5-HT1A Receptor Reduces -induced Respiratory Depression, Analgesia, and Sedation in Rats

Jun Ren, Ph.D., Xiuqing Ding, B.Sc., John J. Greer, Ph.D.

ABSTRACT

Background: There is an unmet clinical need to develop a pharmacological therapy to counter -induced respiratory depression without interfering with analgesia or behavior. Several studies have demonstrated that 5-HT1A receptor alleviate opioid-induced respiratory depression in rodent models. However, there are conflicting reports regarding their effects on analgesia due in part to varied agonist receptor selectivity and presence of anesthesia. Therefore the authors performed a Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/2/424/267207/20150200_0-00031.pdf by guest on 27 September 2021 study in rats with befiradol (F13640 and NLX-112), a highly selective 5-HT1A receptor agonist without anesthesia. Methods: Respiratory neural discharge was measured using in vitro preparations. Plethysmographic recording, nociception testing, and righting reflex were used to examine respiratory ventilation, analgesia, and sedation, respectively. Results: Befiradol (0.2 mg/kg, n = 6) reduced fentanyl-induced respiratory depression (53.7 ± 5.7% of control minute ven- tilation 4 min after befiradol vs. saline 18.7 ± 2.2% of control, n = 9; P < 0.001), duration of analgesia (90.4 ± 11.6 min vs. saline 130.5 ± 7.8 min; P = 0.011), duration of sedation (39.8 ± 4 min vs. saline 58 ± 4.4 min; P = 0.013); and induced baseline hyperventilation, hyperalgesia, and “behavioral syndrome” in nonsedated rats. Further, the befiradol-induced alleviation of opioid-induced respiratory depression involves sites or mechanisms not functioning in vitro brainstem–spinal cord and medul- lary slice preparations. Conclusions: The reversal of opioid-induced respiratory depression and sedation by befiradol in adult rats was robust, whereas involved mechanisms are unclear. However, there were adverse concomitant decreases in fentanyl-induced analgesia and altered baseline ventilation, nociception, and behavior. (Anesthesiology 2015; 122:424-34)

UPPRESSION of central respiratory drive by opioid What We Already Know about This Topic S is a clinical problem for which improved therapeutic treatments and safety are needed.1 Balancing • Reversal of opioid-induced respiratory depression without re- versal of analgesia is an important unmet clinical need the trade-off between analgesia and respiratory depression • Studies suggest that 5-HT1A receptor agonists may provide is one of the major challenges of anesthesiology, pain, and such selective reversal, at least in some animal models intensive care medicine. Current approaches to reversing opioid-induced respiratory depression, such as naloxone, What This Article Tells Us That Is New can be effective short-term, but only at the price of impaired • In conscious rats, administration of the 5-HT1A receptor ago- analgesia. Thus, there is a need to develop a safe and effective nist, befiradol, reversed fentanyl-induced respiratory depres- sion, but also antinociception, and caused abnormal behav- pharmacological therapy to counter -induced respi- iors that may limit clinical ratory depression that does not interfere with analgesia or significantly modulate baseline cardiorespiratory parameters. Recent advances in understanding the neurochemical including 5-HT released from the raphé nuclei that control of respiration have provided the foundation for potently alters the excitability of respiratory motoneurons, targeting specific neurotransmitter receptor systems for the preBötzinger complex (preBötC) and other brainstem potential drug therapies. A major component of excitatory respiratory nuclei. Several studies have demonstrated that synaptic drive necessary for respiratory rhythmogenesis and activation of 5-HT1A receptors (5-HT1AR) with the agonists activation of respiratory motoneurons is via the amino-3-hy- 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), droxy-5-methyl-4-isoxazolepropionate (AMPA) subclass of or alleviates opioid-induced respira- glutamate receptors.2–5 Those studies provided the founda- tory depression in rodent models.11–15 However, there are tion for the investigations of ampakine (positive modulators conflicting reports regarding the effects of these agents on of AMPA receptors) therapy to alleviate drug-induced respi- analgesia, sedation, baseline nociception, and cardiorespira- ratory depression.6–10 Further conditioning of respiratory tory parameters.12–24 Contradictory findings in those pre- activity is provided by a diverse group of neuromodulators, vious reports likely reflect varied drug receptor specificity,

Submitted for publication June 24, 2014. Accepted for publication September 4, 2014. From the Department of Physiology, Neuroscience, and Mental Health Institute, University of Alberta, Edmonton, Alberta, Canada (J.R., X.D., J.J.G.); and the Alberta Innovates Health Sciences Foundation, Edmonton, Alberta, Canada (J.J.G.). Copyright © 2014, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. All Rights Reserved. Anesthesiology 2015; 122:424-34

