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While most deaths from asphyxia related to buprenorphine (BUP) overdose have been reported in males, higher plasma concentrations of BUP and its toxic metabolite norbuprenorphine (NBUP) have been observed in females. We previously demonstrated that P-glycoprotein (P-gp) modulation at the blood-brain barrier (BBB) contributes highly to BUP-related respiratory toxicity, by limiting NBUP entrance into the brain. In this work, we sought to investigate the role of P-gp-mediated transport at the BBB in gender and strain-related variability of BUP and NBUP-induced respiratory effects in mice. Ventilation was studied using plethysmography, P-gp expression using western blot, and transport at the BBB using in situ cerebral perfusion. In male Fvb and Swiss mice, BUP was responsible for ceiling respiratory effects. NBUP-related reduction in minute volume was dose-dependent but more marked in Fvb (p<0.01 at 1mg/kg NBUP and p<0.001 at 3 and 9mg/kg NBUP) than in Swiss mice (p<0.001 at 9mg/kg NBUP). Female Fvb mice were more susceptible to BUP than males with significantly increased inspiratory time (p<0.05) and to NBUP with significantly increased expiratory time (p<0.01). Following BUP administration, plasma BUP concentrations were significantly higher (p<0.01) and plasma NBUP concentrations significantly lower (p<0.001) in Fvb mice compared to Swiss mice. Plasma BUP concentrations were significantly higher (p<0.05) and plasma NBUP concentrations significantly lower (p<0.01) in male compared to female Fvb mice. In contrast, following NBUP administration, comparable plasma NBUP concentrations were observed in both genders and strains. No differences in P-gp expression or BUP and NBUP transport across the BBB were observed between male and female Fvb mice as well as between Swiss and Fvb mice. Our results suggest that P-gp-mediated transport across the BBB does not play a key-role in gender and strain-related variability in BUP and NBUP-induced respiratory toxicity in mice. Both gender- and strain-related differences in respiratory effects of BUP could be attributed to BUP itself rather than to its metabolite, NBUP., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objectives: Deaths due to asphyxia as well as following acute poisoning with severe respiratory depression have been attributed to buprenorphine in opioid abusers. However, in human and animal studies, buprenorphine exhibited ceiling respiratory effects, whereas its metabolite, norbuprenorphine, was assessed as being a potent respiratory depressor in rodents. Recently, norbuprenorphine, in contrast to buprenorphine, was shown in vitro to be a substrate of human P-glycoprotein, a drug-transporter involved in all steps of pharmacokinetics including transport at the blood-brain barrier. Our objectives were to assess P-glycoprotein involvement in norbuprenorphine transport in vivo and study its role in the modulation of buprenorphine-related respiratory effects in mice., Setting: University-affiliated research laboratory, INSERM U705, Paris, France., Subjects: Wild-type and P-glycoprotein knockout female Friend virus B-type mice., Interventions: Respiratory effects were studied using plethysmography and the P-glycoprotein role at the blood-brain barrier using in situ brain perfusion., Measurements and Main Results: Norbuprenorphine(≥ 1 mg/kg) and to a lesser extent buprenorphine (≥ 10 mg/kg) were responsible for dose-dependent respiratory depression combining increased inspiratory (TI) and expiratory times (TE). PSC833, a powerful P-glycoprotein inhibitor, significantly enhanced buprenorphine-related effects on TI (p < .01) and TE (p < .05) and norbuprenorphine-related effects on minute volume (VE, p < .05), TI, and TE (p < .001). In P-glycoprotein-knockout mice, buprenorphine-related effects on VE (p < .01), TE (p < .001), and TI (p < .05) and norbuprenorphine-related effects on VE (p < .05) and TI (p < .001) were significantly enhanced. Plasma norbuprenorphine concentrations were significantly increased in PSC833-treated mice (p < .001), supporting a P-glycoprotein role in norbuprenorphine pharmacokinetics. Brain norbuprenorphine efflux was significantly reduced in PSC833-treated and P-glycoprotein-knockout mice (p < .001), supporting P-glycoprotein-mediated norbuprenorphine transport at the blood-brain barrier., Conclusions: P-glycoprotein plays a key-protective role in buprenorphine-related respiratory effects, by allowing norbuprenorphine efflux at the blood-brain barrier. Our findings suggest a major role for drug-drug interactions that lead to P-glycoprotein inhibition in buprenorphine-associated fatalities and respiratory depression.

