently, novel 5-HT1A receptor ‘biased agonists,’ F15599 (a.k.a. NLX-101) and F13714 have been reported that exhibit distinctive properties for in vitro signaling, neurochemical, electrophysiological effects and in brain imaging. The present study characterized their effects in rat models of anxiety (elevated plus-maze and Vogel test), in depressive-like behavior (forced swim test) and on the induction of the three serotonergic behaviors (forepaw treading, flat body posture, and lower lip retraction). The prototypical 5-HT1A receptor ligands, (±)8-OH-DPAT and buspirone were tested as comparators. In the elevated plus-maze, F15599, F13714 and (±)8-OH-DPAT dose-dependently increased the amount and percentage of time spent in the open arms with minimal effective doses (MED) of 5 mg/kg p.o., 2.5 mg/kg p.o. and 1.25 mg/kg s.c., respectively. The effects of the three agonists were abolished by pretreatment with the selective 5-HT1A receptor antagonist, WAY100635 (0.63 mg/kg s.c.). Buspirone did not show significant activity in the EPM. In contrast, in the Vogel test only buspirone was active, significantly increasing the number of licks and shocks accepted (active dose: 1.25 mg/kg s.c.). However, WAY100635 failed to reverse the effects of buspirone in this test, suggesting that they were not 5-HT1A receptor-mediated. In the forced swim test, F15599, F13714 and (±)8-OH-DPAT were potently active, abolishing immobility (MED: 0.63 mg/kg p.o., 0.63 mg/kg p.o. and 0.16 mg/kg s.c., respectively). Buspirone was not active. In measures of serotonergic behavior, F13714 and (±)8-OH-DPAT robustly elicited all three signs of serotonergic behaviors, whereas F15599 and buspirone elicited only lower-lip retraction. Taken together, these observations highlight the distinct profiles of activity of 5-HT1A agonists and suggest that the novel biased agonist F15599 combines pronounced activity in a test of anxiety (elevated plus-maze) with potent antidepressant-like effects and low propensity to induce serotonergic behaviors. These data suggest that selective biased agonists could constitute promising pharmacotherapeutics for mood disorders.

Keywords: Forced swimming test, 5-HT syndrome, 5-HT1A, F15599, F13714 Abbreviations: Fore-paw treading, FPT; Flat Body Posture, FBP; Lower Lip Retraction, LLR; Elevated Plus Maze, EPM; Forced Swim Test, FST Running Title: “Selective 5-HT1A agonists and behavior”