Anesthesiology, V 122 • No 2 424 February 2015 PAIN MEDICINE

route of administration, dose range, and critically, the and data acquisition software (Axon Instruments AxoScope; absence, presence, and type of anesthesia. In this study, we Molecular Devices). performed a systematic analysis of the effects on ventilation and analgesia of a novel agonist, 4-piperidinemethanamine, Whole Body Plethysmographic Recordings 1-(3-chloro-4-fluorobenzoyl)-4-fluoro-N-(5-methyl-2- Measurements from unrestrained newborn and adult pyridinyl)-methyl,(2E)-2-butenedioate (befiradol, also Sprague–Dawley male rats were performed in whole body, known as F13640 and NLX-112), that has high selectivity plexiglass plethysmographs that had inflow and outflow ports and agonist efficacy at 5-HT1AR located at both presynap- for the continuous delivery of fresh room air and removal of tic and postsynaptic sites,23,25,26 and has shown efficacy in expired carbon dioxide. The plethysmograph volumes were rodent models of neuropathic, inflammatory, and surgical 50 and 2,000 ml for measures of respiratory parameters of pain.27 We performed these studies without the confound- neonatal (3 to 4 days after birth) and adult (290 to 360 g) male Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/2/424/267207/20150200_0-00031.pdf by guest on 27 September 2021 ing effects of anesthesia that may interfere with detection rats with a flow rate of 50 ml/min and 1 l/min, respectively. of drug-induced changes in important baseline parameters For newborns, the plethysmograph was contained within an related to ventilation, pain sensitivity, and arousal. infant incubator (Isolette, model C-86; Air-Shields/Dräger Medical, Hatboro, PA) to maintain the ambient temperature Materials and Methods at the approximate nest temperature of 32°C. For newborn experiments, fentanyl citrate (35 μg/kg; Sandoz, Boucher- Brainstem–Spinal Cord and Medullary Slice Neonatal ville, QC, Canada) was injected into subcutaneously the neck Preparations fat pad. For adult infusion experiments, adult rats were anes- All experimental procedures were approved by the Faculty thetized with 3% in an induction chamber and of Medicine and Dentistry Animal Welfare Committee at maintained with 2% isoflurane anesthesia during tail vein the University of Alberta (Edmonton, Alberta, Canada). cannulation (P10 size tubing, with both vein cannulated). Neonatal (3 to 4 days after birth) Sprague–Dawley rats were The chamber had an additional port to allow exteriorization anesthetized with metofane, decerebrated and the brain- of the tail for iv drug infusion via an infusion pump (KD Sci- stem–spinal cord (BSSC) dissected as previously reported.28 entific, Hollistan, MA). With the infusion approach, all drug The neuraxis was continuously perfused at 27° ± 1°C (per- deliveries can be performed with continuous monitoring fusion rate, 5 ml/min; chamber volume, 3 ml) with modi- of plethysmographic recordings without physical handling fied Kreb’s solution that contained 128 mM NaCl, 3.0 mM of the animal. Pressure changes were detected with a pres-

KCl, 1.5 mM CaCl2, 1.0 mM MgSO4, 24 mM NaHCO3, sure transducer (model DP 103; Validyne, Northridge, CA), d 0.5 mM NaH2PO4, and 30 mM -glucose equilibrated with signal conditioner (CD-15; Validyne), analog-digital board 95% O2–5% CO2 (pH 7.4). The μ-opioid receptor agonist (Digidata 1322A), and data acquisition (Axoscope) and d-Ala2, N-MePhe4, Gly-ol-enkephalin (DAMGO, 300 nM; analysis (Clampfit) software (Axon Inst., Molecular Devices, Sigma Canada, Markham, Ontario, Canada) was added to Sunnyvale, CA). A pulse oximeter (Norin 8600V, Plymouth, the bathing medium to induce respiratory depression in vitro. MN) was placed on the tail to monitor oxygen saturation o BSSC preparations isolated from newborn rats were pinned (Sa 2) levels and heart rate in adult rats. Body temperature down, ventral surface upward, on a paraffin coated block.3 was measured using a rectal probe (Dual thermometer; Fisher The block was mounted in the vise of a vibratory micro- Scientific, Ottawa, ON, Canada). tome (VT1000 S; Leica Microsystems, Wetzlar, Germany). It should be noted that our plethysmograph is effective for

The brainstem was sectioned serially in the transverse plane, studying respiratory frequency (fR) and detection of apneas. It starting from the rostral medulla to within approximately is not designed for precise quantification of tidal volume (VT, 150 μm of the rostral boundary of the preBötC, as judged by ml/g). The physical principle underlying whole-body plethys- the appearance of the inferior olive. A single transverse slice mography is the detection of pressure changes in the chamber containing the preBötC and more caudal reticular formation resulting from the heating and humidification of inspired gas. regions was then cut (600 μm thick), transferred to a record- However, tidal volume measurements may also be influenced ing chamber, and pinned down onto Sylgard elastomer by gas compression effects related to the airway resistance. (Dow Corning, Midland, MI). The medullary slice was con- Because of these limitations, our whole-body plethysmo- tinuously perfused with a bathing solution identical to that graphic system only provided semiquantitative measurements used for BSSC preparation with the exception that the KCl of VT from which we report changes relative to the control concentration was increased to 9 mM to facilitate long-term state. As a result, our measurements of VE (minute ventila- 3 −1 −1 generation of stable rhythm by these preparations. Record- tion: ml min g ), which equates to fR × VT, are also semi- ings from the fourth ventral cervical nerve roots of BSSC quantitative and only reported relative to control. An apnea or hypoglossal nerve roots of medullary slice preparations is defined as the absence of airflow for a period equivalent or were amplified, rectified, lowpass filtered, and recorded to a greater than two complete respiratory cycles. computer, using an analog–digital converter (Axon Instru- For the determination of baseline breathing parameters in ments Digidata 1200; Molecular Devices, Sunnyvale, CA) freely moving adult rats in experiments that did not require

