Baclofen-Induced Neuro-Respiratory Toxicity in the Rat: Contribution of Tolerance and Characterization of Withdrawal Syndrome

Baclofen-Induced Neuro-Respiratory Toxicity in the Rat: Contribution of Tolerance and... Abstract Baclofen, a γ-amino-butyric acid type-B receptor agonist with exponentially increased use at high-dose to facilitate abstinence in chronic alcoholics, is responsible for increasing poisonings. Tolerance and withdrawal syndromes have been reported during prolonged treatment but their contribution to the variability of baclofen-induced neurotoxicity in overdose is unknown. We studied baclofen-induced effects on rat sedation, temperature, and ventilation and modeled baclofen pharmacokinetics and effect/concentration relationships aiming to investigate the consequences of repeated baclofen pretreatment and to characterize withdrawal syndrome. Baclofen-induced dose-dependent sedation (p <0.01), hypothermia (p <.001) and respiratory depression (p <.01) were altered in repeatedly baclofen-pretreated rats (p <.05). Repeatedly baclofen-pretreated rats did not exhibit respiratory depression following baclofen overdose due to limitations on baclofen-induced increase in inspiratory (p <.01) and expiratory times (p <.01). Only slight hypoxemia without respiratory acidosis was observed. Baclofen discontinuation resulted in hyperlocomotion and non-anxiogenic withdrawal symptoms. Regarding pharmacokinetics, repeated baclofen pretreatment increased the peak concentration (p <.05) and absorption constant rate (p <.05) and reduced the distribution volume (p <.0001) and elimination half-life (p <.05). Analysis of the effect/concentration relationships indicated that plasma baclofen concentration decreases more rapidly than all studied neuro-respiratory effects, in tolerant and non-tolerant rats. Taken together, our findings supported the role of brain distribution in baclofen-induced neurotoxicity expression and its probable involvement in tolerance-related attenuation in addition to physiological adaptations of ventilation. In conclusion, repeated pretreatment attenuates baclofen-attributed neurotoxicity in overdose and results in post-discontinuation withdrawal syndrome. Our findings suggest both pharmacodynamic and pharmacokinetic mechanisms whose relative contributions to the variability of baclofen-induced neurotoxicity in overdose remain to be established. baclofen, tolerance, respiratory depression, pharmacokinetics, poisoning, withdrawal Baclofen is a selective γ-amino-butyric acid type-B receptor (GABAB-R) agonist approved for the treatment of spasticity (Kumar et al., 2013). Recently, despite the absence of solid evidence to support its efficiency, baclofen has been used at high-dose to facilitate abstinence in dependent chronic alcoholics (Liu and Wang 2017; Reynaud et al., 2017). Following the publication in 2008 of “Le Dernier Verre” [The Last Glass], a book by Dr. Amsein (2008) reporting the successful self-management of his alcohol dependence with extremely high-dose baclofen (up to 270 mg/day), off-labeled prescriptions exponentially increased with an estimated 213 000 treated patients between 2009 and 2015 in France (French National Agency for Medicines and Health Products Safety, 2017). Although clinical studies showed that high-dose baclofen can be used with manageable side-effects (Müller et al., 2015; Reynaud et al., 2017; Rigal et al., 2015), the French health authorities reported dose-dependent risk of hospitalization (15% and 46% increase at 75–180 and >180 mg daily doses, respectively) and death (1.5- and 2.27-fold increase for the same dose intervals) as compared to other approved medications used to treat ethanol dependence (French National Agency for Medicines and Health Products Safety, 2017). With the increasing use of high-dose baclofen, accidental and self-poisonings dramatically rose. From January 2008 to December 2013, 294 baclofen toxic exposures in alcohol-dependent patients were reported to the French Poison control Centers with 132 severe poisonings and 9 fatalities (Pelissier et al., 2017). Baclofen overdose is responsible for life-threatening central nervous system (CNS) toxicity including coma, respiratory depression, flaccidity, areflexia, hypothermia, hypotension, and seizures (Franchitto et al., 2014; Léger et al., 2017). In addition, severe electroencephalographic abnormalities including isoelectric signal mimicking brain death have been reported (Sullivan et al., 2012). Baclofen poisoning has been reported in previously untreated (‘acute poisoning’) and treated patients (’acute-on-chronic poisoning’). Whether the severity of acute poisoning depends on previous baclofen treatment has not been established, although tolerance development was suggested under chronic baclofen treatment with the onset of transient side-effects like somnolence, asthenia, and confusion appearing during the titration period to reach the targeted dose and disappearing later on (Müller et al., 2015; Rigal et al., 2015; Reynaud et al., 2017). In addition, symptoms suggestive of withdrawal syndrome including hallucinations, agitation, delirium, psychosis, seizures, and even features mimicking neuroleptic malignant syndrome have been observed in chronically baclofen-treated patients (Peng et al., 2008; Richter et al., 2016). However, the time-course of such symptoms and their relationships to the discontinuation of prior baclofen treatment still raise questions. The exact mechanisms of baclofen-induced neuro-respiratory toxicity in overdose have been poorly investigated. The contribution of tolerance to the expression of acute toxicity remains unknown. We thus designed an experimental study in the rat aiming to (1) describe baclofen-induced neuro-respiratory toxicity in overdose; (2) investigate the consequences of tolerance acquisition on baclofen-related toxicity, and (3) characterize withdrawal syndrome resulting from the discontinuation following prior prolonged baclofen administration. MATERIALS AND METHODS Experimental protocols were carried out within the ethical guidelines established by the National Institute of Health and approved by Paris-Descartes University ethics committee for animal experimentation (No. APAFIS#5285-2016050321031432v2). Animals Five- to seven-week-old male Sprague Dawley rats (Janvier-labs, France) weighing 250–350 g during experimentation were used and housed for 7 days before experimentation in an environment maintained at 21 ± 0.5°C with controlled humidity and a light–dark cycle. Food and water were provided ad libitum. Chemicals and Drugs Baclofen (Sigma-Aldrich, France) was diluted in saline to obtain a 2.5 mg/ml solution [used for intraperitoneal administration] or in 10% Tween (Tween-20 diluted in 0.9% NaCl) to obtain a 10 mg/ml solution [used for intragastric administration]. Femoral Artery and Jugular Venous Catheterization The day before the experiment, animals were anesthetized with intraperitoneal 70 mg/kg ketamine (Clorketam 1000) and 10 mg/kg xylazine (Rompum) then placed on a warming blanket with regulating thermostat. The femoral artery (to allow arterial blood gas measurement) or the jugular vein (to allow plasma baclofen concentration measurement) was catheterized using 30 and 20-cm silastic tubing, respectively, with external and internal diameters of 0.94 and 0.51 mm, respectively (Dow Corning Co., Midland, MI). The arterial or venous catheter was subcutaneously tunneled and fixed at the back of the neck. Heparinized saline (100 UI/ml) was injected into the catheter to avoid thrombosis and catheter obstruction. Rats were then returned to their individual cages for a minimum of 24-h recovery period, to allow complete anesthesia washout. On the day of experimentation, rats were placed in horizontal Plexiglas cylinders (6.5 cm-internal diameter, up to 20 cm-adjustable length) (Harvard Apparatus, Inc., Holliston, MA). Before the drug administration, the catheter was exteriorized, purged, and its permeability checked. Clinical Parameters Temperature, sedation, seizures, and withdrawal signs were monitored by 2 researchers blinded to the rat group allocation. Temperature was measured using intraperitoneally implanted temperature transmitters for the purpose of plethysmography study. Sedation level based on a 4-stage scale from 0 (awake) to 3 (coma) was assessed (Pirnay et al., 2008). At stage 0, rats were completely awake and their gait and righting reflexes were intact. At stage 1, rats had reduced activity, showed light impairment of gait and an intact righting reflex with diminished muscle tonus. At stage 2, rats were asleep or static and showed a reduced righting reflex. At stage 3, rats were comatose and did not have any righting reflex. Seizure severity was graded according to the modified Racine Score (Racine, 1972). At stage 1, rats were immobile, with eyes closed, twitching of vibrissae and facial clonus. At stage 2, rats had head nodding associated with more severe facial clonus. At stage 3, rats had clonus of one forelimb. At stage 4, rats had rearing, often accompanied by bilateral forelimb clonus. At stage 5, rats had rearing with loss of balance and falling accompanied by generalized clonic seizures. All these parameters were measured before (T0) and at 5, 10, 15, 30, 60, 90, 120, 240, 360, 480, and 1440 min post-baclofen administration. Following chronic treatment discontinuation, physical dependence to baclofen was evaluated using Gellert’s withdrawal score (Table 1) (Gellert and Holtzman, 1978). Withdrawal signs were noted during a 5-min observation period each day (from days 1 to 12) after discontinuation of repeated baclofen. Table 1. Withdrawal Signs and Their Corresponding Weighting Factors (Signs Noted Simply as Present or Absent) Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Table 1. Withdrawal Signs and Their Corresponding Weighting Factors (Signs Noted Simply as Present or Absent) Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Behavioral Measurements Locomotor activity The open field (OPF) test used an enclosed white Plexiglas chamber open at the top, divided into 4 equal-sized areas (50 ×50 × 35 cm3) and maintained under low illumination (10 lx). At the experiment time, 1 rat was placed in each area and recorded during 15 min. The chamber was fitted with an infrared floor connected to a miniature overhead infrared video-camera and a computer that used automated video-tracking software (ViewPoint, Videotrack, France) to determine the rat horizontal locomotor activity (traveled distance in cm). Locomotor activity was measured on days 1, 5, and 12 after repeated baclofen discontinuation. Anxiety-like behavior The elevated plus-maze (EPM) test used a central platform (10 × 10 cm2) formed by black Plexiglas boards surrounded by 2 opposed highly illuminated (180 lx) open arms (50 × 10 cm2) and 2 opposed faintly illuminated (10 lx) enclosed arms (50 × 10 cm2 with 45 cm-high walls). The maze was placed 50 cm above an infrared floor. At the experiment time, each rat was placed in the central platform facing the open arm away from the experimenter. The rat behavior was recorded by a miniature overhead infrared video-camera over a 5 min-period. The number of entries and time spent in each arm were tracked using Viewpoint software. Results are expressed as the percentage of number of entries and time spent in the open arms. Anxiety-like behavior was measured on day 6 after discontinuation of repeated baclofen. Whole Body Plethysmography Four days before the study, temperature transmitters (TA-F10, DSI, the Netherlands) were implanted in the peritoneal cavity. Ventilatory parameters were recorded in a whole body plethysmograph by the barometric method described and validated in the rat (Bartlett and Tenney, 1970). The first measurements were performed after a 30-min period of accommodation at −30, −15, and −5 min, while the rat was quiet and not in deep or rapid eye movement sleep, as roughly estimated from their behavior, response to noise, and pattern of breathing. Then, the rat was gently removed from the chamber for baclofen intragastric administration at T0, and replaced in the chamber for the remaining measurements. Ventilation was recorded at 5, 10, 15, 30, 60, 90, 120, 240, 360, 480, and 1440 min, each record lasting about 60 s. The following parameters, ie, the tidal volume (VT), the inspiratory time (TI) and the expiratory time (TE), were measured using ELPHY software (ElectroPhysiological Software Version 3.1.0.38, CNRS-UNIC, France). Additional parameters including the respiratory frequency (f = 1/(TI + TE)) and the minute volume (VM = VT × f) were calculated. Arterial Blood Gas and Lactate Measurement To investigate arterial blood gas, 200 µl of arterial blood were sampled from catheterized rats and immediately analyzed using the Vetstat system (IDEXX, France). To measure lactate, a blood drop was deposited on a lactate testing strip and immediately analyzed using Lactate Scout (ERF diagnostic, France). Arterial pH, PaO2, PaCO2, bicarbonate, and lactate concentrations were measured before (T0) and at 1, 2, 4, 6, and 8 h after intragastric baclofen administration to study its effects on ventilation as well as on the acid-base balance. Since significant changes in the rat core temperature were observed, arterial blood gases corrected at the actual temperature of the animals were interpreted. 