Research Article pubs.acs.org/chemneuro
Studies To Examine Potential Tolerability Differences between the 5‑HT2C Receptor Selective Agonists Lorcaserin and CP-809101 Guy A. Higgins,*,†,‡ Leonardo B. Silenieks,† Amy Patrick,† Ines A. M. De Lannoy,† Paul J. Fletcher,§,∥ Linda A. Parker,⊥ Neil J. MacLusky,# Laura C. Sullivan,∇ Teresa A. Chavera,∇ and Kelly A. Berg∇ †
InterVivo Solutions Inc., 120 Carlton Street, Toronto, Ontario M5A 4K2, Canada Department of Pharmacology & Toxicology, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada § Centre for Addiction and Mental Health, 250 College St, Toronto, Ontario M5T 1L8, Canada ∥ Department of Psychology & Psychiatry, University of Toronto, 100 St. George Street, Toronto, Ontario M5S 3G3, Canada ⊥ Department of Psychology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada # Ontario Veterinary College, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada ∇ Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, United States ‡
ABSTRACT: Lorcaserin (LOR) is a selective 5-HT2C receptor agonist that has been FDA approved as a treatment for obesity. The most frequently reported side-effects of LOR include nausea and headache, which can be dose limiting. We have previously reported that in the rat, while LOR produced unconditioned signs characteristic of nausea/ malaise, the highly selective 5-HT2C agonist CP-809101 (CP) produced fewer equivalent signs. Because this may indicate a subclass of 5-HT2C agonists having better tolerability, the present studies were designed to further investigate this apparent difference. In a conditioned gaping model, a rodent test of nausea, LOR produced significantly higher gapes compared to CP consistent with it having higher emetogenic properties. Subsequent studies were designed to identify features of each drug that may account for such differences. In rats trained to discriminate CP-809101 from saline, both CP and LOR produced full generalization suggesting a similar interoceptive cue. In vitro tests of functional selectivity designed to examine signaling pathways activated by both drugs in CHO (Chinese hamster ovary) cells expressing h5-HT2C receptors failed to identify evidence for biased signaling differences between LOR and CP. Thus, both drugs showed similar profiles across PLC, PLA2, and ERK signaling pathways. In studies designed to examine pharmacokinetic differences between LOR and CP, while drug plasma levels correlated with increasing dose, CSF levels did not. CSF levels of LOR increased proportionally with dose; however CSF levels of CP plateaued from 6 to 12 mg/kg. Thus, the apparently improved tolerability of CP likely reflects a limit to CNS levels attained at relatively high doses. KEYWORDS: Serotonin, 5-HT, lorcaserin, CP-809101, nausea, biased signaling, pharmacokinetics, CSF compartment, plasma compartment, conditioned gaping, drug discrimination, rat
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INTRODUCTION
Occupancy and subsequent activation of central 5-HT2C receptors is considered essential to the therapeutic properties of lorcaserin;1−5,9 however the degree of occupancy for the approved dosage form is currently unknown due to lack of validated biomarkers for the 5-HT2C receptor. Without knowing target occupancy, there is a risk of underestimating the efficacy of a drug if this level is suboptimal.10−12 We previously reported that in comparison to lorcaserin, the highly selective 5-HT2C agonist CP-80910113,14 may induce fewer signs of malaise or nausea in rats.15 Specifically, we observed
Clinical studies with the 5-HT2C receptor agonist lorcaserin (LOR) have demonstrated efficacy in the treatment of obesity and possibly smoking cessation.1−4 In 2012, the FDA approved lorcaserin for the treatment of obesity with concomitant lifestyle management. The approved lorcaserin dosage for obesity of 10 mg b.i.d. is based on a consideration of plasma levels likely free from off-target activity (notably the 5-HT2A and 5-HT2B receptors)5 and issues of safety and tolerability. A meta-analysis of Phase 3 obesity trials1,2 showed that nausea, headache, and dizziness are among the most common side effects, and their incidence increases considerably at supratherapeutic doses.6 Nausea is one of the most common doselimiting side-effects for CNS based therapeutics.7,8 © XXXX American Chemical Society