Anesthesiology 2015; 122:424-34 425 Ren et al. Befiradol Reduces Opioid-induced Apnea and Analgesia

tail vein infusions, the Buxco plethysmographic system In Vivo Drug Administration and Experimental Protocols (Buxco Research System, Wilmington, NC) was used. The Fentanyl citrate (Sandoz, Boucherville, QC, Canada) was Buxco plethysmograph was supplied with fresh room air by infused intravenously into one tail vein and a bolus of befi- the bias flow regulator (2.5 l/min), and signals were ampli- radol or saline was injected intravenously into the other tail fied by the MAX II preamplifier. Respiratory parameters vein. The overall fentanyl experimental protocol is depicted in μ (fR, VT, and VE) were recorded by Biosystem XA software figure 1. Fentanyl (60 g/kg per 20 min) infusion commenced (Buxco Research System, Wilmington, NC). approximately 5 min after the plethysmographic chamber was flushed with room air to remove the residual ambient isoflu- Nociception Testing and Righting Reflex rane. Righting reflex testing started approximately 4 min after Measures of analgesia were performed by examining fentanyl administration and continued until the animal awoke responses to tail clamping with forceps (with consistent from fentanyl-induced sedation (note that this is not from Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/2/424/267207/20150200_0-00031.pdf by guest on 27 September 2021 pressure) at 3- to 5-min intervals. A positive response to residual isoflurane). Saline or befiradol (provided by Dr. Mark tail clamping was indicated by obvious alteration in at least Varney, Ph.D., Neurolixis Inc., San Diego, CA, dissolved in two of the following parameters: (1) breathing variability a 0.9% saline, 0.1 to 0.6 mg/kg, iv, bolus) was administered (by plethysmograph), (2) heart rate (by pulse oximetry), at approximately 6 min after fentanyl infusion. The concen- (3) oxygen saturation (by pulse oximetry), and (4) body tration of befiradol administered in vitro and in vivo in our movement (visual observation). Further, thermal nocicep- study was based on the demonstration that befiradol activates 23,25,26 tion was measured by a plantar test apparatus (Ugo Basile, 5-HT1AR with an EC50 in the nM range. Tail clamp- Comerio VA, Italy), consisting of an infrared heat source ing started from approximately 11 min after fentanyl infusion positioned directly beneath the hind paw, 20 mm below until a positive response was observed. Tail clamping was only the chamber floor.6 When the rat perceived pain and with- tested in some animals (six of nine vehicle groups and five of drew its paw, the instrument automatically detected the six 0.2 mg/kg befiradol group, but not in other dosage of befir- withdrawal latency to the nearest 0.1 s. The heat stimulus adol groups). When the animal regained righting reflex, it was was automatically terminated if a withdrawal response was taken out from the recording chamber for thermal nociceptive not observed within 20 s of its onset to avoid the tissue testing. Thermal nociception testing was repeated every 2 to damage. Paw withdrawal latencies were measured after the 5 min until a positive paw withdrawal response of less than 8 animal began to recover from the fentanyl-induced seda- s (upper-limit of response time on baseline) was observed on tion, as assessed by duration of loss of righting reflex. Loss two consecutive tests. The time from the beginning of fentanyl of righting reflex was defined as the rat’s inability to right administration to the positive paw withdrawal of less than 8 itself into the prone position after the animal was placed s was arbitrarily defined as the duration of analgesia, since supine by repositioning the plethysmographic chamber. we were unable to measure the onset of analgesia by thermal Although loss of righting reflex is used frequently as a nociception testing in this study. For experiments measuring marker for the hypnotic state (unconsciousness), the exact baseline parameters, the nociception testing and behavioral state of consciousness could not be determined without observation started 10 min before, and 10 min postinjection definitive electroencephalography. Therefore we used the of drugs (subcutaneous) and was repeated every 3 min for a term sedation. All animals tested in this study were unable total of three times outside of Buxco chamber, followed by to right when placed supine at approximately 4 min after 30 min of measurement of breathing parameters via plethys- fentanyl infusion. We did not measure the onset of loss of mography in the Buxco system. righting reflex, therefore duration of loss of righting reflex in this study was arbitrarily defined as the time interval Statistical Analysis from the beginning of fentanyl administration to recovery Data are expressed as mean ± SEM (Sigmaplot 11 Systat of righting reflex. Software Inc., San Jose, CA). Sample sizes were used based on previous experience. Randomization methods were used Behavioral Observations to assign units to experimental condition. Blind testing is We measured four types of behavior: head weaving, forepaw used where one person administered the drug, and second treading, flat body posture, and lower lip retraction. Behav- person observed, rated the behavior, and analyzed the data iors were visually scored during observation periods of 45 s without knowledge of drug administration. There was no on a 4-point ranked intensity scale (0 = absent, 1 = equivo- missing for the data used for statistical analysis. Respiratory cal, 2 = definite, and 3 = intense).29 Scores for each behavior parameters were calculated by an average of 2-min, 1-min were averaged over three observation periods in the 10 min continuous recording data in vitro, in vivo, respectively. before drug administration and then three more starting The respiratory parameters fR, VT, and VE were reported as 10 min after drug administration (neck subcutaneously). means relative to control values (before fentanyl administra- Only those animals in which the scores across all behaviors tion in vivo, DAMGO in vitro, control values as 1). For in averaged greater than 2 were defined as displaying “behav- vitro experiments, the significance of changes in respiratory ioral syndrome.” parameters before and after befiradol administration to the