200 ml of heparinized 0.9% NaCl were administered every hour to the rat via the arterial catheter to reduce the risk of catheter clotting and to compensate for volume loss. Plasma Baclofen Concentration Measurement Following high-dose baclofen administration, 300 µl of blood were sampled from catheterized rats before (T0) and at 5, 15, and 30 min and at 1, 2, 4, 6, 8, 10, 24, and 28 h after intragastric baclofen administration. Blood samples were collected in Eppendorf tubes containing 30 µl of sodium heparin (Panpharma, Sanofi Winthrop, France). After centrifugation (5000 rpm, 10 min), plasma was separated and stored at −20°C before analysis. Plasma baclofen concentration was measured using liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS) following a simple one-step protein precipitation (Labat et al., 2016). Chromatographic separation was performed on an Accucore PFP column (100 × 2.1 mm; 2.6 μm, Thermo Scientific, Belgium) at 30-Celsius degrees using gradient elution with a mobile phase consisting of water and acetonitrile (0.1% formic acid). MS analysis was performed on Quantum Ultra® (Thermo Fisher Scientific, Germany) using electrospray ionization with quantification transition m/z 213.9/196.9 for baclofen and 217.9/200.9 for baclofen-d4 (internal standard). The method was validated in plasma with linear response for concentrations ranging between 10 and 2000 µg/l (r2 > .999) and precisions and accuracies below 11.5%. Modeling of Baclofen Pharmacokinetics Plasma baclofen pharmacokinetic parameters after intragastric administration were assessed using one-compartment analysis (WinNonlin v.5.3, Certara, NJ) including the peak plasma concentration (Cmax), the time to achieve the Cmax (Tmax), the area under the concentration-time curve from 0 to infinity (AUC0→∞), the absorption rate constant (ka), the volume of distribution (VD/F), the total clearance (Cl/F), the terminal elimination rate constant (ke) and the elimination half-life (t1/2). Study Design Study 1—Baclofen-induced neuro-respiratory effects Rats were randomized in 3 groups (N = 6 rats/group) to receive 43.5, 72.5, and 116 mg/kg baclofen by gavage, corresponding to 30%, 50%, and 80% of the lethal dose-50% (LD50), ie, 145 mg/kg (Cayman chemicals, 2015). Subsequently, baclofen-induced effects on temperature, sedation, ventilation (using plethysmography), and blood lactate concentrations were measured. Study 2—Baclofen-induced tolerance Rats were randomized in 2 groups (N = 7–8 rats/group) to receive repeated intraperitoneal saline (acute poisoning group) or baclofen (acute-on-chronic poisoning group) 3 times per day for 15 consecutive days, with increasing baclofen doses every 3 days ranging from 5 to 15 mg/kg. On the morning of day 16, rats received 116 mg/kg baclofen by gavage. Subsequently, baclofen-induced effects on temperature, sedation, ventilation (using plethysmography and arterial blood gas analysis), and blood lactate concentrations were measured and plasma baclofen concentrations determined. Study 3—Baclofen-induced neurobehavioral effects during withdrawal Rats were randomized in 2 groups (N = 8 rats/group) to receive repeated intraperitoneal saline (control group) or baclofen (baclofen group) 3 times per day for 15 consecutive days, with increasing baclofen doses as previously described. Treatment was stopped on day 16. Baclofen withdrawal syndrome was investigated with the Gellert’s score and behavioral tests. Statistical Analysis Results are expressed as mean ± SEM and as mean ± SD for the PK study. For plethysmography, the value at T0 was the mean of the 3 baseline measurements. For each sedation, temperature, lactate concentrations, plethysmography, and blood gas measurement, baseline values (T0) were compared to the values at each sampling time using two-way analyses of variance (ANOVA) for repeated measures followed by multiple comparisons using Sidak’s correction. To permit the simultaneous analysis of the effect of time and treatments, we calculated for each animal and for each studied parameter, the area under the curve from T0 to the completion of the measurement (1440 min) using the trapezoid method (Studies 1 and 2). Thereafter, we compared the AUCs using linear regression (Study 1) and Mann-Whitney tests (Study 2). Two-way ANOVA for repeated measurements followed by multiple comparisons using Sidak’s correction were performed to compare body weight, sedation, temperature, lactate concentrations, plethysmography, and blood gas parameters (Study 2) and withdrawal scores (Study 3) between the study groups. Unpaired Student’s t tests were used to compare PK parameters (Study 2) and Mann-Whitney tests to compare locomotor activity and anxiety-like behavior (Study 3) at each measurement day between the study groups. All tests were performed using Prism version 6.0 (GraphPad Software). P-value < .05 was considered as significant. RESULTS Baclofen-Induced Neuro-Respiratory Effects (Study 1) Baclofen was responsible for significant dose-dependent early-onset sedation (p = .004) and hypothermia (p = .0002) in the rat (Figure 1). No seizures were observed. Based on plethysmography, baclofen induced a significant dose-dependent increase in TI (p = .0003), TE (p = .02) and VT (p = .004) resulting in a dose-dependent decrease in VM (p = .001) (Figure 2). Interestingly, no dose-effect relationship was observed regarding blood lactate measurements following baclofen administration. Figure 1. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A) and temperature (B) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Figure 1. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A) and temperature (B) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Figure 2. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on inspiratory time (A), expiratory time (B), tidal volume (C) and minute volume (D) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Figure 2. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on inspiratory time (A), expiratory time (B), tidal volume (C) and minute volume (D) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Baclofen-Induced Tolerance (Study 2) Clinical observations during repeated baclofen treatment Repeated baclofen administration significantly reduced weight gain along with food intake in comparison to the control (p < .05; Figure 3A). Significant decrease in sedation was observed on the third day of each baclofen dose step with no additional sedation at the 5th dose step in comparison to the 4th step (Figure 3B). Figure 3. View largeDownload slide Effects of repeated intraperitoneal administration of saline (dashed grey line) and baclofen (full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days in Sprague Dawley rats on body weight (A) and sedation (B) (N = 12/group). The arrow represents the first day for each dose step. Results are expressed as mean ± SEM. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. ##p <.01, ###p <.001, ####p <.0001. Figure 3. View largeDownload slide Effects of repeated intraperitoneal administration of saline (dashed grey line) and baclofen (full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days in Sprague Dawley rats on body weight (A) and sedation (B) (N = 12/group). The arrow represents the first day for each dose step. Results are expressed as mean ± SEM. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. ##p <.01, ###p <.001, ####p <.0001. Effects on baclofen-induced sedation In comparison to the baseline, baclofen significantly deepened sedation from 60 to 360 min in the acute-on-chronically versus 30–480 min in the acutely poisoned rats (p < .0001; Figure 4A). Sedation differed significantly between these 2 groups at 30, 360, and 480 min (p < .01) as supported by the AUC analysis (p < .001; Figure 4B). No seizures were observed in either rat group. Figure 4. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A, C), temperature (B, D) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A, B) and areas under the effect-time curve (AUC) determined (C, D). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. **p <.01, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001, ####p <.0001. Figure 4. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A, C), temperature (B, D) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A, B) and areas under the effect-time curve (AUC) determined (C, D). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. **p <.01, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001, ####p <.0001. Effects on baclofen-induced hypothermia Baclofen significantly reduced body temperature at 480 min in both groups (p < .01) without significant differences at each measurement time between the 2 groups (Figure 4C). However, temperature decrease was less marked in the acute-on-chronically poisoned rats, as evidenced by the AUC analysis (p < .05; Figure 4D). Effects on baclofen-induced respiratory depression Baclofen significantly prolonged TI from 60 to 480 min in the acute-on-chronically (p < .001) versus 60 min to 24 h in the acutely poisoned rats (p < .01; Figure 5A). TE significantly increased at 240 min in the acute-on-chronically but from 240 to 480 min in the acutely poisoned rats (p < .05; Figure 5B). VT significantly increased from 60 to 360 min in the acute-on-chronically (p < .01) versus 60–480 min in the acutely poisoned rats (p < .05; Figure 5C). Interestingly, VM was not significantly reduced in the acute-on-chronically as compared to the acutely poisoned rats exhibiting delayed VM reduction at 360 and 480 min (p < .05; Figure 5D). These effects were significantly more marked at 480 min and 24 h for TI and at 360 and 480 min for TE and VT (p < .01). VM significantly differed between the 2 groups at 480 min (p < .001). Consistently, baclofen pretreatment significantly reduced intensity and duration of all acute baclofen-induced effects on ventilation as demonstrated by the AUC analysis (p < .01; Figs. 5E–H). Figure 5. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on the plethysmography parameters including inspiratory time (A, E), expiratory time (B, F), tidal volume (C, G) and minute volume (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *P <.05, **P <.01, ***P <.001, ****P <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #P <.05, ##P <.01, ###P <.001, ####P <.0001. Figure 5. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on the plethysmography parameters including inspiratory time (A, E), expiratory time (B, F), tidal volume (C, G) and minute volume (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *P <.05, **P <.01, ***P <.001, ****P <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #P <.05, ##P <.01, ###P <.001, ####P <.0001. Significant decrease in PaO2 was observed from 120 to 240 min in the acute-on-chronically (p < .01) versus 240–360 min in the acutely poisoned rats (p < .0001; Figure 6A). PaCO2 did not vary significantly in the acute-on-chronically poisoned rats while significantly increased from 240 to 360 min in the acutely poisoned rats (p < .001; Figure 6B). Similarly, arterial pH did not vary significantly in the acute-on-chronically but was significantly reduced from 240 to 360 min in the acutely poisoned rats (p < .01; Figure 6C). Blood bicarbonate concentration significantly increased at 240 min in the acute-on-chronically (p < .05) versus at 360 min in the acutely poisoned rats (p < .01; Figure 6D). These effects were significantly more marked at 240 and 360 min for PaO2 and pH (p < .01), at 240 min for bicarbonate (p < .01) and at 360 min for PaCO2 (p < .01). Consistently, baclofen pretreatment significantly diminished intensity and shortened duration of acute baclofen-induced effects on arterial blood gases as demonstrated by the AUC analysis (p < .01; Figs. 6E–H). No significant difference was observed in blood lactate concentration between the two rat groups based on the AUC analysis (data not shown). Figure 6. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on arterial blood gas parameters including PaO2 (A, E), PaCO2 (B, F), pH (C, G) and bicarbonate concentration (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *p <.05, **p <.01, ***p <.001, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001. Figure 6. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on arterial blood gas parameters including PaO2 (A, E), PaCO2 (B, F), pH (C, G) and bicarbonate concentration (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *p <.05, **p <.01, ***p <.001, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001. Effects on plasma baclofen pharmacokinetics and concentration-effect relationships The time-course of plasma baclofen concentrations is shown in Figure 7 and pharmacokinetic parameters in Table 2. When comparing the acute-on-chronically versus acutely poisoned rats, Cmax and ka were significantly increased (p < .05), VD/F reduced (p < .0001) and t1/2 shortened (p < .05). In contrast, AUCs and Cl/F were not significantly altered. Table 2. Pharmacokinetic Parameters Following Intragastric 116 mg/kg Baclofen Administration in Sprague Dawley Rats Pretreated With Repeated Intraperitoneal Saline (Acute Poisoning Group) or Baclofen (Acute-on-Chronic Poisoning Group) At Increasing Doses From 5 to 15 mg/kg Every 3 days During 15 Days Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters were determined using one-compartment model (N = 7/group) Values are expressed as mean ± SD. Comparisons between acute and acute-on-chronic poisoning groups were performed using Mann-Whitney tests, # p < .05, ## p < .01, #### p < .0001. Cmax, peak plasma concentration; Tmax, time to achieve the Cmax; ka, absorption rate constant; ke, elimination rate constant; VD/F, volume of distribution; Cl/F, total clearance; t1/2, terminal elimination half-life; AUC0→∞, area under the concentration-time curve from 0 to infinity. Table 2. Pharmacokinetic Parameters Following Intragastric 116 mg/kg Baclofen Administration in Sprague Dawley Rats Pretreated With Repeated Intraperitoneal Saline (Acute Poisoning Group) or Baclofen (Acute-on-Chronic Poisoning Group) At Increasing Doses From 5 to 15 mg/kg Every 3 days During 15 Days Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters were determined using one-compartment model (N = 7/group) Values are expressed as mean ± SD. Comparisons between acute and acute-on-chronic poisoning groups were performed using Mann-Whitney tests, # p < .05, ## p < .01, #### p < .0001. Cmax, peak plasma concentration; Tmax, time to achieve the Cmax; ka, absorption rate constant; ke, elimination rate constant; VD/F, volume of distribution; Cl/F, total clearance; t1/2, terminal elimination half-life; AUC0→∞, area under the concentration-time curve from 0 to infinity. Figure 7. View largeDownload slide Plasma baclofen concentration-time profiles following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats showing the observed (squares and circles) and predicted (dashed lines) data using one-compartment model (N = 7/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SD. Figure 7. View largeDownload slide Plasma baclofen concentration-time profiles following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats showing the observed (squares and circles) and predicted (dashed lines) data using one-compartment model (N = 7/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SD. For both rat groups, an anticlockwise hysteresis loop was obtained for sedation and a clockwise hysteresis loop for body temperature in relation to plasma baclofen concentrations (Figure 8). Regarding plethysmography parameters, anticlockwise hysteresis loops were obtained for TI, TE, and VT and a clockwise hysteresis loop for VM in the 2 groups. Regarding arterial blood gas parameters, anticlockwise hysteresis loops were obtained for PaO2 and arterial pH and clockwise hysteresis loops for PaCO2 and bicarbonate in the 2 groups. Figure 8. View largeDownload slide Effect/concentration relationships following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats for sedation (A), temperature (B), inspiratory time (C), expiratory time (D), tidal volume (E), minute volume (F), PaO2 (G), PaCO2 (H), arterial pH (I) and bicarbonate concentration (J) (N = 7–8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SEM. Figure 8. View largeDownload slide Effect/concentration relationships following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats for sedation (A), temperature (B), inspiratory time (C), expiratory time (D), tidal volume (E), minute volume (F), PaO2 (G), PaCO2 (H), arterial pH (I) and bicarbonate concentration (J) (N = 7–8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SEM. Baclofen-Induced Neurobehavioral Effects During Withdrawal (Study 3) Withdrawal score No withdrawal symptoms were noticed in control rats. In contrast, discontinuation after repeated baclofen administration was responsible for marked withdrawal scores (peak values on days 5 and 6) with significant differences in comparison to the controls on each day from days 1 to 9 post-baclofen discontinuation (p < .0001). Symptoms such as facial fasciculations, chromodacryorrhea, and escape attempts were present as early as the first day post-discontinuation while wet shakes, swallowing movements, diarrhea, and ptosis were observed on the following days. Locomotor activity The distance traveled based on the OPF test significantly decreased on day 1 (p < .01), increased on day 5 (p < .05) but did not significantly differ on day 12 between the baclofen-pretreated and the control rats (Figure 9A). The distance traveled was significantly increased in the baclofen-pretreated rats (p < .01) and decreased in the controls on day 5 as compared to day 1. On day 12, no significant difference was noted in the baclofen-pretreated rats as compared to day 1, whereas significant decrease was still observed in the controls (p < .001). Figure 9. View largeDownload slide Effects of baclofen discontinuation in Sprague Dawley rats after repeated intraperitoneal saline (crisscrossed grey lines) or baclofen at increasing doses from 5 to 15 mg/kg every 3 days (black) during 15 days on the distance traveled in the open-field (A) and on the percentage of entries (B) and time spent (C) in the open arms of the EPM (N = 8/group). Results are expressed as mean ± SEM. Mann-Whitney tests were performed to compare results between each day of discontinuation with the first day **p <.01, ***p <.001 and between the two groups ##p <.01, ###p <.001. Figure 9. View largeDownload slide Effects of baclofen discontinuation in Sprague Dawley rats after repeated intraperitoneal saline (crisscrossed grey lines) or baclofen at increasing doses from 5 to 15 mg/kg every 3 days (black) during 15 days on the distance traveled in the open-field (A) and on the percentage of entries (B) and time spent (C) in the open arms of the EPM (N = 8/group). Results are expressed as mean ± SEM. Mann-Whitney tests were performed to compare results between each day of discontinuation with the first day **p <.01, ***p <.001 and between the two groups ##p <.01, ###p <.001. Anxiety-like behavior Based on the EPM test, the percentage of number of entries (Figure 9B) and the time spent (Figure 9C) in the open arms were not significantly different between the 2 groups on day 6 post-baclofen discontinuation. DISCUSSION Baclofen overdose is responsible for dose-dependent sedation, hypothermia, and respiratory depression in the rat. Repeated pre-treatment induces tolerance to baclofen-induced neuro-respiratory effects in overdose and results in withdrawal syndrome post-discontinuation. Our rat model reliably reproduced life-threatening baclofen toxicity in humans (Franchitto et al., 2014). Behavior, sleep, and electroencephalographic activity impairments have been described in the rat following single or repeated <20 mg/kg baclofen (Beveridge et al., 2013; Hodor et al., 2015). Here, we studied regimens mimicking acute poisoning and prolonged high-dose treatment in humans. Baclofen overdose resulted in CNS depressant effects as previously established in humans (Rolland et al., 2015) and expected from its mechanism of action, mediated by the presynaptic GABAB-R activation which reduces excitatory neurotransmitter release. GABAB-Rs are involved in various structures including the brainstem ventilation control center, temperature-controlling hypothalamic center, and anterior horn α-motoneurons. Baclofen acts mainly centrally to depress ventilation although inspiratory muscle relaxing effects also contributes. Baclofen-mediated GABAB-R stimulation decreases glutamate release preventing N-methyl-d-Aspartate (NMDA) and non-NMDA-receptor stimulation on inspiratory neurons (Pierrefiche et al., 1993). Baclofen inhibits inspiratory neurons of the solitary tract nucleus within the pneumotaxic center involved in the automatic regulation of respiratory volume and prevents phrenic nerve discharges stimulating the diaphragm (Pierrefiche et al., 1993). Interestingly, baclofen was shown to induce selective TI lengthening without simultaneous TE change by modulating the Hering-Breuer inflation and deflation reflexes. Such distinct TI/TE responses are explained by distinct medullary pathway control reflexes with TI-promoting reflex exclusively mediated by high-threshold receptor activation (all-or-nothing response) and TE-promoting reflex depending on high- or low-threshold receptor activation (Seifert and Trippenbach, 1998). Another hypothesis was related to disparate GABAB-R distribution within the respiratory neuronal network. Here, high-dose baclofen induced both TI and TE increase suggesting that, like opioid receptors (Chevillard et al., 2010), GABAB-R-mediated effects on ventilation