Special Issue: Serotonin Research 2016 Received: December 19, 2016 Accepted: March 13, 2017
A
DOI: 10.1021/acschemneuro.6b00444 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 1. Effects of lorcaserin and CP-809101 were investigated in the taste reactivity model (A, C, E) and a one-bottle conditioned taste avoidance test (B, D, F). (A) In experiment 1a, lorcaserin (1−6 mg/kg SC) produced a dose-related increase in the number of gapes; the effect size at 6 mg/kg was equivalent to lithium chloride (LiCl, 127 mg/kg), a known emetogen. (C) In experiment 1b, the effect of lorcaserin (6 mg/kg) on gaping was blocked by pretreatment with SB-242084 (0.5 mg/kg IP). (E) In experiment 1c, CP-809101 (3−12 mg/kg) also induced gaping although the effect size was significantly less than that of lorcaserin (6 mg/kg) and only reached marginal statistical significance compared to vehicle pretreatment at the 12 mg/kg dose. (B) In the one-bottle conditioned taste avoidance test, both lorcaserin and LiCl reduced saccharin intake relative to vehicle pretreatment measured after 0.5 h access. (D) In contrast to the gaping measure, pretreatment with SB-242084 did not significantly reverse the reduced saccharin intake produced by lorcaserin (6 mg/kg). (F) In contrast to gaping, both CP-809101 and lorcaserin reduced saccharin intake relative to vehicle pretreatment measured after 0.5 h access. *P < 0.05 vs vehicle (Veh) pretreatment. #P < 0.05 vs lorcaserin pretreated group (Bonferroni post hoc tests following significant ANOVA).
fewer unconditioned signs of malaise16−18 following CP809101, which suggested a potential for 5-HT2C receptor agonists with improved side-effect profiles and therapeutic value.15 Based on the potential for tolerability differences between CP-809101 and lorcaserin, we adopted four lines of study for further investigation. First, we examined lorcaserin and CP809101 in the conditioned taste reactivity model, a validated rodent test to study nausea.19 Given the likelihood of improved functional selectivity of CP-809101 for the 5-HT2C receptor compared to lorcaserin (at least among the 5-HT2 receptor subclass),9,13 we also wanted to establish whether any effect of lorcaserin in this model was 5-HT2C receptor based. This would help to establish whether the adverse signs of headache and nausea reported for lorcaserin in humans was based on the 5HT2C receptor agonist property as opposed to an off-target activity, which has already been reported for lorcaserin at higher doses (i.e., ≥3 mg/kg) in the rat.20,21 Second, having found evidence to support the initial observation of tolerability differences between lorcaserin and CP-809101, using a drug discrimination procedure, we trained rats to a CP-809101 drug cue to see if both lorcaserin and CP-809101 shared a similar discriminative stimulus property.22 Third, differences between lorcaserin and CP-809101 could be due to their pharmacokinetic properties and CNS penetration. We therefore compared both plasma and CSF levels of each drug at doses relevant to the in vivo studies. Finally, 5-HT2C receptors couple to multiple intracellular signaling pathways, which may be differentially activated by agonists.23−25 This concept of ligand functional selectivity or biased agonism has been proposed as a mechanism by which
agonists, despite sharing the same GPCR target, may have distinct physiological and behavioral properties. In turn this could result in improved therapeutic potential.26−30 For example, Valant et al.29 have described an adenosine A1 agonist with target based efficacy but without a target based side effect due to biased agonism at the A1 receptor. We therefore compared the signaling profiles of lorcaserin and CP809101 at h5-HT2C receptors to determine whether differences between these two compounds were potentially due to differential activation of signaling cascades.