Anesthesiology 2015; 122:424-34 426 Ren et al. PAIN MEDICINE

Fig. 1. Graphic outline of the experimental protocol to evaluate the effects of befiradol on the fentanyl-induced respiratory ­depression, analgesia, and sedation. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/2/424/267207/20150200_0-00031.pdf by guest on 27 September 2021

Fig. 2. Befiradol does not alleviate DAMGO-induced respiratory depression in vitro. A representative rectified and integrated ­recording of the respiratory discharge of fourth cervical ventral nerve roots (C4) produced by a postnatal day (P) 3 brainstem–spi- nal cord preparation in (A) control, (B) with bath application of DAMGO (300 nM), and (C) subsequent bath application of ­befiradol (10 μM) in the continued presence of DAMGO (300 nM). (D) Population data with brainstem–spinal cord (300 nM DAMGO, 1 to 30 μM befiradol, n = 6 each) or medullary slice (300 nM DAMGO, 3 to 30 μM befiradol, n = 5 each). There is no significant dif- ference in respiratory frequency after befiradol using one-way repeated-measures ANOVA (Tukey method). DAMGO =D -Ala2, N-MePhe4, Gly-ol-enkephalin. bathing medium was compared in the absence or presence of neurochemical control of respiration.30,31 Similar to past DAMGO with paired t test, or one-way repeated-measures studies, the respiratory frequency was markedly suppressed by ANOVA (Tukey method; fig. 2), respectively. The nature the bath application of DAMGO (300 nM) to 31.9 ± 6.8% of the hypothesis testing is two tailed. For in vivo fentanyl (n = 6, P < 0.001; fig. 2) of control levels observed before 32 experiments, the significance of changes in fR, VT, VE, and DAMGO. Subsequent bath application of befiradol (1 to o Sa 2 after treatment was compared with two-way repeated- 30 μM) in the presence of DAMGO did not alleviate the measures ANOVA (dose × time; Holm–Sidak method; suppression of respiratory frequency (31.1 ± 7.1% of control, figs. 3 and 4). The significance of changes in the duration P = 0.99, n = 6; fig. 2). The amplitude of motor nerve dis- of fentanyl-induced analgesia (fig. 5) or sedation (fig. 6) charge generated by BSSC preparations was not affected by was compared between saline- and befiradol-treated groups DAMGO nor subsequent application of befiradol (n = 6). with t test. To examine if the effects of befiradol on fentanyl- The administration of befiradol on its own did not signifi- induced respiratory depression (or sedation) were correlated cantly alter the control values of respiratory frequency or with its effects on analgesia, we used the Pearson product amplitude of motor nerve discharge generated by BSSC moment test (figs. 5 and 6). For experiments examining the preparations (1 to 30 μM, n = 5). This is consistent with effects of befiradol on baseline parameters, the significance previous studies of the 5-HT1AR agonist, 8-OH-DPAT, in V or latency of paw withdrawal response to 33–35 of changes in E rat and mouse in vitro preparations. thermal nociceptive stimulus was compared between saline- The medullary slice preparation is a derivative of the and befiradol-treated group with t test (fig. 7). P valueless BSSC preparation.2 Similarly to past studies, the frequency than 0.05 or critical level was taken as significant difference of rhythmic respiratory discharge in medullary slice prepara- for two comparison (t test, paired t test, and Pearson product tions was markedly suppressed by the bath application of moment test), multiple comparisons (ANOVA), respectively. DAMGO (300 nM, n = 5) to 41.1 ± 5.4% of control levels before DAMGO (P < 0.001), consistent with our previous Results study.32 Subsequent bath application of befiradol (3 to 30 Befiradol Has No Effects on the Fentanyl-induced μM, 38.8 ± 6.3% of control before DAMGO, n = 5) in the Respiratory Depression or Baseline Respiratory Rhythm presence of DAMGO did not alleviate the respiratory depres- In Vitro sion (P = 0.96, fig. 2D). The amplitude of inspiratory motor We initiated our study using standard in vitro newborn rat nerve discharge generated by medullary slice preparations preparations that have been used extensively to examine the was not affected by DAMGO and subsequent application of

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Fig. 3. Effects of befiradol on fentanyl-induced respiratory depression in newborn rats. (A and B) Representative continuous whole body plethysmographic recordings from two postnatal day (P) 3 pups. Administration of fentanyl (35 μg/kg, neck subcu- taneously) caused a marked depression of respiratory frequency and tidal volume 5 min within fentanyl. Respiratory variables were measured before fentanyl, and 5 to 35 min after administration of fentanyl. Top trace: before fentanyl administration; middle trace: 5 min after fentanyl; and bottom trace: 10 min after fentanyl with saline (A) or befiradol (0.6 mg/kg, neck subcutaneously) (B), injected 5 min after administration of fentanyl. (C) Population data showing the time course of relative respiratory frequency