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Introduction The identification of subtypes of serotonin (5-hydroxytryptamine, 5-HT) receptors 1, 2 led to widespread interest in the role of 5-HT in a variety of CNS functions, including mood, cognition, sleep, sexual behavior and anxiety. Among 5-HT receptors, the 5-HT1A receptor subtype has profound influence on the control of anxiety and depression by influencing serotonergic neurotransmission in multiple brain regions. These include the Raphe nuclei, where it is expressed as an somatodendritic autoreceptor, and in other brain regions such as cortex and hippocampus where it is expressed as a heteroreceptor on GABAergic and glutamatergic neurons 3, 4. In accordance with the broad therapeutic potential of 5-HT1A receptors, drugs targeting this receptor have been developed and commercialized. Two of the best known are the azapirones, buspirone (Buspar®) and tandospirone (Sediel®), which are commercialized for the treatment of anxiety, providing a non-sedative and nonaddictive therapeutic alternative to the use of benzodiazepines 5, 6. Buspirone and other 5-HT1A-targeted compounds, such as the prototypical agonist, (±)8-OH-DPAT, exhibit substantial limitations including poor receptor selectivity, partial agonist efficacy and/or poor pharmacokinetic properties leading to the generation of metabolites that are active on other receptor systems. Thus, improved therapeutic benefit could be achieved if novel chemical entities were available that exhibited higher agonist efficacy, increased selectivity and a lack of active metabolites. There has therefore been continued interest in identifying and developing therapeutics that target 5-HT1A receptors. Recently-described drugs that target 5-HT1A receptors include the antidepressants vilazodone (Viibryd®) and vortioxetine (Brintellix®), as well as the antipsychotics, aripiprazole (Abilify®) brexpiprazole (Rexulti®), and a pharmacotherapeutic for female sexual interest/arousal disorder, flibanserin (Addyi®). However, these drugs also act as partial agonists at 5HT1A receptors and lack selectivity for 5-HT1A receptors, binding to various other targets, such as dopamine D2/D3/D4, noradrenergic alpha1, serotonin 5-HT7 and/or 5-HT reuptake sites 3. Consequently, the potential clinical utility of a highly selective, fully efficacious 5-HT1A receptor agonist as a treatment for anxiety and other mood disorders remains to be investigated. In addition to the above, the serotonergic neurobiology underlying the anxiolytic effects of buspirone and related compounds is still a matter of debate and substantial contradictory evidence has been reported. For example, buspirone’s anxiolytic-like effects in the elevated plus maze (EPM) have been variously attributed to activation of 5-HT1A receptors in the dentate gyrus 7 or in the nucleus incertus 8 . One study concluded that the modest anxiolytic-like effect of buspirone in the EPM was mediated by its principal metabolite, 1-PP (1-(2-pyrimidinyl)-piperazine), which has noradrenergic alpha2 receptor antagonist properties 9. However, this contradicted a previous study, which found no anxiolytic activity in the EPM for 1-PP or for another alpha2 receptor antagonist, idazoxan 10. Buspirone has also been reported to be inactive in the EPM 11 or even to exhibit anxiogenic effects 12. In the case of (±)8-OH-DPAT, some researchers reported the absence of activity in the EPM 13, whereas others found that it was, in fact, active 12. Such divergent findings illustrate the need for clarification of the activity of 5-HT1A receptor agonists in the EPM and other models of anxiety. A novel strategy to achieve this could be to exploit the emerging concept of ‘biased agonism’ (also known as functional selectivity 14) by differential pharmacological targeting of 5-HT1A receptor subpopulations in specific brain regions 15. Indeed, by preferentially activating 5-HT1A receptors coupled to different intracellular signaling mechanisms, such as specific G-protein subtypes16, biased agonists can target distinct subpopulations of 5-HT1A receptors that have different effects in behavioral paradigms. For example, activation of postsynaptic 5-HT1A receptors is associated with antidepressant-like effects, whereas activation of 5-HT1A



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autoreceptors delays the antidepressant activity of 5-HT reuptake inhibitors 3. Other studies suggest that activation of 5-HT1A receptors in specific brain regions differentially influences cognitive function and can serve as a functional biomarker of memory / dementia 15, 17, 18. The identification of 5-HT1A receptor biased agonists therefore raises the possibility of preferentially targeting distinct receptor subpopulations in a more precise, and therefore potentially more therapeutically-beneficial, manner. However, the activity of such biased agonists in models of anxiety has not yet been reported and the present study addressed this issue by testing the biased agonists, F15599 and F13714 in rat experiments. F15599 ((3-chloro-4-fluorophenyl)-[4-fluoro-4-[[(5-methylpyrimidin-2yl)methylamino]methyl]piperidin-1-yl]methanone) is a first-in-class biased agonist that preferentially targets extracellular signal-regulated kinase (ERK) phosphorylation in vitro and 5-HT1A heteroreceptors in cortical brain regions in vivo 19, 20. F15599 mediates a variety of responses that are mediated by post-synaptic 5-HT1A receptors, including activation of c-Fos, increased pyramidal cell firing and stimulation of dopamine release 19, 21. In behavioral tests, F15599 shows striking activity in tests of depression (forced swim test, FST) and of cognition, reversing PCP-induced working memory deficits and increasing pattern recognition performance 22, 23. In microPET and phMRI brain imaging in rat, F15599 preferentially targets cortical regions 24, 25. In contrast to F15599, a close chemical analogue, F13714 (3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methyl-6-methylamino-pyridin-2ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone), shows a strikingly divergent profile: it potently activates several transduction pathways in vitro and 5-HT1A autoreceptors in vivo 20, 26. In behavioral tests, F13714 impairs cognitive performance and induces marked serotonergic behaviors 23. Its brain imaging profile differs strongly from that of F15599, with pronounced labelling of hippocampal and subcortical structures 24, 27. F15599 and F13714 therefore constitute innovative pharmacological tools to explore the role of different 5-HT1A receptor subpopulations and, potentially, develop new therapeutics for CNS disorders associated with serotonergic signaling. However, although these novel ligands have been characterized in a variety of tests, no information is available concerning their effects in classic tests of anxiolytic activity such as the elevated plus maze or the Vogel test. The present study therefore tested F15599 and F13714 in these models of anxiety, in comparison with buspirone and (±)8-OHDPAT. In order to ascertain that the observed effects were specifically mediated by 5-HT1A receptor activation, we examined the effects of the agonists following pre-treatment with the selective 5-HT1A receptor antagonist, WAY100635. In addition, the present study also tested the effects of the agonists on the FST and on serotonergic behaviors to provide comparisons with previous studies and with the anxiety tests carried out in the same laboratory.