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RESULTS AND DISCUSSION Experiment 1: Study of Lorcaserin and CP-809101 in the Taste Reactivity Test. Although rats lack a vomiting reflex, they demonstrate avoidance of a flavor previously paired with an emetogenic stimulus and a conditioned gaping reaction during re-exposure to that flavor.19,31 While the conditioned flavor avoidance is also induced by nonemetogenic, including appetitive, treatments, the conditioned gaping reaction is specifically induced by emetogenic drugs and is believed to be a selective measure of conditioned malaise.19,31 In experiment 1a, the effect of relatively high doses of lorcaserin (1−6 mg/kg SC) were directly compared to the known emetic lithium chloride,19 in the taste reactivity test. Across this dose range, lorcaserin produced a dose related increase in gaping following oral infusion of a saccharin solution to which it had been paired in two conditioning sessions (Figure 1A) (F4,36 = 4.99, p < 0.05). At the 6 mg/kg dose, the magnitude of lorcaserin effect was equivalent to lithium chloride. In the conditioned taste avoidance study phase, all doses of lorcaserin and lithium chloride reduced saccharin B
DOI: 10.1021/acschemneuro.6b00444 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience Table 1. Behavioural Measures and Saccharin Intake from the One Bottle Conditioned Taste Aversion Testa
saccharin intake (mL) treatment
dose (mg/kg)
vehicle LiCl lorcaserin lorcaserin lorcaserin
127 1 3 6
vehicle lorcaserin lorcaserin + SB242084
6 6 + 0.5
vehicle CP809101 CP809101 CP809101 lorcaserin
3 6 12 6
gaping 0 13.8 1.9 5.3 10.2
+ 4.1** ± 0.9 ± 1.9 ± 0.2**
0 9.6 ± 2.6* 2.5 ± 1.0 0 1.3 1.9 2.9 13.9
± ± ± ±
1.1 1.2 1.0 4.9**
hedonic signs 17.4 0.9 10.1 1.6 2.3
± ± ± ± ±
2.8 0.4** 3.0* 0.5** 0.5**
20.8 ± 3.2 2.6 ± 0.7** 4.1 ± 1.3** 24.3 5.6 3.4 4.0 3.0
± ± ± ± ±
5.7 1.4** 1.1** 2.1** 1.3**
aversive signs 0.3 20.1 3.4 10.7 10.8
± ± ± ± ±
0.3 5.1** 1.3 2.6 2.5*
0.2 ± 0.1 13.5 ± 3.0** 3.9 ± 1.7 0.1 1.8 3.9 4.4 16.6
± ± ± ± ±
0.1 1.1 1.5 1.1 5.7**
0.5 h 12.1 0.7 3.8 1.1 1.3
± ± ± ± ±
0.7 0.1* 1.1* 0.4* 0.4*
11.9 ± 0.9 1.2 ± 0.3* 3.7 ± 0.9* 11.8 5.0 2.4 2.5 1.3
± ± ± ± ±
1.7 0.9 0.8 0.6 0.4
2h 20.1 3.2 15.2 5.0 6.4
± ± ± ± ±
2.8 0.9** 2.3 1.4** 1.5**
25.3 ± 2.2 6.6 ± 1.1** 11.6 ± 2.0** 30.4 15.5 10.4 9.9 6.1
± ± ± ± ±
2.5 1.4** 2.9** 1.6** 1.7**
6h 46.1 17.0 37.8 19.4 22.0
± ± ± ± ±
5.0 3.8** 4.8 2.3** 2.0**
58.6 ± 4.3 25.6 ± 1.9** 34.2 ± 6.3** 69.9 41.5 29.7 36.7 30.6
± ± ± ± ±
4.1 3.6** 4.0** 4.9** 2.5**
a Hedonic signs (i.e. tongue protrusions, mouth movements) and aversive signs (i.e. gaping, chin rubbing, paw treading) were measured. *P < 0.05 vs. vehicle pretreatment in respective experiment.
