5-HT1A Agonist Properties Contribute to a Robust Response to Vilazodone in the Novelty Suppressed Feeding Paradigm

Background: Differences in 5-HT1A receptor function have been implicated in vulnerability to depression and in response to treatment. Adding 5-HT1A partial agonists to selective serotonin reuptake inhibitors has been touted as a strategy to increase their efficacy. Here we use the novelty suppressed feeding paradigm to compare the effects of vilazodone, a high-potency selective serotonin reuptake inhibitor, with high affinity for 5-HT1A receptors to the reference selective serotonin reuptake inhibitor fluoxetine across several mouse strains that differ in their response to selective serotonin reuptake inhibitors. Methods: To confirm 5-HT1A agonist activity, body temperature was measured after acute administration of vilazodone or fluoxetine, as administration of 5-HT1A agonists induces hypothermia. We next used 3 strains of mice to examine the effects of the drugs on latency in the novelty suppressed feeding, a paradigm generally sensitive to chronic but not acute effects of antidepressants. Results: Vilazodone induces robust hypothermia and blocks stress-induced hyperthermia in a 5-HT1A-dependent manner, consistent with agonist effects at 5-HT1A autoreceptors. In 129SvEv mice, vilazodone (10mg/kg/d) reduces the latency to eat in the novelty suppressed feeding test within 8 days, while no effect of fluoxetine (20mg/kg/d) was detected at that time. In contrast, both vilazodone and fluoxetine are effective at decreasing latency to eat in the novelty suppressed feeding paradigm in a strain with low autoreceptor levels. In mice with higher autoreceptor levels, no significant difference was detected between fluoxetine and vehicle (P=.8) or vilazodone and vehicle (P=.06). Conclusion: In mice, vilazodone may offer advantages in time of onset and efficacy over a reference selective serotonin reuptake inhibitor in the novelty suppressed feeding test.


Introduction
Depression and anxiety are chronic and recurring illnesses that strike early in life and are thus amongst the leading causes of disability worldwide (Murray et al., 2012). While currently approved medications with antidepressant/anxiolytic activity are effective for some patients, many patients do not respond to initial treatment with a first-line medication. In addition, while most first-line treatments target some aspect of monoaminergic modulatory systems, a specific understanding of why individual drugs with antidepressant/anxiolytic activity are effective in some patients but not others remains an unsolved problem. Indeed, a physician's first choice of medication is often influenced more by side effect than by efficacy profiles. As a result, there are often delays in obtaining optimal treatment, resulting in excess morbidity and suffering. The ability to select a medication both on considerations of likely efficacy and tolerability would be a great step forward. To this end, an improved understanding of the mechanistic differences between available medications is critical.
Here we compare the effects of vilazodone, a high efficacy serotonin reuptake inhibitor with potent 5-HT 1A partial agonist properties, to fluoxetine, the prototype selective serotonin reuptake inhibitor (SSRI) approved for the treatment of depression, in the novelty suppressed feeding (NSF) paradigm. This paradigm has previously been shown in mice to be sensitive to chronic but not acute treatment with several classes of antidepressants in a variety of mouse strains (Dulawa and Hen, 2005;Samuels and Hen, 2011). Several lines of evidence suggest that adding a 5-HT 1A partial agonist to SSRIs would augment the antidepressant effect (Celada et al., 2004), and there is some evidence to suggest that 5-HT 1A agonists alone may have antidepressant effect (Wilcox et al., 1996;Bielski et al., 2008). In addition, several genetics studies in humans suggest that variation in the gene that encodes the 5-HT 1A receptor may be important in determining both the vulnerability to depression and the response to treatment with antidepressants (Le Francois et al., 2008;Richardson-Jones et al., 2010). These studies are additionally supported by imaging studies in humans that suggest that 5-HT 1A receptor expression patterns may predict response to treatment with antidepressants (Miller et al., 2013). Indeed, previous work in mice has shown that mice with relatively higher levels of 5-HT 1A autoreceptors do not respond to fluoxetine in the NSF paradigm when compared with otherwise identical mice with lower levels of 5-HT 1A autoreceptors (Richardson-Jones et al., 2010). Here we hypothesized that vilazodone would be effective in the NSF paradigm under conditions that are resistant to treatment with fluoxetine.

Animal Husbandry
Animals were housed in groups of 3 to 5 per cage and had ad libitum access to food and water. Animals were maintained on a 12:12 light/dark schedule. Animal protocols were approved by the Institutional Animal Care and Use Committee at the New York State Psychiatric Institute and were conducted in accordance to the NIH Guide for the Care and Use of Laboratory Animals.

Generation of the Conditional 5-HT 1A Autoreceptor Mice
To obtain a regulatable 5-HT 1A autoreceptor mouse, tetO-1A mice were bred to a transgenic mouse line with tTS expression driven from Pet promoter fragments as previously described (Richardson-Jones et al., 2010. tetO-1A+/+/Pet-tTS+ (1A-Hi) mice maintained in the presence of doxycycline (DOX) displayed no receptor suppression and with a pattern of expression that is indistinguishable from Pet-tTs-mice. Mice that have DOX withdrawn at P50 display an approximately 30% decrease in autoreceptor expression after 4 weeks (1A-Low) (Richardson-Jones et al., 2010.