require higher ligand concentration to prolong TE than that which is necessary to prolong TI. We obtained tolerance to baclofen-induced neurotoxicity with repeated pretreatment. Interestingly, in our baclofen-pretreated rats, hypophagic effects resulting in weight gain reduction were observed as previously (Bains and Ebenezer, 2013; Patel and Ebenezer 2010). In obese baclofen-treated subjects, decreased appetite concomitantly with reduced weight, waist circumference, and adipose stores supported by decreased serum leptin were reported (Arima and Oiso, 2010). GABAB-Rs regulate food intake and weight control by inhibiting the potent orexigenic neuropeptide Y and hypothalamic proopiomelanocortin secretion (Bäckberg et al., 2003). Decreased appetite would thus be interesting to study in patients suffering from bulimia, as already suggested by on case report (Weibel et al., 2015). In humans, tolerance to baclofen-induced sedation was described (Rigal et al., 2015). In rats, tolerance was reported with various repeated low-dose regimens, to sedation (Bains and Ebenezer, 2013), hypothermia (Lehmann et al., 2003), hypolocomotion (Beveridge et al., 2013), changes in ankle torque hind limb posture and stretch reflex activity (Oshiro et al., 2010; Wang et al., 2002). Tolerance acquisition was supported by glucose metabolism decrease in the thalamus and raphe nuclei involved in the sleep/wake cycle regulation and the substantia nigra and cerebellum involved in motor skills (Beveridge et al., 2013). Tolerance acquisition was related to reduction in presynaptic GABAB-R activation (Hefferan et al., 2006). Interestingly, GABAB-R desensitization, demonstrated using GTP-γS binding assays (Keegan et al., 2015) was not associated with changes in GABAB-R subunit-coding mRNAs (Sands et al., 2003). However, GABAB-R density and number of binding sites were shown to decrease following at least 21-day baclofen pretreatment suggesting receptor down-regulation, post-transcriptional degradation and/or internalization (Lehmann et al., 2003; Malcangio et al., 1993). Here, tolerance significantly attenuated baclofen-mediated TI and TE increase. Surprisingly, these effects were delayed (480 and 360 min post-administration, respectively). In contrast, Baclofen-induced effects on VT increase, supposed physiologically to counteract the decrease in f, tended to be enhanced earlier (90 min) in the tolerant versus non-tolerant rats, contributing additionally to limit respiratory depression. Only slight hypoxemia without respiratory acidosis was observed in tolerant rats. Moreover, the lack of lactate modification supported the absence of baclofen-induced metabolic or cardiovascular impairment in both non-tolerant and tolerant rats. These observations clearly demonstrated the development of physiological adapting mechanisms but did not rule out additional pharmacokinetic modifications that overcome these pharmacodynamic changes. The earlier increase in VT observed in tolerant rats with the goal to counteract the decreased f, with the limits for extension to humans, could be of paramount importance in the clinical practice. Our pharmacokinetic data with intragastric 116 mg/kg baclofen showed delayed absorption as compared to intragastric 1 mg/kg baclofen (Tmax, 2.0 vs 0.67 h) (Kim et al., 2014), possibly explained by saturation of previously evidenced baclofen-intake transporters at the intestinal barrier, ie, the Large Neutral Amino Acid (LNAA, solute carrier SLC7A5) and β-amino acid (SLC6A6) transporters (Moll-Navarro et al., 1996). In addition VD/F (5.4 vs 2.8 l/kg) and t1/2 (5.6 vs 4.1 h) were increased. Our findings were consistent with baclofen pharmacokinetics in poisoned patients reporting delayed absorption, increased VD/F and prolonged t1/2 despite preserved kidney function (Cléophax et al., 2015). Repeated baclofen pretreatment resulted in pharmacokinetic alterations. Increase in Cmax and ka, despite absence of Tmax modification suggested adapting arrangements possibly mediated by the induction of the above-mentioned transporters. Since attenuated CNS toxicity was observed, despite lack of proof, the decrease in baclofen VD/F suggested a probable reduction in brain distribution possibly related to the saturation of baclofen-mediated influx system at the blood-brain barrier (BBB) involving the LNAA transporter (Km=0.012 µg/l) (Van Bree et al., 1988). Baclofen was shown to be strongly restricted by efflux systems, most likely probenecid-sensitive transporters while its passive diffusion is negligible due to its chemical properties (Deguchi et al., 1995). Baclofen-mediated efflux transporters have not yet been characterized but organic anion transporter (OAT)3, multidrug resistance-associated protein (MRP)1 and MRP4 have been hypothesized (Dalvi et al., 2014). In particular, MRP4 is inducible. These transporters across the BBB may explain the slow baclofen distribution to its targets and thus its prolonged effects, as observed in our study. Interestingly, increase in ke together with shortened t1/2 despite non-increased Cl/F due to reduced VD/F indicate enhanced elimination, more likely by renal transporter induction since baclofen is eliminated by filtration and tubular secretion. Baclofen is secreted by probenecid-sensitive OATs at the renal barrier (Wuis et al., 1989). Baclofen undergoes restricted inactivation metabolism (∼15%) by oxidative deamination. However, since baclofen-metabolizing enzymes have not yet been described, despite the possible involvement of monoamine oxidase and cytochromes P450 (Sanchez-Ponce et al., 2012), metabolism induction remains a theoretical hypothesis to explain the observed enhanced elimination. Anticlockwise hysteresis loops obtained for baclofen-induced stimulating effects (ie, sedation, PaCO2, TI, TE and VT) and clockwise hysteresis loops for inhibitory effects (ie, PaO2 and VM) indicated that plasma baclofen concentrations decrease more rapidly than all resulting neuro-respiratory effects. Our data are supported by animal experiments using radio-labeled baclofen showing that, despite lower concentrations in the brain, the elimination rate from the nerve tissue is much slower than from the blood (Faigle and Keberle, 1972). Taken together, these findings may explain the prolonged period of CNS complications following massive overdose in humans while plasma baclofen concentrations return to the therapeutic range (Ghose et al., 1980). Baclofen-related effects were attenuated in the tolerant versus non-tolerant rats despite no significantly different AUCs and increased Cmax. Reduction in the intensity and duration of baclofen-induced neurotoxicity in overdose is consistent with its decreased distribution at the BBB. In humans, alteration in baclofen distribution following tolerance acquisition is supported by the required steady increase in daily baclofen dose in patients chronically treated for spasticity and the need for oral-to-intrathecal route switch to regain effectiveness (Van der Plas et al., 2011). Conversely, the terminal non-improvement despite intrathecal baclofen dosage increase and non-progression of the underlying spastic disease highly suggests the involvement of additional pharmacodynamic mechanisms in tolerance like GABAB-R desensitization or down-regulation. Rapid-onset withdrawal syndrome occurs in patients if baclofen is suddenly discontinued (Peng et al., 2008). In the rat, hind limb hyperreflexia and increased electromyogram magnitudes were observed post-baclofen discontinuation (Priano et al., 2011; Wang et al., 2002). Here, withdrawal symptoms appeared early, peaked on day 5 and were accompanied by non-anxiogenic hyperlocomotion. The hypolocomotion observed on day 1 probably corresponded to persistent baclofen-related effects on locomotion, attributed to substantia nigra dopaminergic neuron inhibition, as shown (Beveridge et al., 2013). Mechanisms of baclofen-related withdrawal syndrome are not fully understood but involve imbalance between GABA and dopamine systems in the mesolimbic and nigrostriatal regions. Surprisingly, baclofen withdrawal did not induce anxiogenic-like effects in our rats as expected from (1) experimental studies supporting baclofen-induced anxiety reversal, (2) molecular studies supporting limbic GABAB-R contribution to the regulation of emotional behaviors, and (3) observations at the bedside. However, anxiogenic-like effects related to baclofen withdrawal are not consistently reported in clinical series. Therefore, our finding of non-anxiogenic withdrawal in the rat should be confirmed with tests able to evaluate anxiety-like behavior without being influenced by the induced hyperlocomotion. Our study has limitations. Extension to humans of rat findings should be cautious. However, investigating in vivo baclofen-related neurotoxicity represents a major strength. The effects and concentrations were not obtained in the same animals. The effect/concentration relationships were obtained using plasma but not brain concentrations, given the major BBB contribution to limiting baclofen distribution in the brain. However, despite there being no established value to predict neurotoxicity, plasma baclofen concentration remains the only available biomarker in humans. In addition, to better understand the pharmacokinetic alterations after prior repeated baclofen administration as well as their exact contribution to tolerance development, baclofen intestinal absorption, brain distribution and urinary excretion remain to be characterized in the rat. In conclusion, baclofen is responsible for dose-dependent sedation, hypothermia and respiratory depression in the rat. Prolonged baclofen treatment induces tolerance to neurotoxicity in overdose and results in withdrawal syndrome post-baclofen discontinuation. Tolerance to respiratory effects is explained by the significant decrease in baclofen-related TI and TE increase with a relatively early-onset compensatory trend to enhance VT increase. Mechanisms of tolerance partly involve pharmacokinetic alterations mainly at the brain distribution. In acute-on-chronically poisoned patients, the consequences of tolerance-mediated attenuation in neurotoxicity, as suggested by our rat findings, remain to be investigated. ACKNOWLEDGMENTS The authors would like to acknowledge Mrs. Alison Good, Scotland, UK, for her helpful review of this manuscript. The authors would also like to thank the Animal Platform, CRP2—UMS 3612 CNRS—US25 Inserm-IRD – Faculté de Pharmacie de Paris, Université Paris Descartes, Paris, France for the technical assistance during this work. FUNDING Institutional and departmental sources from the Institut national de la santé et de la recherche médicale (INSERM). REFERENCES Amsein O. ( 2008 ). Le Dernier Verre. [The Last Glass]. Denoël Edition, Paris, France. Arima H. , Oiso Y. ( 2010 ). Positive effect of baclofen on body weight reduction in obese subjects: a pilot study . Intern. Med . 49 , 2043 – 2047 . Google Scholar CrossRef Search ADS PubMed Bäckberg M. , Collin M. , Ovesjö M. L. , Meister B. ( 2003 ). 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W. , Dirks M. J. M. , Termond E. F. S. , Vree T. B. , Van der Kleijn E. ( 1989 ). Plasma and urinary excretion kinetics of oral baclofen in healthy subjects . Eur. J. Clin. Pharmacol . 37 , 181 – 184 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Baclofen-Induced Neuro-Respiratory Toxicity in the Rat: Contribution of Tolerance and Characterization of Withdrawal Syndrome

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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10.1093/toxsci/kfy073
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Abstract