Thus, similar to lithium chloride (a known emetic),19 lorcaserin induced a significant gaping response. SB-242084 reversed this effect, confirming it to be 5-HT2C receptor mediated. In contrast, CP-809101 produced a much smaller effect that only reached statistical significance at the 12 mg/kg dose, suggesting that relative to lorcaserin, CP-809101 is a weaker emetic agent. Both drugs produced a significant taste avoidance; however the effect of lorcaserin on this measure was not reversed by SB-242084. This would be consistent with taste avoidance being a more general response to a novel drug stimulus. The results of the gaping studies are therefore consistent with the findings from a previous study where we found that lorcaserin produced more pronounced unconditioned signs of malaise compared to CP-809101.15 This prompted experiments 2−4 designed to investigate features of both drugs that might account for this difference. Experiment 2: Study of Lorcaserin and CP-809101 in Rats Trained to Discriminate a CP-809101 Cue. CP809101 was selected as the training drug for the drug discrimination study since it represents the most selective 5HT2C receptor agonist reported to date13 and is therefore most likely to produce a cue that is pharmacologically selective. Rats required approximately 60 sessions to acquire reliable stimulus control to CP-809101 (1 mg/kg SC). A reduction in response rate was apparent at this dose throughout the course of training (see Figure 2A,B). In tests of substitution, both CP-809101 (0.1−3 mg/kg SC) and lorcaserin (0.1−3 mg/kg SC) completely generalized with similar potency (see Figure 2C, Table 2). However, differences were noted on response rate. Over the dose range tested, lorcaserin produced a monotonic decline with response rate reduced by 95% from baseline at the 3 mg/kg dose (Veh, 1.31 ± 0.14 response/s; LOR, 0.07 ± 0.02 response/s). By comparison, the equivalent doses of CP-809101 only reduced response rate by approximately 45% (Veh, 1.24 ± 0.12 response/s; CP, 0.69 ± 0.11 response/s) (Figure 2D). The effect of the selective 5-HT2A receptor antagonist M10090732 and the selective 5-HT2C receptor antagonist SB24208433 was also examined to better characterize the nature of the CP-809101 cue. SB-242084 (0.002−0.5 mg/kg IP) but not
intake compared to vehicle controls in a one bottle choice test. Thus, a 2 way ANOVA revealed a main effect of treatment (F4,36 = 19.7, P < 0.01), time (F2,72 = 213.0, P < 0.01), and treatment × time (F8,72 = 5.0, P < 0.01) reflecting reduced saccharin intake by all treatment groups at 0.5 h, although at the later time points saccharin intake in the lorcaserin 1 mg/kg group was equivalent to vehicle control (Figure 1B, Table 1). Experiment 1b was conducted to see if the effect of lorcaserin (6 mg/kg) on gaping could be blocked by pretreatment with the selective 5-HT2C antagonist SB-242084 (0.5 mg/kg) during conditioning. A one-way ANOVA revealed a significant main effect (F2,28 = 6.87, p < 0.01) on gaping. Lorcaserin significantly increased gaping relative to vehicle pretreated controls, an effect that was significantly reduced by SB-242084 (Figure 1C, Table 1). In the conditioned taste avoidance study a main effect of treatment (F2,35 = 50.7, P < 0.01) and treatment × time (F4,70 = 5.7, P < 0.01) reflected a significant reduction in saccharin intake in both lorcaserin and lorcaserin/ SB-242084 groups relative to vehicle controls. Comparison between lorcaserin treated animals either cotreated or untreated with SB-242084, showed no significant differences between these groups, although a modest trend for higher consumption in SB-242084 cotreated rats was evident. Thus, SB-242084 pretreatment reliably blocked the conditioned gaping response but not the