Buprenorphine is a partial opioid agonist with a "ceiling effect" for respiratory depression. Despite this, it has been associated with severe overdoses. Conflicting data exist regarding its response in overdose to naloxone. We compared clinical overdose characteristics of buprenorphine with heroin and methadone and assessed responses to naloxone and flumazenil. Patients admitted to two intensive care units with severe opioid overdoses were enrolled into this 4-year prospective study. Urine and blood toxicological screening were performed to identify overdoses involving predominantly buprenorphine, heroin, or methadone. Eighty-four patients with heroin (n = 26), buprenorphine (n = 39), or methadone (n = 19) overdoses were analyzed. In the buprenorphine group, sedative drug coingestions were frequent (95%), whereas in the methadone group, suicide attempts were significantly more often reported (p = .0007). Buprenorphine overdose induced an opioid syndrome not differing significantly from heroin and methadone in mental status (as measured by Glasgow Coma Score) or arterial blood gases. Mental status depression was not reversed in buprenorphine overdoses with naloxone (0.4-0.8 mg) but did improve with flumazenil (0.2-1 mg) if benzodiazepines were coingested. In conclusion, buprenorphine overdose causes an opioid syndrome clinically indistinguishable from heroin and methadone. Although mental status and respiratory depression are often unresponsive to low-dose naloxone, flumazenil may be effective in buprenorphine overdoses involving benzodiazepines., (Copyright 2010 Elsevier Inc. All rights reserved.)

High-dosage buprenorphine (BUP) consumed concomitantly with benzodiazepines (BZDs) including flunitrazepam (FZ) may cause life-threatening respiratory depression despite a BUP ceiling effect and BZDs' limited effects on ventilation. However, the mechanism of BUP/FZ interaction remains unknown. We hypothesized that BUP may alter the disposition of FZ active metabolites in vivo, contributing to respiratory toxicity. Plasma FZ, desmethylflunitrazepam (DMFZ), and 7-aminoflunitrazepam (7-AFZ) concentrations were measured using gas chromatography-mass spectrometry. Intravenous BUP 30 mg/kg pretreatment did not alter plasma FZ and 7-AFZ kinetics in Sprague-Dawley rats infused with 40 mg/kg FZ over 30 min, whereas resulting in a three-fold increase in the area under the curve (AUC) of DMFZ concentrations compared with control (p < 0.01). In contrast, BUP did not significantly modify plasma DMFZ concentrations after intravenous infusion of 7 mg/kg DMFZ, whereas resulting in a similar peak concentration to that generated from 40 mg/kg FZ administration. Regarding the effects on ventilation, BUP (30 mg/kg) as well as its combination with FZ (0.3 mg/kg) significantly increased PaCO(2), whereas only BUP/FZ combination decreased PaO(2) (p < 0.001). Interestingly, FZ (40 mg/kg) but not DMFZ (40 mg/kg) significantly increased PaCO(2) (p < 0.05), whereas DMFZ but not FZ decreased PaO(2) (p < 0.05). Thus, decrease in PaO(2) appears related to BUP-mediated effects on DMFZ disposition, although increases in PaCO(2) relate to direct BUP/FZ additive or synergistic dynamic interactions. We conclude that combined high-dosage BUP and FZ is responsible for increased respiratory toxicity in which BUP-mediated alteration in DMFZ disposition may play a significant role.

Fatalities have been attributed to combinations of high-dose buprenorphine with benzodiazepines. In rats, high-dose buprenorphine combined with midazolam was shown to induce sustained respiratory acidosis, while buprenorphine alone did not. However, the effects of buprenorphine combined with pharmacological doses of benzodiazepines remain unknown. Our objective was to compare the acute effects of four selected benzodiazepines used intravenously at equi-efficacious doses in rats, alone and in combination with buprenorphine on sedation, respiratory rate and arterial blood gases. Buprenorphine (30 mg/kg) did not significantly modify sedation level or respiratory rate, but induced mild and transient effects on pH and PaCO(2) (P < 0.05). Similarly, despite having no effects on respiratory rate, nordiazepam (10 mg/kg), bromazepam (1 mg/kg) and oxazepam (12 mg/kg) mildly and transiently altered pH and PaCO(2) (P < 0.05), whereas clonazepam (5 mg/kg) did not. Buprenorphine combined with each benzodiazepine induced no significant effects on respiratory rate or blood gases, in comparison with buprenorphine alone. However, combinations of oxazepam or nordiazepam with buprenorphine significantly deepened sedation. While both combinations reduced respiratory rate, buprenorphine + 30 mg/kg clonazepam significantly increased PaCO(2) and buprenorphine + 30 mg/kg nordiazepam decreased PaO(2). In conclusion, not all benzodiazepines induce significant respiratory depression at therapeutic doses. We were unable to demonstrate significant effects on rat ventilatory parameters of buprenorphine combined with equi-efficacious pharmacological doses of benzodiazepines in comparison with buprenorphine alone. Our results may suggest that effects of these combinations are rather mild. Respiratory failure may, however, result from the association of buprenorphine with elevated doses of benzodiazepines.