Abstract Baclofen, a γ-amino-butyric acid type-B receptor agonist with exponentially increased use at high-dose to facilitate abstinence in chronic alcoholics, is responsible for increasing poisonings. Tolerance and withdrawal syndromes have been reported during prolonged treatment but their contribution to the variability of baclofen-induced neurotoxicity in overdose is unknown. We studied baclofen-induced effects on rat sedation, temperature, and ventilation and modeled baclofen pharmacokinetics and effect/concentration relationships aiming to investigate the consequences of repeated baclofen pretreatment and to characterize withdrawal syndrome. Baclofen-induced dose-dependent sedation (p <0.01), hypothermia (p <.001) and respiratory depression (p <.01) were altered in repeatedly baclofen-pretreated rats (p <.05). Repeatedly baclofen-pretreated rats did not exhibit respiratory depression following baclofen overdose due to limitations on baclofen-induced increase in inspiratory (p <.01) and expiratory times (p <.01). Only slight hypoxemia without respiratory acidosis was observed. Baclofen discontinuation resulted in hyperlocomotion and non-anxiogenic withdrawal symptoms. Regarding pharmacokinetics, repeated baclofen pretreatment increased the peak concentration (p <.05) and absorption constant rate (p <.05) and reduced the distribution volume (p <.0001) and elimination half-life (p <.05). Analysis of the effect/concentration relationships indicated that plasma baclofen concentration decreases more rapidly than all studied neuro-respiratory effects, in tolerant and non-tolerant rats. Taken together, our findings supported the role of brain distribution in baclofen-induced neurotoxicity expression and its probable involvement in tolerance-related attenuation in addition to physiological adaptations of ventilation. In conclusion, repeated pretreatment attenuates baclofen-attributed neurotoxicity in overdose and results in post-discontinuation withdrawal syndrome. Our findings suggest both pharmacodynamic and pharmacokinetic mechanisms whose relative contributions to the variability of baclofen-induced neurotoxicity in overdose remain to be established. baclofen, tolerance, respiratory depression, pharmacokinetics, poisoning, withdrawal Baclofen is a selective γ-amino-butyric acid type-B receptor (GABAB-R) agonist approved for the treatment of spasticity (Kumar et al., 2013). Recently, despite the absence of solid evidence to support its efficiency, baclofen has been used at high-dose to facilitate abstinence in dependent chronic alcoholics (Liu and Wang 2017; Reynaud et al., 2017). Following the publication in 2008 of “Le Dernier Verre” [The Last Glass], a book by Dr. Amsein (2008) reporting the successful self-management of his alcohol dependence with extremely high-dose baclofen (up to 270 mg/day), off-labeled prescriptions exponentially increased with an estimated 213 000 treated patients between 2009 and 2015 in France (French National Agency for Medicines and Health Products Safety, 2017). Although clinical studies showed that high-dose baclofen can be used with manageable side-effects (Müller et al., 2015; Reynaud et al., 2017; Rigal et al., 2015), the French health authorities reported dose-dependent risk of hospitalization (15% and 46% increase at 75–180 and >180 mg daily doses, respectively) and death (1.5- and 2.27-fold increase for the same dose intervals) as compared to other approved medications used to treat ethanol dependence (French National Agency for Medicines and Health Products Safety, 2017). With the increasing use of high-dose baclofen, accidental and self-poisonings dramatically rose. From January 2008 to December 2013, 294 baclofen toxic exposures in alcohol-dependent patients were reported to the French Poison control Centers with 132 severe poisonings and 9 fatalities (Pelissier et al., 2017). Baclofen overdose is responsible for life-threatening central nervous system (CNS) toxicity including coma, respiratory depression, flaccidity, areflexia, hypothermia, hypotension, and seizures (Franchitto et al., 2014; Léger et al., 2017). In addition, severe electroencephalographic abnormalities including isoelectric signal mimicking brain death have been reported (Sullivan et al., 2012). Baclofen poisoning has been reported in previously untreated (‘acute poisoning’) and treated patients (’acute-on-chronic poisoning’). Whether the severity of acute poisoning depends on previous baclofen treatment has not been established, although tolerance development was suggested under chronic baclofen treatment with the onset of transient side-effects like somnolence, asthenia, and confusion appearing during the titration period to reach the targeted dose and disappearing later on (Müller et al., 2015; Rigal et al., 2015; Reynaud et al., 2017). In addition, symptoms suggestive of withdrawal syndrome including hallucinations, agitation, delirium, psychosis, seizures, and even features mimicking neuroleptic malignant syndrome have been observed in chronically baclofen-treated patients (Peng et al., 2008; Richter et al., 2016). However, the time-course of such symptoms and their relationships to the discontinuation of prior baclofen treatment still raise questions. The exact mechanisms of baclofen-induced neuro-respiratory toxicity in overdose have been poorly investigated. The contribution of tolerance to the expression of acute toxicity remains unknown. We thus designed an experimental study in the rat aiming to (1) describe baclofen-induced neuro-respiratory toxicity in overdose; (2) investigate the consequences of tolerance acquisition on baclofen-related toxicity, and (3) characterize withdrawal syndrome resulting from the discontinuation following prior prolonged baclofen administration. MATERIALS AND METHODS Experimental protocols were carried out within the ethical guidelines established by the National Institute of Health and approved by Paris-Descartes University ethics committee for animal experimentation (No. APAFIS#5285-2016050321031432v2). Animals Five- to seven-week-old male Sprague Dawley rats (Janvier-labs, France) weighing 250–350 g during experimentation were used and housed for 7 days before experimentation in an environment maintained at 21 ± 0.5°C with controlled humidity and a light–dark cycle. Food and water were provided ad libitum. Chemicals and Drugs Baclofen (Sigma-Aldrich, France) was diluted in saline to obtain a 2.5 mg/ml solution [used for intraperitoneal administration] or in 10% Tween (Tween-20 diluted in 0.9% NaCl) to obtain a 10 mg/ml solution [used for intragastric administration]. Femoral Artery and Jugular Venous Catheterization The day before the experiment, animals were anesthetized with intraperitoneal 70 mg/kg ketamine (Clorketam 1000) and 10 mg/kg xylazine (Rompum) then placed on a warming blanket with regulating thermostat. The femoral artery (to allow arterial blood gas measurement) or the jugular vein (to allow plasma baclofen concentration measurement) was catheterized using 30 and 20-cm silastic tubing, respectively, with external and internal diameters of 0.94 and 0.51 mm, respectively (Dow Corning Co., Midland, MI). The arterial or venous catheter was subcutaneously tunneled and fixed at the back of the neck. Heparinized saline (100 UI/ml) was injected into the catheter to avoid thrombosis and catheter obstruction. Rats were then returned to their individual cages for a minimum of 24-h recovery period, to allow complete anesthesia washout. On the day of experimentation, rats were placed in horizontal Plexiglas cylinders (6.5 cm-internal diameter, up to 20 cm-adjustable length) (Harvard Apparatus, Inc., Holliston, MA). Before the drug administration, the catheter was exteriorized, purged, and its permeability checked. Clinical Parameters Temperature, sedation, seizures, and withdrawal signs were monitored by 2 researchers blinded to the rat group allocation. Temperature was measured using intraperitoneally implanted temperature transmitters for the purpose of plethysmography study. Sedation level based on a 4-stage scale from 0 (awake) to 3 (coma) was assessed (Pirnay et al., 2008). At stage 0, rats were completely awake and their gait and righting reflexes were intact. At stage 1, rats had reduced activity, showed light impairment of gait and an intact righting reflex with diminished muscle tonus. At stage 2, rats were asleep or static and showed a reduced righting reflex. At stage 3, rats were comatose and did not have any righting reflex. Seizure severity was graded according to the modified Racine Score (Racine, 1972). At stage 1, rats were immobile, with eyes closed, twitching of vibrissae and facial clonus. At stage 2, rats had head nodding associated with more severe facial clonus. At stage 3, rats had clonus of one forelimb. At stage 4, rats had rearing, often accompanied by bilateral forelimb clonus. At stage 5, rats had rearing with loss of balance and falling accompanied by generalized clonic seizures. All these parameters were measured before (T0) and at 5, 10, 15, 30, 60, 90, 120, 240, 360, 480, and 1440 min post-baclofen administration. Following chronic treatment discontinuation, physical dependence to baclofen was evaluated using Gellert’s withdrawal score (Table 1) (Gellert and Holtzman, 1978). Withdrawal signs were noted during a 5-min observation period each day (from days 1 to 12) after discontinuation of repeated baclofen. Table 1. Withdrawal Signs and Their Corresponding Weighting Factors (Signs Noted Simply as Present or Absent) Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Table 1. Withdrawal Signs and Their Corresponding Weighting Factors (Signs Noted Simply as Present or Absent) Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Sign Weighting Factor Graded signs  Weight loss (for each >1% above the weight lost by the control rat) 1  Number of escape attempts  2–4 1  5–9 2  10 or more 3  Number of abdominal constrictions (each one) 2  Number of wet shakes  1–2 2  3 or more 4 Checked signs  Diarrhea 2  Facial fasciculation/teeth chatter 2  Swallowing movements 2  Profuse salivation 7  Chromodacryorrhea 5  Ptosis 2  Abnormal posture 3  Erection or ejaculation 3  Irritability 3 Behavioral Measurements Locomotor activity The open field (OPF) test used an enclosed white Plexiglas chamber open at the top, divided into 4 equal-sized areas (50 ×50 × 35 cm3) and maintained under low illumination (10 lx). At the experiment time, 1 rat was placed in each area and recorded during 15 min. The chamber was fitted with an infrared floor connected to a miniature overhead infrared video-camera and a computer that used automated video-tracking software (ViewPoint, Videotrack, France) to determine the rat horizontal locomotor activity (traveled distance in cm). Locomotor activity was measured on days 1, 5, and 12 after repeated baclofen discontinuation. Anxiety-like behavior The elevated plus-maze (EPM) test used a central platform (10 × 10 cm2) formed by black Plexiglas boards surrounded by 2 opposed highly illuminated (180 lx) open arms (50 × 10 cm2) and 2 opposed faintly illuminated (10 lx) enclosed arms (50 × 10 cm2 with 45 cm-high walls). The maze was placed 50 cm above an infrared floor. At the experiment time, each rat was placed in the central platform facing the open arm away from the experimenter. The rat behavior was recorded by a miniature overhead infrared video-camera over a 5 min-period. The number of entries and time spent in each arm were tracked using Viewpoint software. Results are expressed as the percentage of number of entries and time spent in the open arms. Anxiety-like behavior was measured on day 6 after discontinuation of repeated baclofen. Whole Body Plethysmography