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Results and Discussion The main finding of the present study is that the biased agonists, F13714 and F15599, displayed distinct behavioral profiles from each other and from those of the prototypical 5-HT1A receptor agonist, (±)8-OH-DPAT or the anxiolytic drug, buspirone, in widely-used rat models of mood disorders. Taken together, the data suggests that preferential targeting of 5-HT1A receptor subpopulations translates to separate patterns of behavioral effects in models of mood disorders and could therefore inform our understanding of anxiety conditions in human.

Effects of 5-HT1A receptor agonists in the EPM The principal observations in the EPM were as follows: F13714 increased the time spent in the open arms (by 3-fold at 0.63 mg/kg and by 5-fold at 2.5 mg/kg, Fig. 1a), the percentage of time spent in open arms (by 5-fold at 2.5 mg/kg, Fig. 1b) and the percentage of entries into open arms number (by about 2.5-fold at 0.63 mg/kg, Fig. 1d). The increased number of entries into the open arms did not reach significance (Fig. 1c). F15599increased the time spent in the open arms (by 3-fold at 0.63 mg/kg, and 6-fold at 5 mg/kg, Fig. 1a), the percentage of time spent in the open arms w (by 5.5-fold at 5 mg/kg, Fig. 1b). and the percentage of entries into the open arms (by about 2-fold at 0.63 and 5 mg/kg, Fig 1.d). The increased number of entries into the open arms did not reach significance (Fig. 1c). The prototypical 5-HT1A receptor agonist, (±)8-OH-DPAT increased the time spent in the open arms (by about 3.5-fold at 1.25 mg/kg, Fig. 1a), and percentage of time spent in open arms (by 3-fold at 1.25 mg/kg, Fig. 1b). The increased number of open-arm entries and percentage of entries into open arms not reach significance (Fig. 1c and 1d). The anxiolytic drug, buspirone, did not achieve statistically-significant effects at any dose tested (Fig 1.). It should be noted that the % of time/entries spent in the open arms was calculated based on measures from open + closed arms, not including time in the center platform. Hence the analysis of the % time/entries in the open arms can slightly differ from that of the amount of time / number of entries in the open arms. The time spent in the closed arms and the number of entries therein decreased at the highest doses of (±)8-OHDPAT, F13714 and F15599 (see supplementary Fig. S1), reflecting the fact that the rats spent more time in the open arms, and that the compounds exerted anxiolytic-like effects. The decreased time in the closed arms does not imply an inhibitory effect on exploratory activity (see below). Overall, these data show that the 5-HT1A biased agonists, F13714 and F15599, as well as (±)8-OHDPAT, exhibit anxiolytic-like activity in the EPM, but buspirone did not. Several points arise from these observations. (i) Although F13714 preferentially activates 5-HT1A autoreceptors and F15599 preferentially activates 5-HT1A heteroreceptors, both compounds appeared similarly active in the present conditions. Indeed, the active doses were well above those that have been previously reported to be selective for 5-HT1A receptor subpopulations 19, 22, 23, 28, and it is therefore likely that the brain region preference of the biased agonists does not constitute a differentiating factor herein. (ii) The activity of (±)8-OH-DPAT, a prototypical agonist not known to be ‘biased’, was similar to that of the biased agonists, reinforcing the interpretation that preferential regional activation of 5-HT1A receptor subpopulations is not the principal mechanism of action underlying activity in the EPM. It may be speculated that simultaneous activation of 5-HT1A receptors in several brain regions may be necessary. (iii) Buspirone failed to show marked anxiolytic-like activity in the present conditions, unlike previous studies where buspirone was reported to be active in rat and mouse (see Introduction). However, buspirone has been described as showing “highly inconsistent effects” in the EPM 29 for reasons which are still unclear, although its lack of selectivity for 5-HT1A receptors over dopamine D2 receptors may be involved 29. In any case, an obvious differentiating factor between the compounds tested herein is their agonist efficacy at 5-HT1A receptors: buspirone only acts as a partial agonist, whereas the other compounds more efficaciously activate this receptor 20, 30, 31. It