In humans, asphyxic deaths and severe poisonings have been attributed to high-dosage buprenorphine, a maintenance therapy for heroin addiction. However, in rats, intravenous buprenorphine at doses up to 90 mg kg(-1) was not associated with significant effects on arterial blood gases. In contrast, norbuprenorphine, the buprenorphine major cytochrome P450 (CYP) 3A-derived metabolite, is a potent respiratory depressant. Thus, our aim was to study the consequences of CYP3A induction on buprenorphine-associated effects on resting ventilation in rats. We investigated the effects on ventilation of 30 mg kg(-1) buprenorphine alone or following cytochrome P450 (CYP) 3A induction with dexamethasone, using whole body plethysmography (N=24) and arterial blood gases (N=12). Randomized animals in 4 groups received sequential intraperitoneal dosing with: (dexamethasone [days 1-3]+buprenorphine [day 4]), (dexamethasone solvent [days 1-3]+buprenorphine [day 4]), (dexamethasone [days 1-3]+buprenorphine solvent [day 4]), or (dexamethasone solvent [days 1-3]+buprenorphine solvent [day 4]). Buprenorphine alone caused a significant rapid and sustained increase in the inspiratory time (P<0.001), without significant effects on the respiratory frequency, the tidal volume, the minute volume, or arterial blood gases. In dexamethasone-pretreated rats, there was no significant alteration in the respiratory parameters, despite CYP3A induction and significant increase of the ratio of plasma norbuprenorphine-to-buprenorphine concentrations. In conclusion, dexamethasone did not modify the effects of 30 mg kg(-1) buprenorphine on rat ventilation. Our results suggest a limited role of drug-mediated CYP3A induction in the occurrence of buprenorphine-attributed respiratory depression in addicts.

A liquid chromatographic-mass spectrometric method with electrospray ionization is presented for the simultaneous determination of buprenorphine, nordiazepam and their pharmacologically active metabolites, norbuprenorphine and oxazepam, in rat plasma. The drugs were extracted from plasma by liquid-liquid extraction and chromatographically separated using a gradient elution of aqueous ammonium formate and acetonitrile. Following electrospray ionization, the analytes were quantified in the single ion storage mode. The assay was validated according to current acceptance criteria for bioanalytical method validation. It was proved to be linear from 0.7 to 200 ng/ml plasma for buprenorphine, 1.0 to 200 ng/ml for norbuprenorphine, 2.0 to 200 ng/ml for nordiazepam, and from 5.0 to 200 ng/ml for oxazepam. The average recoveries of buprenorphine, norbuprenorphine, nordiazepam and oxazepam were 89, 39, 88 and 82%, respectively, with average coefficients of variation ranging from 1.8 to 14.3%. The limits of quantitation for these drugs were 0.7, 1.0, 2.0 and 5.0 ng/ml, respectively, with associated precisions within 17% and accuracies within +/-18% of the nominal values. Both the intra- and inter-assay precision values did not exceed 11.3% for the four analytes. Intra- and inter-assay accuracies lay within +/-15% of the nominal values. The validated method was applied to the determination of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in plasma samples collected from rats at various times after intravenous administration of buprenorphine and nordiazepam.

High dose buprenorphine is used as substitution treatment in heroin addiction. However, deaths have been reported in addicts using buprenorphine. The role of norbuprenorphine, an N-dealkyl metabolite of buprenorphine, was hypothesized to explain these fatal cases. We determined the median intravenous lethal dose (LD(50)) of norbuprenorphine in male Sprague-Dawley rats. The effects of a single intravenous dose of 3 or 9 mg/kg norbuprenorphine alone on arterial blood gases were studied. Finally, the effect of pre- and post-administrations of buprenorphine on norbuprenorphine-induced changes on arterial blood gases were analyzed. Norbuprenorphine's LD(50) was 10 mg kg(-1). Norbuprenorphine 3 mg kg(-1) produces the rapid onset of sustained respiratory depression, as demonstrated at 20 min by a maximal significant increase in PaCO(2) (8.4+/-0.9 versus 5.7+/-0.1 kPa), decrease in arterial pH (7.25+/-0.06 versus 7.44+/-0.01), and hypoxia (8.3+/-0.6 versus 11.1+/-0.2 kPa). Buprenorphine not only protected against the effects of 3 mg kg(-1) norbuprenorphine in a dose-dependent manner but also reversed the effects when given afterward. Binding experiments suggest a role for micro- and to a lesser extent for delta-opioid receptors in buprenorphine protective effect against norbuprenorphine-induced respiratory depression. In conclusion, our data clearly show that norbuprenorphine alone causes important deleterious effects on ventilation in rats. However, buprenorphine protective effect calls into question the role for norbuprenorphine in respiratory toxicity associated with buprenorphine use.