(fR), tidal volume (VT), and minute ventilation (VE) after injection of saline or befiradol (0.6 mg/kg, neck subcutaneously) approxi- mately 5 min after fentanyl infusion. Respiratory variables (fR, VT, and VE,) are presented as % relative to control (i.e., prefentanyl administration). Each animal tested only once with vehicle or befiradol. *Significant difference, compared with vehicle control group using two-way repeated-measures ANOVA (Holm–Sidak methods). befiradol (n = 5). The administration of befiradol on its own of control, P = 0.016), and VE (29.3 ± 3.1% of control vs. did not significantly alter the control values of respiratory saline: 12.4 ± 2.8% of control, P = 0.009). frequency or amplitude of motor nerve discharge generated by medullary slice preparations (3 to 30 μM, n = 5). Befiradol Alleviates the Fentanyl-induced Respiratory Depression in In Vivo Adult Rats Befiradol Alleviates the Fentanyl-induced Respiratory The next series of experiments was performed in in vivo adult Depression in In Vivo Neonatal Rats rats to determine the dose-dependent efficacy of the befiradol, The next stage of the study was to examine befiradol in vivo. to counter the fentanyl-mediated respiratory depression. Fen- Toward correlating with the in vitro studies, we first exam- tanyl (60 μg/kg, iv) was delivered to adult rats over a 20-min ined the effects of befiradol (0.6 mg/kg, fig. 3) on respira- infusion period (fig. 4). This induced a marked suppression tory depression induced by fentanyl (35 μg/kg, n = 5) in of fR (44.8 ± 5.3% of control), VT (49.8 ± 4.2% of control), o neonatal rats in vivo. This induced a marked suppression of VE (22.6 ± 3.2% of control), and Sa 2 (51.6 ± 5.3% of con- fR (31 ± 2% of control), VT (55.4 ± 6.3% of control), and VE trol) at approximately 6 min after fentanyl administration (17.2 ± 2.1% of control) approximately 5 min postadminis- (n = 9). Subsequent injection of saline vehicle (fig. 4A) did tration. The fentanyl-induced respiratory depression lasted not change the course of fentanyl action (fig. 4C–F). In con- for over 35 min after subsequent saline administration. trast, subsequent injection of befiradol (0.1 to 0.4 mg/kg, iv,

Administration of befiradol (0.6 mg/kg, at ~5 min postfen- n = 16, fig. 4B) caused a dose-dependent increase in fR, VT, tanyl) alleviated the fentanyl-induced respiratory depression and VE. Whereas a low dose of befiradol (0.1 mg/kg, n = 5) over 20 min (fig. 3). Specifically, alleviation of fentanyl- alleviated the fentanyl-induced respiratory depression for less induced respiratory depression by befiradol was observed than 10 min, the effects of high doses of befiradol (0.2 mg/ within 5 min after befiradol, as evidenced by increased kg, n = 6 and 0.4 mg/kg, n = 5) lasted beyond the dura- fR (39.2 ± 3.3% of control vs. saline: 24.2 ± 3.4% of control, tion of the fentanyl infusion (fig. 4C–F). Specifically, 4 min P = 0.014), VT (76.1 ± 6.2% of control vs. saline: 52.2 ± 7.5% after befiradol, VE was 37.4 ± 4.8% of control (0.1 mg/kg,

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Fig. 4. Effects of befiradol on fentanyl-induced respiratory depression in adult rats. (A–B) Representative continuous whole body plethysmographic recordings from two adult rats. A 20-min infusion of fentanyl (60 μg/kg, iv) caused a significant depres- sion of respiratory frequency (fR) and tidal volume (VT). (A) Saline or (B) befiradol (0.2 mg/kg, iv, bolus) was injected approxi- mately 6 min after administration of fentanyl. Respiratory variables and oxygen saturation were measured before fentanyl, and 5 to 20 min after administration of fentanyl. Nociceptive testing via tail clamping was performed starting approximately 11 min after fentanyl administration. (C–F) Dose-dependence data showing the relative respiratory frequency (fR, C), tidal volume (VT, D), minute ventilation (VE, E), and oxygen saturation (Sao2, F) after injection of vehicle or increasing doses of befiradol approximately 6 min after fentanyl infusion. Respiratory variables (fR, VT, and VE,) are presented as % relative to control (i.e., prefentanyl administration). Each animal tested only once with vehicle or one dose of befiradol. *Significant difference, com- pared with vehicle control group; #compared between two doses of befiradol-treatment groups; and using two way repeated measures ANOVA (Holm–Sidak methods). n = 5, P < 0.001), 53.7 ± 5.7% of control (0.2 mg/kg, n = 6, ± 0.07°C, n = 5) was not significantly different (P = 0.14) P < 0.001), and 65.8 ± 5.1% of control (0.4 mg/kg, n = 5, from saline treatment (−0.65° ± 0.06°C, n = 4). P < 0.001), significantly increased from 18.7 ± 2.2% of control o in the saline group (n = 9). Four minutes after befiradol, Sa 2 Befiradol Decreases the Fentanyl-induced Analgesia was 59.5 ± 3.5% (0.1 mg/kg, n = 5, P = 0.013), 75.4 ± 3.7% We then determined whether befiradol, at the doses of alle- (0.2 mg/kg, n = 6, P < 0.001), and 78.5 ± 3.1% (0.4 mg/kg, viating the fentanyl-induced respiratory depression, affected n = 5, P < 0.001), significantly increased from 45.7 ± 4.8% in fentanyl-induced analgesia. Figure 4 shows measures of anal- the saline group (n = 9). Note the decrease of body temperature gesia by examining responses to tail clamping with forceps at 4 min after befiradol treatment (0.2 to 0.4 mg/kg, −0.48° at 3 to 5 min intervals starting at approximately 11 min after