Four days before the study, temperature transmitters (TA-F10, DSI, the Netherlands) were implanted in the peritoneal cavity. Ventilatory parameters were recorded in a whole body plethysmograph by the barometric method described and validated in the rat (Bartlett and Tenney, 1970). The first measurements were performed after a 30-min period of accommodation at −30, −15, and −5 min, while the rat was quiet and not in deep or rapid eye movement sleep, as roughly estimated from their behavior, response to noise, and pattern of breathing. Then, the rat was gently removed from the chamber for baclofen intragastric administration at T0, and replaced in the chamber for the remaining measurements. Ventilation was recorded at 5, 10, 15, 30, 60, 90, 120, 240, 360, 480, and 1440 min, each record lasting about 60 s. The following parameters, ie, the tidal volume (VT), the inspiratory time (TI) and the expiratory time (TE), were measured using ELPHY software (ElectroPhysiological Software Version 3.1.0.38, CNRS-UNIC, France). Additional parameters including the respiratory frequency (f = 1/(TI + TE)) and the minute volume (VM = VT × f) were calculated. Arterial Blood Gas and Lactate Measurement To investigate arterial blood gas, 200 µl of arterial blood were sampled from catheterized rats and immediately analyzed using the Vetstat system (IDEXX, France). To measure lactate, a blood drop was deposited on a lactate testing strip and immediately analyzed using Lactate Scout (ERF diagnostic, France). Arterial pH, PaO2, PaCO2, bicarbonate, and lactate concentrations were measured before (T0) and at 1, 2, 4, 6, and 8 h after intragastric baclofen administration to study its effects on ventilation as well as on the acid-base balance. Since significant changes in the rat core temperature were observed, arterial blood gases corrected at the actual temperature of the animals were interpreted. 200 ml of heparinized 0.9% NaCl were administered every hour to the rat via the arterial catheter to reduce the risk of catheter clotting and to compensate for volume loss. Plasma Baclofen Concentration Measurement Following high-dose baclofen administration, 300 µl of blood were sampled from catheterized rats before (T0) and at 5, 15, and 30 min and at 1, 2, 4, 6, 8, 10, 24, and 28 h after intragastric baclofen administration. Blood samples were collected in Eppendorf tubes containing 30 µl of sodium heparin (Panpharma, Sanofi Winthrop, France). After centrifugation (5000 rpm, 10 min), plasma was separated and stored at −20°C before analysis. Plasma baclofen concentration was measured using liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS) following a simple one-step protein precipitation (Labat et al., 2016). Chromatographic separation was performed on an Accucore PFP column (100 × 2.1 mm; 2.6 μm, Thermo Scientific, Belgium) at 30-Celsius degrees using gradient elution with a mobile phase consisting of water and acetonitrile (0.1% formic acid). MS analysis was performed on Quantum Ultra® (Thermo Fisher Scientific, Germany) using electrospray ionization with quantification transition m/z 213.9/196.9 for baclofen and 217.9/200.9 for baclofen-d4 (internal standard). The method was validated in plasma with linear response for concentrations ranging between 10 and 2000 µg/l (r2 > .999) and precisions and accuracies below 11.5%. Modeling of Baclofen Pharmacokinetics Plasma baclofen pharmacokinetic parameters after intragastric administration were assessed using one-compartment analysis (WinNonlin v.5.3, Certara, NJ) including the peak plasma concentration (Cmax), the time to achieve the Cmax (Tmax), the area under the concentration-time curve from 0 to infinity (AUC0→∞), the absorption rate constant (ka), the volume of distribution (VD/F), the total clearance (Cl/F), the terminal elimination rate constant (ke) and the elimination half-life (t1/2). Study Design Study 1—Baclofen-induced neuro-respiratory effects Rats were randomized in 3 groups (N = 6 rats/group) to receive 43.5, 72.5, and 116 mg/kg baclofen by gavage, corresponding to 30%, 50%, and 80% of the lethal dose-50% (LD50), ie, 145 mg/kg (Cayman chemicals, 2015). Subsequently, baclofen-induced effects on temperature, sedation, ventilation (using plethysmography), and blood lactate concentrations were measured. Study 2—Baclofen-induced tolerance Rats were randomized in 2 groups (N = 7–8 rats/group) to receive repeated intraperitoneal saline (acute poisoning group) or baclofen (acute-on-chronic poisoning group) 3 times per day for 15 consecutive days, with increasing baclofen doses every 3 days ranging from 5 to 15 mg/kg. On the morning of day 16, rats received 116 mg/kg baclofen by gavage. Subsequently, baclofen-induced effects on temperature, sedation, ventilation (using plethysmography and arterial blood gas analysis), and blood lactate concentrations were measured and plasma baclofen concentrations determined. Study 3—Baclofen-induced neurobehavioral effects during withdrawal Rats were randomized in 2 groups (N = 8 rats/group) to receive repeated intraperitoneal saline (control group) or baclofen (baclofen group) 3 times per day for 15 consecutive days, with increasing baclofen doses as previously described. Treatment was stopped on day 16. Baclofen withdrawal syndrome was investigated with the Gellert’s score and behavioral tests. Statistical Analysis Results are expressed as mean ± SEM and as mean ± SD for the PK study. For plethysmography, the value at T0 was the mean of the 3 baseline measurements. For each sedation, temperature, lactate concentrations, plethysmography, and blood gas measurement, baseline values (T0) were compared to the values at each sampling time using two-way analyses of variance (ANOVA) for repeated measures followed by multiple comparisons using Sidak’s correction. To permit the simultaneous analysis of the effect of time and treatments, we calculated for each animal and for each studied parameter, the area under the curve from T0 to the completion of the measurement (1440 min) using the trapezoid method (Studies 1 and 2). Thereafter, we compared the AUCs using linear regression (Study 1) and Mann-Whitney tests (Study 2). Two-way ANOVA for repeated measurements followed by multiple comparisons using Sidak’s correction were performed to compare body weight, sedation, temperature, lactate concentrations, plethysmography, and blood gas parameters (Study 2) and withdrawal scores (Study 3) between the study groups. Unpaired Student’s t tests were used to compare PK parameters (Study 2) and Mann-Whitney tests to compare locomotor activity and anxiety-like behavior (Study 3) at each measurement day between the study groups. All tests were performed using Prism version 6.0 (GraphPad Software). P-value < .05 was considered as significant. RESULTS Baclofen-Induced Neuro-Respiratory Effects (Study 1) Baclofen was responsible for significant dose-dependent early-onset sedation (p = .004) and hypothermia (p = .0002) in the rat (Figure 1). No seizures were observed. Based on plethysmography, baclofen induced a significant dose-dependent increase in TI (p = .0003), TE (p = .02) and VT (p = .004) resulting in a dose-dependent decrease in VM (p = .001) (Figure 2). Interestingly, no dose-effect relationship was observed regarding blood lactate measurements following baclofen administration. Figure 1. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A) and temperature (B) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Figure 1. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A) and temperature (B) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Figure 2. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on inspiratory time (A), expiratory time (B), tidal volume (C) and minute volume (D) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Figure 2. View largeDownload slide Dose-effect of intragastric 43.5, 72.5, and 116 mg/kg baclofen administration in Sprague Dawley rats on inspiratory time (A), expiratory time (B), tidal volume (C) and minute volume (D) (N = 6/group). Areas under the effect-time curve (AUC) were determined. Results are expressed as mean ± SEM. Comparisons were performed using linear regression. Baclofen-Induced Tolerance (Study 2) Clinical observations during repeated baclofen treatment Repeated baclofen administration significantly reduced weight gain along with food intake in comparison to the control (p < .05; Figure 3A). Significant decrease in sedation was observed on the third day of each baclofen dose step with no additional sedation at the 5th dose step in comparison to the 4th step (Figure 3B). Figure 3. View largeDownload slide Effects of repeated intraperitoneal administration of saline (dashed grey line) and baclofen (full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days in Sprague Dawley rats on body weight (A) and sedation (B) (N = 12/group). The arrow represents the first day for each dose step. Results are expressed as mean ± SEM. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. ##p <.01, ###p <.001, ####p <.0001. Figure 3. View largeDownload slide Effects of repeated intraperitoneal administration of saline (dashed grey line) and baclofen (full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days in Sprague Dawley rats on body weight (A) and sedation (B) (N = 12/group). The arrow represents the first day for each dose step. Results are expressed as mean ± SEM. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. ##p <.01, ###p <.001, ####p <.0001. Effects on baclofen-induced sedation In comparison to the baseline, baclofen significantly deepened sedation from 60 to 360 min in the acute-on-chronically versus 30–480 min in the acutely poisoned rats (p < .0001; Figure 4A). Sedation differed significantly between these 2 groups at 30, 360, and 480 min (p < .01) as supported by the AUC analysis (p < .001; Figure 4B). No seizures were observed in either rat group. Figure 4. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A, C), temperature (B, D) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A, B) and areas under the effect-time curve (AUC) determined (C, D). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. **p <.01, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001, ####p <.0001. Figure 4. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on sedation (A, C), temperature (B, D) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A, B) and areas under the effect-time curve (AUC) determined (C, D). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. **p <.01, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001, ####p <.0001. Effects on baclofen-induced hypothermia Baclofen significantly reduced body temperature at 480 min in both groups (p < .01) without significant differences at each measurement time between the 2 groups (Figure 4C). However, temperature decrease was less marked in the acute-on-chronically poisoned rats, as evidenced by the AUC analysis (p < .05; Figure 4D). Effects on baclofen-induced respiratory depression Baclofen significantly prolonged TI from 60 to 480 min in the acute-on-chronically (p < .001) versus 60 min to 24 h in the acutely poisoned rats (p < .01; Figure 5A). TE significantly increased at 240 min in the acute-on-chronically but from 240 to 480 min in the acutely poisoned rats (p < .05; Figure 5B). VT significantly increased from 60 to 360 min in the acute-on-chronically (p < .01) versus 60–480 min in the acutely poisoned rats (p < .05; Figure 5C). Interestingly, VM was not significantly reduced in the acute-on-chronically as compared to the acutely poisoned rats exhibiting delayed VM reduction at 360 and 480 min (p < .05; Figure 5D). These effects were significantly more marked at 480 min and 24 h for TI and at 360 and 480 min for TE and VT (p < .01). VM significantly differed between the 2 groups at 480 min (p < .001). Consistently, baclofen pretreatment significantly reduced intensity and duration of all acute baclofen-induced effects on ventilation as demonstrated by the AUC analysis (p < .01; Figs. 5E–H). Figure 5. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on the plethysmography parameters including inspiratory time (A, E), expiratory time (B, F), tidal volume (C, G) and minute volume (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *P <.05, **P <.01, ***P <.001, ****P <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #P <.05, ##P <.01, ###P <.001, ####P <.0001. Figure 5. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on the plethysmography parameters including inspiratory time (A, E), expiratory time (B, F), tidal volume (C, G) and minute volume (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *P <.05, **P <.01, ***P <.001, ****P <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #P <.05, ##P <.01, ###P <.001, ####P <.0001. Significant decrease in PaO2 was observed from 120 to 240 min in the acute-on-chronically (p < .01) versus 240–360 min in the acutely poisoned rats (p < .0001; Figure 6A). PaCO2 did not vary significantly in the acute-on-chronically poisoned rats while significantly increased from 240 to 360 min in the acutely poisoned rats (p < .001; Figure 6B). Similarly, arterial pH did not vary significantly in the acute-on-chronically but was significantly reduced from 240 to 360 min in the acutely poisoned rats (p < .01; Figure 6C). Blood bicarbonate concentration significantly increased at 240 min in the acute-on-chronically (p < .05) versus at 360 min in the acutely poisoned rats (p < .01; Figure 6D). These effects were significantly more marked at 240 and 360 min for PaO2 and pH (p < .01), at 240 min for bicarbonate (p < .01) and at 360 min for PaCO2 (p < .01). Consistently, baclofen pretreatment significantly diminished intensity and shortened duration of acute baclofen-induced effects on arterial blood gases as demonstrated by the AUC analysis (p < .01; Figs. 6E–H). No significant difference was observed in blood lactate concentration between the two rat groups based on the AUC analysis (data not shown). Figure 6. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on arterial blood gas parameters including PaO2 (A, E), PaCO2 (B, F), pH (C, G) and bicarbonate concentration (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *p <.05, **p <.01, ***p <.001, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001. Figure 6. View largeDownload slide Effects of intragastric 116 mg/kg baclofen administration in Sprague Dawley rats on arterial blood gas parameters including PaO2 (A, E), PaCO2 (B, F), pH (C, G) and bicarbonate concentration (D, H) (N = 8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Time course of the effects were represented (A–D) and areas under the effect-time curve (AUC) determined (E–H). Results are expressed as mean ± SEM. Comparisons with the baseline values (T0) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. *p <.05, **p <.01, ***p <.001, ****p <.0001. Comparisons between the two groups were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Sidak’s correction. AUC comparisons between the two groups were performed using Mann-Whitney tests. #p <.05, ##p <.01, ###p <.001. Effects on plasma baclofen pharmacokinetics and concentration-effect relationships The time-course of plasma baclofen concentrations is shown in Figure 7 and pharmacokinetic parameters in Table 2. When comparing the acute-on-chronically versus acutely poisoned rats, Cmax and ka were significantly increased (p < .05), VD/F reduced (p < .0001) and t1/2 shortened (p < .05). In contrast, AUCs and Cl/F were not significantly altered. Table 2. Pharmacokinetic Parameters Following Intragastric 116 mg/kg Baclofen Administration in Sprague Dawley Rats Pretreated With Repeated Intraperitoneal Saline (Acute Poisoning Group) or Baclofen (Acute-on-Chronic Poisoning Group) At Increasing Doses From 5 to 15 mg/kg Every 3 days During 15 Days Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters were determined using one-compartment model (N = 7/group) Values are expressed as mean ± SD. Comparisons between acute and acute-on-chronic poisoning groups were performed using Mann-Whitney tests, # p < .05, ## p < .01, #### p < .0001. Cmax, peak plasma concentration; Tmax, time to achieve the Cmax; ka, absorption rate constant; ke, elimination rate constant; VD/F, volume of distribution; Cl/F, total clearance; t1/2, terminal elimination half-life; AUC0→∞, area under the concentration-time curve from 0 to infinity. Table 2. Pharmacokinetic Parameters Following Intragastric 116 mg/kg Baclofen Administration in Sprague Dawley Rats Pretreated With Repeated Intraperitoneal Saline (Acute Poisoning Group) or Baclofen (Acute-on-Chronic Poisoning Group) At Increasing Doses From 5 to 15 mg/kg Every 3 days During 15 Days Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters Unit Acute Poisoning Acute-On-Chronic Poisoning Estimation CV (%) Estimation CV (%) Tmax H 2.0 0 1.9 18.9 Cmax µg/l 28860.9 32.9 46493.1# 23.3 ka h 1 29.7 1.5# 26.7 ke h 0.1 27.6 0.2#### 4.8 VD/F l/kg 5.4 11.5 3.1#### 14.4 Cl/F l/kg/h 0.5 19.7 0.6 10.9 t1/2 h 5.6 27.6 3.5# 4.8 AUC0→∞ µg h/l 178 771 19.7 201 456 10.9 Parameters were determined using one-compartment model (N = 7/group) Values are expressed as mean ± SD. Comparisons between acute and acute-on-chronic poisoning groups were performed using Mann-Whitney tests, # p < .05, ## p < .01, #### p < .0001. Cmax, peak plasma concentration; Tmax, time to achieve the Cmax; ka, absorption rate constant; ke, elimination rate constant; VD/F, volume of distribution; Cl/F, total clearance; t1/2, terminal elimination half-life; AUC0→∞, area under the concentration-time curve from 0 to infinity. Figure 7. View largeDownload slide Plasma baclofen concentration-time profiles following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats showing the observed (squares and circles) and predicted (dashed lines) data using one-compartment model (N = 7/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SD. Figure 7. View largeDownload slide Plasma baclofen concentration-time profiles following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats showing the observed (squares and circles) and predicted (dashed lines) data using one-compartment model (N = 7/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SD. For both rat groups, an anticlockwise hysteresis loop was obtained for sedation and a clockwise hysteresis loop for body temperature in relation to plasma baclofen concentrations (Figure 8). Regarding plethysmography parameters, anticlockwise hysteresis loops were obtained for TI, TE, and VT and a clockwise hysteresis loop for VM in the 2 groups. Regarding arterial blood gas parameters, anticlockwise hysteresis loops were obtained for PaO2 and arterial pH and clockwise hysteresis loops for PaCO2 and bicarbonate in the 2 groups. Figure 8. View largeDownload slide Effect/concentration relationships following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats for sedation (A), temperature (B), inspiratory time (C), expiratory time (D), tidal volume (E), minute volume (F), PaO2 (G), PaCO2 (H), arterial pH (I) and bicarbonate concentration (J) (N = 7–8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SEM. Figure 8. View largeDownload slide Effect/concentration relationships following intragastric 116 mg/kg baclofen administration in Sprague Dawley rats for sedation (A), temperature (B), inspiratory time (C), expiratory time (D), tidal volume (E), minute volume (F), PaO2 (G), PaCO2 (H), arterial pH (I) and bicarbonate concentration (J) (N = 7–8/group). Rats were pretreated with repeated intraperitoneal saline (acute poisoning group, dashed grey line) or baclofen (acute-on-chronic poisoning group, full black line) at increasing doses from 5 to 15 mg/kg every 3 days during 15 days. Results are expressed as mean ± SEM. Baclofen-Induced Neurobehavioral Effects During Withdrawal (Study 3) Withdrawal score No withdrawal symptoms were noticed in control rats. In contrast, discontinuation after repeated baclofen administration was responsible for marked withdrawal scores (peak values on days 5 and 6) with significant differences in comparison to the controls on each day from days 1 to 9 post-baclofen discontinuation (p < .0001). Symptoms such as facial fasciculations, chromodacryorrhea, and escape attempts were present as early as the first day post-discontinuation while wet shakes, swallowing movements, diarrhea, and ptosis were observed on the following days. Locomotor activity The distance traveled based on the OPF test significantly decreased on day 1 (p < .01), increased on day 5 (p < .05) but did not significantly differ on day 12 between the baclofen-pretreated and the control rats (Figure 9A). The distance traveled was significantly increased in the baclofen-pretreated rats (p < .01) and decreased in the controls on day 5 as compared to day 1. On day 12, no significant difference was noted in the baclofen-pretreated rats as compared to day 1, whereas significant decrease was still observed in the controls (p < .001). Figure 9. View largeDownload slide Effects of baclofen discontinuation in Sprague Dawley rats after repeated intraperitoneal saline (crisscrossed grey lines) or baclofen at increasing doses from 5 to 15 mg/kg every 3 days (black) during 15 days on the distance traveled in the open-field (A) and on the percentage of entries (B) and time spent (C) in the open arms of the EPM (N = 8/group). Results are expressed as mean ± SEM. Mann-Whitney tests were performed to compare results between each day of discontinuation with the first day **p <.01, ***p <.001 and between the two groups ##p <.01, ###p <.001. Figure 9. View largeDownload slide Effects of baclofen discontinuation in Sprague Dawley rats after repeated intraperitoneal saline (crisscrossed grey lines) or baclofen at increasing doses from 5 to 15 mg/kg every 3 days (black) during 15 days on the distance traveled in the open-field (A) and on the percentage of entries (B) and time spent (C) in the open arms of the EPM (N = 8/group). Results are expressed as mean ± SEM. Mann-Whitney tests were performed to compare results between each day of discontinuation with the first day **p <.01, ***p <.001 and between the two groups ##p <.01, ###p <.001. Anxiety-like behavior Based on the EPM test, the percentage of number of entries (Figure 9B) and the time spent (Figure 9C) in the open arms were not significantly different between the 2 groups on day 6 post-baclofen discontinuation. DISCUSSION Baclofen overdose is responsible for dose-dependent sedation, hypothermia, and respiratory depression in the rat. Repeated pre-treatment induces tolerance to baclofen-induced neuro-respiratory effects in overdose and results in withdrawal syndrome post-discontinuation. Our rat model reliably reproduced life-threatening baclofen toxicity in humans (Franchitto et al., 2014). Behavior, sleep, and electroencephalographic activity impairments have been described in the rat following single or repeated <20 mg/kg baclofen (Beveridge et al., 2013; Hodor et al., 2015). Here, we studied regimens mimicking acute poisoning and prolonged high-dose treatment in humans. Baclofen overdose resulted in CNS depressant effects as previously established in humans (Rolland et al., 2015) and expected from its mechanism of action, mediated by the presynaptic GABAB-R activation which reduces excitatory neurotransmitter release. GABAB-Rs are