Behavioral and Physiological Testing
All animals used for behavioral testing were age matched within 2 weeks. Animals were males and initially tested at 14 to 16 weeks of age. For 8-OH-DPAT and stress-induced hyperthermia, drugs were given via i.p. injection. For the NSF test, drugs were given via gavage for up to 28 days. Testing in the NSF occurred on day 8 and 28 of treatment.

8-OH-DPAT-Induced Hypothermia
Mice were singly housed in clean cages for 10 minutes and 3 baseline body temperature measurements were taken. Ten minutes after the third baseline measurement, animals received different drugs i.p. Change in core temperature was assessed using a rectal probe every 10 minutes for 60 minutes as previously described (Richardson-Jones et al., 2010. 8-OH-DPAT is the prototypical 5-HT 1A full agonist. Although it does have some activity at 5-HT 7 receptors (Landry et al., 2006), 8-OH-DPAT-induced hypothermia in mice is dependent on functional 5-HT 1A autoreceptors (Richardson-Jones et al., 2011).

Stress-Induced Hyperthermia
Stress-induced hyperthermia was performed as previously described (Richardson-Jones et al., 2010). Briefly, animals in their home cages were moved to a testing room and allowed to acclimate for 1 hour. One animal per cage was removed, and a baseline body temperature was measured rectally immediately prior to administration of the appropriate drug. Each animal was then placed in a novel, clean cage for 10 minutes, after which a second body temperature was recorded.

NSF
The NSF paradigm is a test of hyponeophagia that measures the latency of a mouse to consume food placed in the middle of a brightly lit, aversive arena (Bodnoff et al., 1988). Testing was performed as previously described (David et al., 2009;Richardson-Jones et al., 2010). Briefly, animals were food restricted for 24 hours, and the latency (dependent measure) to begin chewing a food pellet placed on a white piece of filter paper (12.5cm diameter) in the center of brightly lit arena was recorded (40-× 60-cm arena with 2 cm of new corn cob bedding; 800-900 lux). The trial was terminated either when an animal began chewing or 300 seconds transpired. Immediately after terminating the trial, animals were placed in their home cage, and the amount of food consumed in 5 minutes was measured (home cage consumption), followed by an assessment of postrestriction weight. Percentage body weight lost during food deprivation prior to the testing was assessed to ensure both groups lost similar amounts of weight. Home cage consumption immediately after testing was assessed as a relative measure of hunger.

Statistical Analysis
Results from data analyses were expressed as mean ± SEM. Onetailed P < .05 was used as the threshold for significance. Twoway ANOVA with repeated measures in 1 factor was used for 8-OH-DPAT-induced hypothermia where drugs were expected to decrease temperatures more than vehicle (Richardson-Jones et al., 2011). ANOVAs were followed up with Fisher's protected least squares difference posthoc comparisons. For NSF, the hypothesis was that drugs would decrease the latency to feed (Richardson-Jones et al., 2010). Data in several groups were right censored and thus analyzed using a Kaplan-Meier survival analysis with the Gehan-Breslow-Wilcoxon method. Bonferroni corrected P values are presented.

Vilazodone Elicits an Accelerated Effect in the NSF Paradigm Compared with Fluoxetine
The NSF paradigm has 2 features that have made it useful in modeling the response to antidepressants: (1) It is generally sensitive to chronic (>3 weeks) but not acute administration of drugs with antidepressant efficacy (Wang et al., 2008), and (2) the response is affected by the genetic background of the mice (Lucki et al., 2001), with some strains not responding to SSRIs in this paradigm (Ibarguen-Vargas et al., 2008). Here, using the 129SvEv inbred mouse strain, we tested the effect of several doses of vilazodone. After 8 days, no effect of treatment was detected for fluoxetine at 20 mg/kg (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .816). The effect of vilazodone was not significant at 3 mg/kg (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .069) but was significant at 10 mg/kg (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .01; n = 15 mice/group) ( Figure 3A-B). After 28 days of treatment, the expected effect of 20 mg/kg fluoxetine was not significant (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .079), while vilazodone elicited a significant effect relative to vehicle at both 3 mg/kg and 10 mg/kg (Gehan-Breslow-Wilcoxon Bonferroni corrected P < .001 for both; n = 15 mice/ group) ( Figure 3C-D). The effect on latency cannot be attributed to changes in hunger between groups, as we did not detect any change in home cage consumption after the 8-or 28-day tests (supplementary Figure 2A-B). Thus, in the 129SvEv strain, vilazodone elicits a faster and more robust response than fluoxetine in the NSF paradigm.