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Fig. 5. Effects of befiradol on fentanyl-induced analgesia in adult rats. (A) The duration (min) of analgesia-caused by fentanyl (60 μg/kg for 20-min iv infusion) was measured from the beginning of fentanyl administration to the time when a positive response to nociceptive stimulus occurred. A positive response to pinching the tail with forceps was based on observing obvious changes in at least two of following: heart rate, oxygen saturation, respiratory frequency, and body movement. Positive paw withdrawal response to thermal nociceptive stimulus is defined as a withdrawal in less than 8 s. Saline or befiradol (0.2 mg/kg, iv, bolus) was given approximately 6 min after fentanyl. *P < 0.05, significant difference between two groups using t test. Each data point from left to right bars is from 6, 5, 9, and 6 animals, respectively. (B) Correlation between fentanyl-induced analgesia and respiratory depression: duration of fentanyl-induced analgesia plotted against the fentanyl-induced suppression of minute ventilation (VE at ~10 min after fentanyl, relative to control, and before fentanyl) in saline-treated (n = 9) or befiradol-treated (0.1 to 0.6 mg/kg, iv, bo- lus, n = 12) groups. Correlation coefficients were 0.67, P = 0.048 and 0.74, P = 0.006 for saline and befiradol groups, respectively.

Fig. 6. Effects of befiradol on fentanyl-induced sedation in adult rats. (A) The duration (min) of sedation caused by fen- Fig. 7. Effects of befiradol on baseline breathing and noci- tanyl (60 μg/kg for 20 min iv infusion) was measured from the ception in normal adult rats. Minute ventilation (VE) and no- beginning of fentanyl administration to the time when the ani- ciception (latency of paw withdrawal response to thermal mal regained a righting reflex from the prone to supine posi- nociceptive stimulus) relative to control (i.e., before saline or tion. Saline (n = 9) or befiradol (0.2 mg/kg, iv, bolus, n = 6) befiradol 0.2 mg/kg) were measured. *P < 0.05, significant was administered approximately 6 min after fentanyl. *P < difference between two groups, with t test. Each data point 0.05, significant difference between two groups using t test. is six animals. (B) Correlation between fentanyl-induced analgesia and seda- tion: duration of fentanyl-induced analgesia plotted against = 0.011, fig. 5A). To establish whether there is a correla- the duration of fentanyl-induced sedation in saline-treated tion between fentanyl-induced respiratory depression and (n = 9) or befiradol-treated (0.1 to 0.6 mg/kg, iv, bolus, n = 12) analgesia, we plotted suppression of V against duration of groups. Correlation coefficients were 0.75, P = 0.019 and 0.76, E P = 0.004 for saline and befiradol groups, respectively. analgesia in vehicle groups (fig. 5B). The correlation coef- ficient was 0.67 (n = 9, P = 0.048, Pearson product moment fentanyl administration. No positive response to tail clamp- test), suggesting that fentanyl-induced respiratory depres- ing was observed during fentanyl administration in saline- sion and analgesia increase together. We then plotted the treated rats (fig. 4A, n = 6). However, a positive response to same relationship based on data obtained in the presence of tail clamping was observed during fentanyl administration befiradol to determine if 5HT1AR antagonism changes the in two of five befiradol (0.2 mg/kg, fig. 3B)-treated rats. The correlation (fig. 5B). The correlation coefficient was 0.74 onset of a positive response to tail clamping was signifi- for befiradol (0.1 to 0.6 mg/kg, n = 12, P = 0.006, Pear- cantly shortened by befiradol (0.2 mg/kg, 28.4 ± 6.8 min, son product moment test). All data demonstrated that the n = 5 vs. saline 52.4 ± 5.2 min, n = 6; P = 0.019 fig. 5A). positive correlation between fentanyl-induced respiratory The duration of analgesia based on paw withdrawal data depression and analgesia remains in the presence of befira- was also significantly shortened by befiradol (0.2 mg/kg, dol; that is, that befiradol reduces respiratory depression but 90.4 ± 11.6 min, n = 6 vs. saline 130.5 ± 7.8 min, n = 9; P also reduces analgesia.