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therefore appears that high efficacy 5-HT1A receptor activation, such as that of F13714, F15599 and (±)8-OH-DPAT, is necessary for activity in the EPM in the present conditions. The anxiolytic-like properties of the latter agonists are clearly 5-HT1A receptor-dependent. Indeed, an interaction experiment carried out with the 5-HT1A receptor antagonist, WAY100635 (0.63 mg/kg s.c.) showed that the latter, given alone, had no effect in EPM test (Fig. 2) but significantly reversed the anxiolytic-like activity of F13714 (0.63 mg/kg p.o.), of F15599 (0.63 mg/kg p.o.) and of (±)8-OH-DPAT (1.25 mg/kg s.c.) (Fig. 2).

Effects of 5-HT1A receptor agonists on exploratory activity Fig. 3 illustrates the exploratory activity of rats that was measured using the automated version of the EPM (measures were made simultaneously with those of anxiolytic-like activity). Post-hoc statistical analysis found no significant effects for any dose of F13714, F15599 or (±)8-OH-DPAT when given alone. In combination with WAY100635 (Fig 3b), (±)8-OH-DPAT, at dose of 1.25 mg/kg, statistically increased total distance travelled by 42%. Buspirone decreased locomotor activity at the highest dose tested (5 mg/kg), suggesting possible sedative activity. These observations indicate that the activity of the biased agonists on the anxiety tests is not influenced by confounding locomotor effects. This is also important in the FST, where false positives could be observed, as would be the case, for example, for psychostimulants that could reduce immobility by increasing locomotor activity. In contrast, the present data highlight the fact that buspirone does reduce exploratory / motor activity, likely due to its antagonist properties at dopamine D2 receptors 32.