conditioned taste aversion to lorcaserin (Figure 1D, Table 1). In experiment 1c, the effect of CP-809101 was studied on taste reactivity and compared to that of lorcaserin (6 mg/kg). A main effect of treatment group on gaping (F4,43 = 8.01, p < 0.01) was recorded, although in contrast to lorcaserin, CP809101 produced a significantly lower incidence of gaping, for example, CP-809101 12 mg/kg, 2.9 ± 0.9 gapes; lorcaserin 6 mg/kg, 13.9 ± 4.9 gapes (Figure 1E). In the conditioned taste avoidance study, a main effect of treatment (F4,35 = 22.9; P < 0.01) and treatment × time (F8,70 = 9.8, P < 0.01) interaction confirmed that all treatment groups reduced saccharin intake relative to vehicle controls, effects evident at each time point (see Figure 1F, Table 1). In contrast to the gaping measure, there was no difference in the magnitude of decreased saccharin consumption between the two drugs. C
DOI: 10.1021/acschemneuro.6b00444 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 2. Rats were trained to discriminate the selective 5-HT2C receptor agonist CP-809101 (1 mg/kg SC) from saline in a two-lever food reinforced drug discrimination procedure. Acquisition of the CP-809101 cue is presented in terms of drug lever discrimination (A) and response rate (responses/s) (B). Once stable discrimination was acquired, the rats were tested for substitution with various doses of CP-809101 and lorcaserin, and these data are shown for % drug lever responding (C) and response rate (D). Tests of antagonism against the cue maintained by CP-809101 (1 mg/kg SC) were also conducted using the selective 5-HT2C receptor antagonist SB-242084 (0.002−0.5 mg/kg IP) and the selective 5-HT2A receptor antagonist M100907 (0.001−1 mg/kg SC). % drug lever responding is shown in panel E and rate of responding in panel F. Over the dose ranges tested, SB-242084 but not M100907 blocked both the cueing and rate decreasing effect of CP-809101 (1 mg/kg).
Table 2. Summary of Findings from Tests of Substitution and Antagonism Conducted in Rats Trained to Discriminate the Selective 5-HT2C Receptor Agonist CP-809101 (1 mg/kg SC) from Saline treatment details
substitution
drug
dose range (mg/kg), route
pretreatment time (min)
CP-809101 lorcaserin Ro 60-0175 mCPP d-fenfluramine SB-242084 M100907
0.1−3, SC 0.1−3, SC 0.1−3, SC 0.1−2, SC 0.3−3, IP 0.004−0.5, IP 0.001−1, SC
30 15 15 15 30 30 30
ED50 substitution (mg/kg) 0.27 0.15 0.73 0.26 1.27 >0.5 >1
(0.13−0.52) (0.07−0.33) (0.30−1.78) (0.04−1.71) (0.33−4.77)
M100907 (0.001−1 mg/kg SC) blocked both the discrim-
max. substitution (%) 100 87 ± 10 89 ± 6 83 ± 7 100 0 0
antagonism ED50 rate (mg/kg) 0.78 0.74 1.01 0.58 2.18 >0.5 >1
ID50 (mg/kg)
(0.44−1.37) (0.45−1.24) (0.16−6.35) (0.37−0.92) (0.96−4.94) 0.011 (0.004−0.026) >1
substitution tests with Ro 60-0175, mCPP, and d-fenfluramine are reported in Table 2. Lorcaserin fully substituted for CP-809101 over an identical dose range to CP-809101 itself, suggesting both drugs share a
inative cue and the decrease in response rate produced by CP809101 (see Figure 2E,F, Table 2). The outcomes from D
DOI: 10.1021/acschemneuro.6b00444 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Research Article
ACS Chemical Neuroscience
Figure 3. (A) Increasing doses of lorcaserin (0.3−6 mg/kg SC) resulted in a dose proportional increase in drug concentration in both plasma and CSF compartments. CSF/plasma concentration ratio was similar (0.12−0.15) over the 1−6 mg/kg doses at a 1 h time point corresponding to changes in behavioral measures. Lorcaserin in the dose range of 0.3−1 mg/kg SC resulted in maximum plasma concentrations of 28−88 ng/mL. (B) Plasma concentrations of CP-809101 were significantly less than those of lorcaserin; maximum plasma concentrations at 1 mg/kg were