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Befiradol Alleviates the Fentanyl-induced Sedation Discussion We found that 0.6 mg/kg befiradol caused a rapid (within The major findings of this study were that befiradol: (1) 2 min after injection, at ~6 min after fentanyl) loss of fen- dose dependently countered fentanyl-induced respiratory tanyl-induced sedation in two of four rats. These two rats depression; (2) reduced fentanyl-induced analgesia; (3) had full recovery of ventilation with 2 min after injection. reduced fentanyl-induced sedation; and (4) induced hyper- However, the loss of sedation prevented any further data col- ventilation, hyperalgesia, and “behavioral syndrome” in lection from those two rats due to disruption of continuous nonsedated rats. Further, the befiradol-induced alleviation infusion of fentanyl. Fentanyl-induced duration of seda- of opioid-mediated respiratory depression involves sites or tion was significantly shortened in befiradol-treated group mechanisms not present or functional in the isolated brain- (0.2 mg/kg, 39.8 ± 4 min, n = 6 vs. saline 58 ± 4.4 min, n = stem–spinal cord and medullary slice preparations. 9; P = 0.013, t test, fig. 6A). We then determined whether The role of 5-HT in modulating respiration has been Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/2/424/267207/20150200_0-00031.pdf by guest on 27 September 2021 there is a correlation between fentanyl-induced respiratory extensively studied and its relative importance debated.36 sedation and analgesia. Figure 6B shows fentanyl-induced Although the contention that 5-HT is essential for eupnea duration of sedation plotted against duration of analge- and gasping has been disproven,37,38 modulation sia (measured with paw withdrawal to thermal stimulus) of ventilation is very potent and warrants consideration as a in vehicle groups. The correlation coefficient was 0.75 (n prime target for development of pharmacological therapies = 9, P = 0.019, Pearson product moment test), suggest- to counter respiratory depression. For example, the 5-HT1AR ing that fentanyl-induced respiratory sedation and analge- agonist F15599 stabilizes respiratory rhythm in mouse mod- sia tended to increase in parallel. Further, we plotted the 39 els of Rett syndrome. Early reports of the 5-HT4AR agonist same relationship based on data obtained in the presence countering opioid-induced respiratory depression in mouse of befiradol to determine if 5HT R agonism changes the 40 1A models showed promise. However, studies with a 5-HT4A correlation (fig. 6B). The correlation coefficient was 0.76 agonist suitable for clinical use, , did not show for befiradol (0.1 to 0.6 mg/kg, n = 12, P = 0.004). All efficacy in rodent models and a human study.41 That line of data demonstrated that the positive correlation between investigation may require development of improved 5-HT4A fentanyl-induced sedation and analgesia remains in the agonists. The results with 5-HT1AR agonists, as discussed in presence of befiradol; that is, that befiradol reduces seda- the Introduction, have been mixed in rodent models. Fur- tion but also reduces analgesia. ther, a high dose of bisporone (60 mg, oral) was ineffective at reversing -induced respiratory depression in a Befiradol Causes Hyperalgesia, Hyperventilation, and human study.10 However, with the emergence of more selec- Behavioral Syndrome in Normal Adult Rats tive and efficacious 5-HT1AR agonists, such as befiradol and The final series of experiments focused on the effects on related compounds, there is an impetus for further preclini- baseline behaviors of befiradol (0.2 mg/kg, neck subcuta- cal and clinical studies. neously). With thermal nociceptive testing, the latency of Alleviation of fentanyl-induced respiratory depression by paw withdrawal was 5.4 ± 0.5 s before saline (n = 6) treat- befiradolin vivo in neonatal rats was robust. That is why the ment. When the animals were treated with saline (n = 6), lack of effect of befiradol on the DAMGO-induced respira- there was no difference in the latency of paw withdrawal to tory depression observed in vitro was unexpected. thermal stimulus (99.1 ± 5.4% relative to that before saline, act at multiple sites within the central nervous system to P = 0.79, paired t test). When the animals were treated depress respiratory drive,42 including directly via μ-opiate with befiradol (n = 6), the latency of paw withdrawal was receptors within the preBötC in vitro and in vivo.32,43 The shortened (79.1 ± 6.8% relative to that before befiradol, P other major class of drugs used to counter opiate-induced = 0.018, paired t test). There was a significant difference respiratory depression, ampakines, induces a clear reversal between the two groups after befiradol treatment versus of DAMGO-induced respiratory depression in vitro.6 Data saline treatment (P = 0.045, t test, fig. 7). With Buxco examining the interactions between 5-HT1AR agonists and plethsymographic recording, there was no difference in VE opioids, which are limited to analysis of in situ working after saline treatment (101.7 ± 3.9% relative to that before heart brainstem preparation and in vivo (often anesthetized) saline, n = 6, P = 0.82, paired t test). But befiradol-treated models, show a clear 5-HT1AR-mediated reversal of opiate- animals had an increased VE (n = 6; 119.3 ± 6.8% relative induced respiratory depression. Within the brain, 5-HT1ARs to that before befiradol, P = 0.018, paired t test; P = 0.048 are mainly expressed in the hippocampus, lateral septum, compared with saline treatment, t test, fig. 7). All befiradol- cortical regions, raphe nuclei, hypoglossal nuclei, solitary treated animals (n = 6) had at least two aspects of “behav- tract nuclei, and respiratory regions, with a higher level of 44–46 ioral syndrome,” characterized by head weaving (two of 5-HT1ARs in neonatal period (i.e., hypoglossal nuclei). six), forepaw treading (two of six), flat body posture (three The mechanisms proposed include 5-HT1AR agonist actions of six), and lower lip retraction (four of six). None of six on post-I or late expiratory neurons via –coupled saline-treated animals displayed characteristics consistent inwardly rectifying potassium channels, and glycinergic with “behavioral syndrome.” inhibitory neurons within the preBötC.47,48 Why those