Effects of 5-HT1A receptor agonists in the Vogel test Strikingly, the pattern of effects in the Vogel test was the opposite to that seen in the EPM: buspirone showed anxiolytic-like activity (at the dose of 1.25 mg/kg, Fig. 4)33 whereas F13714, F15599 and (±)8-OH-DPAT did not. In the case of buspirone, the present findings resemble those reported previously when it was found to be inactive in the EPM but active in the Vogel test 34. However, in the case of the other agonists, the higher doses tested (F13714 and (±)8-OH-DPAT from 0.63 mg/kg and F15599 at 2.5 mg/kg) significantly decreased number of licks and accepted shocks (Fig. 4), possibly due to behavioral interference related to induction of serotonergic behaviors (notably fore-paw treading, FPT, and flat body posture, FBP, see below). Thus, caution should be exercised when interpreting the present data. Ideally, the compounds would be tested at higher doses without the ‘masking’ effect of FPT and FBP, something that could potentially be achieved by testing the compounds following a chronic administration regimen. Indeed, it is known that repeated administration of high-efficacy 5-HT1A agonists elicits tachyphylaxis to FPT and FBP 35, 36. Nevertheless, it is possible that the different biased agonists may be better suited to treating different types of anxiety states because the Vogel test is based on learned responses to a punished conflict situation (i.e. a conditioned operant conflict test), whereas the EPM test measures the ethological response to an unconditioned situation based on unlearned fear/avoidance behavior. A previous study reported that activation of pre- or post-synaptic 5-HT1A receptors differentially influenced fear conditioning in mice 37. Hence, it may be speculated that some 5-HT1A receptor agonists may preferentially attenuate specific anxiety behaviors through activation of distinct 5-HT1A receptor subpopulations. However, additional investigation of this issue is warranted because the underlying mechanisms appear complex. Some studies found that two buspirone analogues, tandospirone and ipsapirone, were active in the Vogel conflict test via activation of post-synaptic 5HT1A receptors 38, 39. This suggests that F15599 could have been expected to also be active in this test because it is a preferential post-synaptic 5-HT1A receptor biased agonist. However, contrary to expectations, this was not the case. Conversely, other studies concluded that the anxiolytic activity of


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the 5-HT1A agonists, buspirone, (±)8-OH-DPAT and S15535, in the Vogel test were mediated by preferential activation of presynaptic 5-HT1A receptors and was associated with inhibition of 5-HT release in the hippocampus 40, 41. If this was the case, F13714 would be expected to show activity in the Vogel test, because it is a preferential autoreceptor agonist that potently inhibits 5-HT release 26. Once again, contrary to expectations, activity was not observed at the doses tested. Furthermore, although 5-HT1A receptor agonists such as 8-OH-DPAT are known to show analgesic activity 42 (and could therefore be expected to reduce pain elicited by electric shock in the Vogel test), this did not appear to be relevant here as the number of licks was not increased by 8-OH-DPAT or the other highefficacy agonists. Interestingly, the selective antagonist, WAY100635, failed to block the anxiolyticlike activity of buspirone (Fig. 5) suggesting that its effects were not mediated by 5-HT1A receptors but by effects at other targets (e.g. blockade of alpha2 adrenoceptors by the metabolite, 1-PP). Overall, it appears that activation of 5-HT1A receptors by buspirone or brain region-preferential biased agonists does not clearly account for the present observations in the Vogel test (at least upon acute administration) and additional investigations would be desirable to investigate their activity, notably by carrying out the tests following chronic administration of the compounds.