involved in various structures including the brainstem ventilation control center, temperature-controlling hypothalamic center, and anterior horn α-motoneurons. Baclofen acts mainly centrally to depress ventilation although inspiratory muscle relaxing effects also contributes. Baclofen-mediated GABAB-R stimulation decreases glutamate release preventing N-methyl-d-Aspartate (NMDA) and non-NMDA-receptor stimulation on inspiratory neurons (Pierrefiche et al., 1993). Baclofen inhibits inspiratory neurons of the solitary tract nucleus within the pneumotaxic center involved in the automatic regulation of respiratory volume and prevents phrenic nerve discharges stimulating the diaphragm (Pierrefiche et al., 1993). Interestingly, baclofen was shown to induce selective TI lengthening without simultaneous TE change by modulating the Hering-Breuer inflation and deflation reflexes. Such distinct TI/TE responses are explained by distinct medullary pathway control reflexes with TI-promoting reflex exclusively mediated by high-threshold receptor activation (all-or-nothing response) and TE-promoting reflex depending on high- or low-threshold receptor activation (Seifert and Trippenbach, 1998). Another hypothesis was related to disparate GABAB-R distribution within the respiratory neuronal network. Here, high-dose baclofen induced both TI and TE increase suggesting that, like opioid receptors (Chevillard et al., 2010), GABAB-R-mediated effects on ventilation require higher ligand concentration to prolong TE than that which is necessary to prolong TI. We obtained tolerance to baclofen-induced neurotoxicity with repeated pretreatment. Interestingly, in our baclofen-pretreated rats, hypophagic effects resulting in weight gain reduction were observed as previously (Bains and Ebenezer, 2013; Patel and Ebenezer 2010). In obese baclofen-treated subjects, decreased appetite concomitantly with reduced weight, waist circumference, and adipose stores supported by decreased serum leptin were reported (Arima and Oiso, 2010). GABAB-Rs regulate food intake and weight control by inhibiting the potent orexigenic neuropeptide Y and hypothalamic proopiomelanocortin secretion (Bäckberg et al., 2003). Decreased appetite would thus be interesting to study in patients suffering from bulimia, as already suggested by on case report (Weibel et al., 2015). In humans, tolerance to baclofen-induced sedation was described (Rigal et al., 2015). In rats, tolerance was reported with various repeated low-dose regimens, to sedation (Bains and Ebenezer, 2013), hypothermia (Lehmann et al., 2003), hypolocomotion (Beveridge et al., 2013), changes in ankle torque hind limb posture and stretch reflex activity (Oshiro et al., 2010; Wang et al., 2002). Tolerance acquisition was supported by glucose metabolism decrease in the thalamus and raphe nuclei involved in the sleep/wake cycle regulation and the substantia nigra and cerebellum involved in motor skills (Beveridge et al., 2013). Tolerance acquisition was related to reduction in presynaptic GABAB-R activation (Hefferan et al., 2006). Interestingly, GABAB-R desensitization, demonstrated using GTP-γS binding assays (Keegan et al., 2015) was not associated with changes in GABAB-R subunit-coding mRNAs (Sands et al., 2003). However, GABAB-R density and number of binding sites were shown to decrease following at least 21-day baclofen pretreatment suggesting receptor down-regulation, post-transcriptional degradation and/or internalization (Lehmann et al., 2003; Malcangio et al., 1993). Here, tolerance significantly attenuated baclofen-mediated TI and TE increase. Surprisingly, these effects were delayed (480 and 360 min post-administration, respectively). In contrast, Baclofen-induced effects on VT increase, supposed physiologically to counteract the decrease in f, tended to be enhanced earlier (90 min) in the tolerant versus non-tolerant rats, contributing additionally to limit respiratory depression. Only slight hypoxemia without respiratory acidosis was observed in tolerant rats. Moreover, the lack of lactate modification supported the absence of baclofen-induced metabolic or cardiovascular impairment in both non-tolerant and tolerant rats. These observations clearly demonstrated the development of physiological adapting mechanisms but did not rule out additional pharmacokinetic modifications that overcome these pharmacodynamic changes. The earlier increase in VT observed in tolerant rats with the goal to counteract the decreased f, with the limits for extension to humans, could be of paramount importance in the clinical practice. Our pharmacokinetic data with intragastric 116 mg/kg baclofen showed delayed absorption as compared to intragastric 1 mg/kg baclofen (Tmax, 2.0 vs 0.67 h) (Kim et al., 2014), possibly explained by saturation of previously evidenced baclofen-intake transporters at the intestinal barrier, ie, the Large Neutral Amino Acid (LNAA, solute carrier SLC7A5) and β-amino acid (SLC6A6) transporters (Moll-Navarro et al., 1996). In addition VD/F (5.4 vs 2.8 l/kg) and t1/2 (5.6 vs 4.1 h) were increased. Our findings were consistent with baclofen pharmacokinetics in poisoned patients reporting delayed absorption, increased VD/F and prolonged t1/2 despite preserved kidney function (Cléophax et al., 2015). Repeated baclofen pretreatment resulted in pharmacokinetic alterations. Increase in Cmax and ka, despite absence of Tmax modification suggested adapting arrangements possibly mediated by the induction of the above-mentioned transporters. Since attenuated CNS toxicity was observed, despite lack of proof, the decrease in baclofen VD/F suggested a probable reduction in brain distribution possibly related to the saturation of baclofen-mediated influx system at the blood-brain barrier (BBB) involving the LNAA transporter (Km=0.012 µg/l) (Van Bree et al., 1988). Baclofen was shown to be strongly restricted by efflux systems, most likely probenecid-sensitive transporters while its passive diffusion is negligible due to its chemical properties (Deguchi et al., 1995). Baclofen-mediated efflux transporters have not yet been characterized but organic anion transporter (OAT)3, multidrug resistance-associated protein (MRP)1 and MRP4 have been hypothesized (Dalvi et al., 2014). In particular, MRP4 is inducible. These transporters across the BBB may explain the slow baclofen distribution to its targets and thus its prolonged effects, as observed in our study. Interestingly, increase in ke together with shortened t1/2 despite non-increased Cl/F due to reduced VD/F indicate enhanced elimination, more likely by renal transporter induction since baclofen is eliminated by filtration and tubular secretion. Baclofen is secreted by probenecid-sensitive OATs at the renal barrier (Wuis et al., 1989). Baclofen undergoes restricted inactivation metabolism (∼15%) by oxidative deamination. However, since baclofen-metabolizing enzymes have not yet been described, despite the possible involvement of monoamine oxidase and cytochromes P450 (Sanchez-Ponce et al., 2012), metabolism induction remains a theoretical hypothesis to explain the observed enhanced elimination. Anticlockwise hysteresis loops obtained for baclofen-induced stimulating effects (ie, sedation, PaCO2, TI, TE and VT) and clockwise hysteresis loops for inhibitory effects (ie, PaO2 and VM) indicated that plasma baclofen concentrations decrease more rapidly than all resulting neuro-respiratory effects. Our data are supported by animal experiments using radio-labeled baclofen showing that, despite lower concentrations in the brain, the elimination rate from the nerve tissue is much slower than from the blood (Faigle and Keberle, 1972). Taken together, these findings may explain the prolonged period of CNS complications following massive overdose in humans while plasma baclofen concentrations return to the therapeutic range (Ghose et al., 1980). Baclofen-related effects were attenuated in the tolerant versus non-tolerant rats despite no significantly different AUCs and increased Cmax. Reduction in the intensity and duration of baclofen-induced neurotoxicity in overdose is consistent with its decreased distribution at the BBB. In humans, alteration in baclofen distribution following tolerance acquisition is supported by the required steady increase in daily baclofen dose in patients chronically treated for spasticity and the need for oral-to-intrathecal route switch to regain effectiveness (Van der Plas et al., 2011). Conversely, the terminal non-improvement despite intrathecal baclofen dosage increase and non-progression of the underlying spastic disease highly suggests the involvement of additional pharmacodynamic mechanisms in tolerance like GABAB-R desensitization or down-regulation. Rapid-onset withdrawal syndrome occurs in patients if baclofen is suddenly discontinued (Peng et al., 2008). In the rat, hind limb hyperreflexia and increased electromyogram magnitudes were observed post-baclofen discontinuation (Priano et al., 2011; Wang et al., 2002). Here, withdrawal symptoms appeared early, peaked on day 5 and were accompanied by non-anxiogenic hyperlocomotion. The hypolocomotion observed on day 1 probably corresponded to persistent baclofen-related effects on locomotion, attributed to substantia nigra dopaminergic neuron inhibition, as shown (Beveridge et al., 2013). Mechanisms of baclofen-related withdrawal syndrome are not fully understood but involve imbalance between GABA and dopamine systems in the mesolimbic and nigrostriatal regions. Surprisingly, baclofen withdrawal did not induce anxiogenic-like effects in our rats as expected from (1) experimental studies supporting baclofen-induced anxiety reversal, (2) molecular studies supporting limbic GABAB-R contribution to the regulation of emotional behaviors, and (3) observations at the bedside. However, anxiogenic-like effects related to baclofen withdrawal are not consistently reported in clinical series. Therefore, our finding of non-anxiogenic withdrawal in the rat should be confirmed with tests able to evaluate anxiety-like behavior without being influenced by the induced hyperlocomotion. Our study has limitations. Extension to humans of rat findings should be cautious. However, investigating in vivo baclofen-related neurotoxicity represents a major strength. The effects and concentrations were not obtained in the same animals. The effect/concentration relationships were obtained using plasma but not brain concentrations, given the major BBB contribution to limiting baclofen distribution in the brain. However, despite there being no established value to predict neurotoxicity, plasma baclofen concentration remains the only available biomarker in humans. In addition, to better understand the pharmacokinetic alterations after prior repeated baclofen administration as well as their exact contribution to tolerance development, baclofen intestinal absorption, brain distribution and urinary excretion remain to be characterized in the rat. In conclusion, baclofen is responsible for dose-dependent sedation, hypothermia and respiratory depression in the rat. Prolonged baclofen treatment induces tolerance to neurotoxicity in overdose and results in withdrawal syndrome post-baclofen discontinuation. Tolerance to respiratory effects is explained by the significant decrease in baclofen-related TI and TE increase with a relatively early-onset compensatory trend to enhance VT increase. Mechanisms of tolerance partly involve pharmacokinetic alterations mainly at the brain distribution. 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Toxicological SciencesOxford University Press

Published: Mar 21, 2018

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