Effect of 5-HT 1A Autoreceptor Levels on Response to Vilazodone in the NSF Paradigm
We next examined the effects of vilazodone in 2 strains of mice that are identical except for their 5-HT 1A autoreceptor expression profile. The strains are engineered to allow for DOX-dependent temporal control of 5-HT 1A autoreceptor expression (Richardson-Jones et al., 2010). In the presence of DOX, (5-HT 1A Hi) 5-HT 1A autoreceptor levels in the engineered animals are indistinguishable from their nontransgenic littermates. In the absence of DOX, 5-HT 1A expression levels are decreased by about 30% (5-HT 1A Low). We have previously demonstrated that 5-HT 1A Hi mice do not respond to fluoxetine in the NSF paradigm. Here, no effect of treatment is observed at 8 days for either fluoxetine or vilazodone (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .64 and P = .15, respectively; n = 14-15 mice/group) ( Figure 4A-B). At 28 days, no effect of fluoxetine was detected (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .88). The vilazodone effect at 10 mg/kg missed the cutoff for significance (Gehan-Breslow-Wilcoxon Bonferroni corrected P = .06; n = 12-15 mice/group) ( Figure 4C-D). Home cage consumption was decreased in the fluoxetine group after 8 days, but not in the other groups (supplemental Figure 3A-B).

Discussion
The efficacy of vilazodone as an antidepressant is hypothesized to be a result of its combined ability to block the reuptake of serotonin and its ability to act as a partial agonist at 5-HT 1A receptors. Here we use a 5-HT 1A agonist-induced hypothermia paradigm to demonstrate that vilazodone has significant in vivo effects at 5-HT 1A autoreceptors above and beyond those that would be expected from increased serotonin due to 5-HT transporter blockade. In addition, we demonstrate the ability of vilazodone to block stress-induced hyperthermia in a 5-HT 1A -dependent manner. Our behavioral data demonstrate that chronic treatment with vilazodone at doses that exhibit significant 5-HT 1A agonism results in decreased latencies to eat in the NSF paradigm that differ in both timing and effect size to the response to fluoxetine in 3 selected mouse strains. Consistent with previous studies, we saw 3 distinct patterns of response in the NSF to fluoxetine in the strains tested. The 5-HT 1A Low mice were highly responsive to fluoxetine with effects seen both subchronically at 8 days and chronically at 28 days. The 129SvEv strain was moderately responsive with no effect seen subchronically and an effect usually emerging by 4 weeks. In this particular cohort, this effect narrowly missed the cutoff for significance. Finally, the 5-HT 1A Hi strain was completely unresponsive in this paradigm (Santarelli et al., 2003;Richardson-Jones et al., 2010). Interestingly, in the case of vilazodone, both the 129SvEv and the 5-HT 1A Low mice were highly responsive, with the 10 mg/kg of vilazodone eliciting an effect as early as 8 days into treatment. The 5-HT 1A Hi mice were least affected by treatment with vilazodone, with decreased mean latencies that were not statistically distinct from the vehicle group. Interestingly, in the 5-HT 1A Hi mice, the distribution of latencies in the NSF in response to vilazodone at both 8 days and at 28 days appeared bimodal. About one-half of the animals exhibited a very low latency, raising the possibility that a subset of animals in this group may be responding. Such a bimodal distribution in the NSF, with responders and nonresponders within the same strain has previously been described. Finally, the absolute mean latencies of the vilazodone-treated animals were lower than the respective means of both the vehicle and fluoxetine-treated animals in all cohorts. Taken together, these data suggest that vilazodone has a more robust and potentially earlier effect in the NSF across multiple strains compared with fluoxetine.
One explanation that has been given for the delayed response to treatment with SSRI drugs in depression and in some behavioral assays is the need to desensitize 5-HT 1A autoreceptors before a treatment effect can be observed (Blier et al., 1998). The accelerated effect of vilazodone relative to fluoxetine in the NSF in the 129SvEv strain might be interpreted in the context of the results with the 5-HT 1A Low mice, where fluoxetine also acts in an accelerated manner. We previously demonstrated that as a result of low autoreceptor levels, the 5-HT 1A Low mice behave functionally as if they have desensitized autoreceptors (Richardson-Jones et al., 2010), providing a rationale for an early response to SSRIs in this strain. In the case of the 129SvEv strain, it is possible that vilazodone but not fluoxetine is able to generate a subacute response at 8 days, because vilazodone has been shown to promote rapid desensitization of 5-HT 1A receptors (Ashby et al., 2013). A rationale for the relative resistance of the 5-HT 1A Hi mice is less clear, although the potential bimodal distribution of latencies in vilazodone treated mice merits further investigation.
In general, it has been difficult to predict when one treatment for depression might work over another treatment in any given patient. Indeed, this lack of a personalized approach has generally led to the prescription of antidepressants using other criteria such as cost or side effect profiles. Together with data from imaging and genetic studies in humans, our data in the NSF model in mice support a hypothesis in which vilazodone and SSRIs may have equivalent effects in populations with lower levels of 5-HT 1A autoreceptors, while vilazodone may have an advantage in patients that do not respond to SSRIs. Furthermore, our data are consistent with the hypothesis that higher levels of autoreceptors increase resistance to SSRIs (Le Francois et al., 2008).