Effects of 5-HT1A receptor agonists in the FST F13714, F15599 and (±)8-OH-DPAT each potently and almost completely abolished immobility in the FST (Fig. 6). This is a compelling demonstration of the antidepressant-like (or possibly ‘stresscoping’43) potential of high agonist-efficacy 5-HT1A receptor agonists which far exceeds that of monoamine reuptake inhibitors in this model: imipramine and paroxetine tested under similar conditions, only weakly and partially reversed immobility (ED50 values > 40 mg/kg p.o.; maximal effect < 50 %) 44. However, the present findings in the FST differ from those seen previously for F13714 and F15599 in this test 22. Indeed, they have previously been shown to have potent activity (ED50 ~0.1 mg/kg p.o. 22) with dose-response curves that were essentially superimposable. In contrast, notable differences in the dose-response relationship of the two compounds were observed herein. F13714 had a very steep dose-response curve with no effect on immobility at 0.16 mg/kg but almost complete suppression of immobility at the next dose (0.63 mg/kg). In contrast, F15599 exhibited a progressive dose-response with a partial effect being observed at 0.63 mg/kg, increased effect at 2.5 mg/kg and a full reversal of immobility at 5 mg/kg (Fig. 5). The reasons for the different dose-response relationship seen herein is unclear, particularly as the present experimental procedures were modeled closely on those described previously 22. Nevertheless, the present study used Wistar rats that were bred in-house at Jagiellonian University (JU) whereas Assié et al. 22 used Sprague-Dawley rats provided commercially by Charles River. It is therefore possible that the rat strains may have some differences in their sensitivity to 5-HT1A heteroreceptor activation by F15599 - although such a hypothesis is speculative and would require additional investigation. In any case, some clear messages emerge from the FST results: firstly, the three high-efficacy agonists were active whereas the partial agonist, buspirone, was not, highlighting the need for high agonist efficacy to achieve substantial antidepressant-like activity, in agreement with other studies 30. Secondly, activity of F13714, F15599 and (±)8-OH-DPAT in the FST is observed at doses that are somewhat lower than those for the EPM, suggesting that these agonists preferentially target depression-like behaviors rather than the anxiety-like behaviors. Whether such distinctions translate to therapeuticallymeaningful differences at a clinical level remains, however, to be demonstrated because no selective, high efficacy agonist has yet been developed as a therapeutic for mood disorders.



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Effects of 5-HT1A receptor agonists on serotonergic behaviors The influence on serotonergic behaviors was a highly differentiating element between the 5-HT1A receptor agonists (Fig. 7). Indeed, compounds with high efficacy at 5-HT1A receptors are known produce fore paw treading and flat body posture in rats, sometimes referred to as ‘serotonin behavioral syndrome’, upon acute (but not chronic) dosing, whereas low efficacy agonists / antagonists do not 30, 45. However, herein, F13714 and (±)8-OH-DPAT both elicited pronounced FPT and FBP, whereas F15599 elicited little or none, even at doses which were markedly active in the other behavioral tests (e.g. FST). In the case of buspirone, although only LLR was observed at the dose of 5 and 10 mg/kg (Fig. 7), more pronounced effects were observed with buspirone 15 min after p.o. administration (data not shown), suggesting that its rapid metabolism influences its low propensity to elicit serotonergic behaviors at later time-points. The stark contrast between the compounds may be partly ascribed to differences in agonist efficacy, i.e. the partial agonist properties of buspirone at 5-HT1A receptors are not enough to elicit FBP or FPT. However, this interpretation is insufficient to account for the lack of effects of F15599. Indeed, like F13714 and (±)8-OH-DPAT, F15599 acts as a full agonist in various signal transduction measures in vitro 20, and its absence of FBP or FPT induction is therefore more likely attributable to its biased agonist profile. Electrophysiological and neurochemical studies indicate that F15599 preferentially targets cortical 5-HT1A heteroreceptor subpopulations 19, rather than subcortical 5-HT1A receptors targeted by F13714 or (±)8-OH-DPAT 15. As argued previously 22, this implies that specific serotonergic pathways and 5-HT1A receptor subpopulations can be separately targeted in an agonist-specific manner 20. Indeed, compelling evidence of this was recently provided by pharmacoMRI imaging studies that showed that F15599 specifically stimulated blood oxygen level dependence (BOLD) signal in cortical regions whereas F13714 and(±)8-OH-DPAT had more extensive effects in subcortical and other brain regions 24. Taken together, these data support the interpretation that high efficacy 5HT1A agonists can be identified which do not elicit serotonin behavioral syndrome’, thanks to their preferential biased agonism at 5-HT1A receptor subpopulations.