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mechanisms might not be functional in vitro is not clear. The in untreated animals suggests that the effect on fentanyl- medullary slice contains only a subset of medullary respira- treated animals may be mechanistically unrelated to the tory nuclei and a limited repertoire of neuronal populations opioid effect. The lack of effect of befiradol on spinal and based on discharge patterns. Further, chloride-mediated medullary respiratory function with in vitro preparations conductances are likely altered in medullary slice prepara- would seem to support this conclusion. However, these data tions due to the elevated extracellular potassium ion con- do not allow for determination of the mechanisms underly- centration typically used to enhance neuronal and network ing robust reversal of opioid-induced respiratory depression, excitability.31,49 In contrast, the BSSC preparation is main- analgesia, and sedation by befiradol. tained under physiological potassium ion concentration conditions. Detailed extracellular recordings of neuronal Clinical Implications activity in the ventrolateral reticular formation, includ- Developing alternatives to naloxone for reversing opioid- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/122/2/424/267207/20150200_0-00031.pdf by guest on 27 September 2021 ing the preBötC region, demonstrated at least five distinct induced respiratory depression is a clear unmet clinical need. classes of respiratory neurons that are active during either I Ampakines are effective at reducing opioid-induced respira- or E phases.28 Anatomically, the BSSC preparation has the tory depression without altering analgesia or baseline car- full complement of medullary nuclei. However, the extent diorespiratory parameters in all in vitro, in situ, and in vivo to which neuronal populations within the medulla are func- rodent models studied.6,7 Further, those findings translated tional is not clear, given the varied oxygen and pH profile in a human study of alfentanyl-induced respiratory depres- within the tissue.50 Further studies across multiple experi- sion.55 A limitation of ampakines is that only oral formula- mental models will be necessary to clarify what mechanistic tions are available for clinical trials and they are associated substrate of 5-HT1AR action is absent in vitro. with a significant delay in ampakines reaching plasma thera- In contrast, the dose-dependent alleviation of fentanyl- peutic levels (>1 h). Although useful for a variety of clinical induced respiratory depression by befiradol in vivo was settings, it will be important to have injectable formula- clear. There was also a reduction in the duration of fentanyl- tions for acute intervention. An injectable formulation of induced sedation by befiradol that is similar to findings from the ampakine CX1942 is under development, but it has not advanced beyond the preclinical stage of testing. The addi- previous studies of 5-HT1AR agonists and opioids (8-OH- DPAT).51 There are some clinical situations where that could tional development of serotonergic agents for drug-induced be clearly advantageous.52 However, some actions of befira- and disease-related (e.g., Rett syndrome) is clearly warranted. dol are likely to be problematic. The analgesia induced by There may be cases where targeting of AMPA or 5-HT recep- fentanyl was reduced by befiradol. Although not as complete tor systems is more effective for stabilizing central respiratory as that caused by naloxone, the depreciation of analgesia is drive. Data from this study of the highly selective 5-HT1AR a counter indication. This finding is consistent with previ- agonist befiradol demonstrates its efficacy in reversing respi- ratory depression, but also the partial loss of analgesia. The ous reports of 5-HT1AR agonists attenuating opioid-induced analgesia in unanesthetized rodents.16–19 potential problems of altered baseline respiratory function, Additional challenges associated with using befiradol for behavior, and hyperalgesia are additional concerns that will the therapeutic alleviation of fentanyl-induced respiratory require monitoring. Future studies of related compounds, depression include the alteration in baseline respiration, F13714 and F15599,56,57 that preferentially bind to pre- and nociception, and behavior. Befiradol induced an approximate postsynaptically located 5-HT1ARs, respectively, may have 20% increase in baseline ventilation that would be experi- an enhanced therapeutic profile. enced by patients if befiradol was given (1) before fentanyl to decrease the ensuing severity of respiratory depression or Acknowledgments (2) subsequent to fentanyl to alleviate respiratory depression The authors thank Monica Gorassini, Ph.D. (Department of if the befiradol action persisted longer than those of fentanyl. Biomedical Engineering), and Karim Fouad, Ph.D. (Faculty The potential for the induction of “behavioral syndrome” of Rehabilitation Medicine), University of Alberta (Edmon- ton, Alberta, Canada), for the loan of essential equipment. was consistent with studies examining 5-HT1AR agonist Befiradol was generously provided by Mark Varney, Ph.D. 53 8-OH-DPAT. An additional increase in nociception (Neurolixis Inc., San Diego, California). observed would also be problematic. Our finding of an early Supported by the Canadian Institutes of Health (Edmon- pronociceptive effect of befiradol is consistent with previous ton, Alberta, Canada). studies in rat that reported an initial 2-h phase of hyperal- gesia followed by hypoalgesia 8 h later.23,24,54 However, only Competing Interests the initial 2 h of hyperalgesia was related to the current study The authors declare no competing interests. of acute effects of 5-HT1AR agonists on nociception in nor- mal rats. Notably, there have been reports of an increase in Correspondence baseline nociception in unanesthetized rodent models after Address correspondence to Dr. Greer: Department of Physi- 17,22,23 administration of other 5-HT1AR agonists. The find- ology, Neuroscience, and Mental Health Institute, University ing that befiradol induced hyperventilation and hyperalgesia of Alberta, 4–142 Katz Bldg, Edmonton, Alberta, Canada T6G

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