Conclusions Overall, the present data demonstrate that the novel, highly-selective 5-HT1A receptor biased agonists possess profiles of activity in rat behavioral tests that sharply differentiate them from previously-tested compounds such as (±)8-OH-DPAT and buspirone. In particular, F15599, which preferentially targets 5-HT1A heteroreceptors, exhibits activity in the EPM and marked activity in the FST, whilst not eliciting serotonergic behaviors or modifying locomotor activity. In contrast, F13714, which potently activates 5-HT1A autoreceptors, is active in the EPM and FST but also induces marked serotonergic behaviors. These data suggest a clear rationale to pursue the identification of novel biased agonists with improved specificity for 5-HT1A receptor subpopulations mediating therapeuticlike effects. In the case of F15599, its profile of activity suggests that it could exhibit a superior profile for treatment of mood disorders including depression and anxiety. Nevertheless, some limitations of the present study should be noted: neither F15599, F13714 or (±)8-OH-DPAT elicited anxiolytic-like effects in the Vogel test, and the activity of buspirone was not blocked by the 5-HT1A receptor antagonist, WAY100635, suggesting that the present conditions are not suited to detecting anxiolytic-like activity of 5-HT1A agonists in this model. Secondly, all experiments in the present study were based on acute dosing and the question remains as to whether the compounds would maintain the same profiles of activity following a chronic administration schedule. Thirdly, the precise molecular mechanisms that underlie the differential profiles of the biased agonists remain to be elucidated. Indeed, although F15599 has been shown to preferentially activate specific G-protein


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subtypes and phosphorylation of ERK in vitro and ex vivo 20, the relevance of these signaling pathways for in vivo biased agonism has not yet been demonstrated. Nevertheless, the pace of studies on biased agonism is accelerating, and the present data add to a body of literature suggesting that preferential targeting of postsynaptic 5-HT1A receptors is a promising strategy to identify compounds with novel profiles of activity and, ultimately, to achieve improved therapy of mood disorders with fewer associated side-effects.

Methods Animals The experiments were performed on male Wistar rats (170-200 g) obtained from an accredited animal facility at the Jagiellonian University Medical College, Krakow, Poland. The animals were housed in group of four in controlled environment (ambient temperature 21±2 °C; relative humidity 50-60%; 12-h light/dark cycles (lights on at 8:00). Standard laboratory food (LSM-B) and filtered water were freely available. Animals were housed for a period of 6 days in polycarbonate Makrolon type 3 cages (dimensions 26.5 x 15 x 42 cm, ‘open top’) without enrichment environment (only wooden shavings litter). Each animal was assigned randomly to treatment groups and only used once (no repeated use of animals). All the experiments were performed by two observers unaware of the treatment applied between 9:00 and 14:00 on separate groups of animals. All procedures involving animals and their care were conducted in accordance with current European Community and Polish legislation on animal experimentation. Additionally, efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures described in this manuscript were approved by the Local Ethics Commission (no 108/2016), complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were in accordance with the 1996 NIH Guide for the Care and Use of Laboratory Animals.

Drugs The following drugs were used: (±)8-OH-DPAT ((±)8-hydroxydipropylaminotryptamine hydrobromide, Tocris UK), WAY100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2pyridinyl)cyclohexanecarboxamide maleate; from Tocris UK), buspirone (buspirone hydrochloride: from Carbosynth Ltd., UK). F15599 (3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methylpirymidin-2ylmethyl)-amino]-methyl}-piperidin-1-yl)-methadone fumarate) and F13714 (3-chloro-4fluorophenyl-(4-fluoro-4-{[(5-methyl-6-methyloamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin1-yl-methadone glicolate) were synthetized at the Department of Medicinal Chemistry of Jagiellonian University Medical College in Krakow. All drugs were dissolved in distilled water immediately before administration in a volume of 2 ml/kg. The choice of doses was based on previously-published information on the compounds. The routes of administration depended on the compounds: F13714 and F15599 are known to be highly bioavailable 22(and unpublished observations). In addition, they have been previously tested by oral route in the FST 22 so this route was used herein, as was also the case for buspirone (compounds administered 60 min before testing). In contrast, (±)8-OH-DPAT is very poorly bioavailable by oral route (

Activity of Serotonin 5-HT1A Receptor Biased Agonists in Rat

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