Buprenorphine is an opioid agonist-antagonist with a 'ceiling effect' for respiratory depression. Compared with methadone, its unique pharmacology offers practical advantages and enhanced safety when prescribed as recommended and supervised by a physician. Buprenorphine has been approved in several countries as an efficient and safe maintenance therapy for heroin addiction. Its use resulted in a salutary effect with a reduction in heroin overdose-related deaths in countries that implemented office-based buprenorphine maintenance. In France, however, where high-dose buprenorphine has been marketed since 1996, several cases of asphyxic deaths were reported among addicts treated with buprenorphine. Death resulted from buprenorphine intravenous misuse or concomitant sedative drug ingestion, such as benzodiazepines. In these situations of abuse, misuse, or in association with elevated doses of psychotropic drugs, buprenorphine may cause severe respiratory depression. Unlike other opiates, the respiratory effects from buprenorphine are not responsive to naloxone. However, the exact mechanism of buprenorphine-induced effects on ventilation is still unknown. The role of norbuprenorphine, the main N-dealkylated buprenorphine metabolite with potent respiratory depressor activity, also remains unclear. Experimental studies investigating the respiratory effects of combinations of high doses of buprenorphine and benzodiazepines suggested that this drug-drug interaction may result from a pharmacodynamic interaction. A pharmacokinetic interaction between buprenorphine and flunitrazepam is also considered. As there are many questions regarding the possible dangers of death or respiratory depression associated with buprenorphine use, we aimed to present a comprehensive critical review of the published clinical and experimental studies on buprenorphine respiratory effects.

Deaths have been reported among heroin addicts related to combined buprenorphine and flunitrazepam use. The aim of this study was to determine the existence of a drug-drug interaction during the distribution phase of buprenorphine. Arterial blood gases were measured after intravenous administration of buprenorphine alone (30 mg/kg), flunitrazepam alone (40 mg/kg) or both drugs in rats. Buprenorphine kinetics was studied in plasma and in striatum using cerebral microdialysis, both alone and after rat pretreatment with flunitrazepam. In contrast to buprenorphine or flunitrazepam alone, buprenorphine in combination with flunitrazepam induced a significant, rapid and sustained respiratory depression. Arterial PCO2 was increased at 1.5 min (6.7+/-0.2 versus 5.4+/-0.3 and 5.5+/-0.3 kPa, respectively, P=0.04) (mean+/-S.E.M.), and arterial pH decreased (7.37+/-0.02 versus 7.45+/-0.02 and 7.45+/-0.01, respectively, P=0.03). Plasma buprenorphine kinetics was well described by a three-compartment linear model, with a distribution half-life of 7.4+/-2.7 min and an elimination half-life of 463.9+/-152.3 min. However, neither plasma nor striatal buprenorphine kinetics were significantly altered by pre-administration of flunitrazepam. The adverse interaction between flunitrazepam and buprenorphine cannot be explained by a pharmacokinetic drug-drug interaction during the distribution phase of buprenorphine.

Aims: To assess the trends in the number, mortality and the nature of forensic cases involving toxicological detection of buprenorphine or methadone among toxicological investigations performed in Paris from June 1997 to June 2002., Design: Retrospective, 5 year study with review of premortem data, autopsy, police reports, hospital data, and post-mortem toxicological analyses., Setting and Participants: 34 forensic cases of buprenorphine and 35 forensic cases of methadone detection among 1600 toxicological investigations performed at the Laboratory of Toxicology in the Medical Examiner's Office in Paris., Measurements and Results: Therapeutic, toxic or lethal drug concentrations were defined based upon the results of blood analyses and the published literature. Drug concentrations were cross-referenced with other available ante- and post-mortem data. Subsequently, we classified a 'clear responsibility', 'possible responsibility' or 'not causative' role for buprenorphine or methadone in the death process, or 'no explanation of death'. Buprenorphine and methadone can be regarded as being directly implicated in, respectively, four of 34 death cases (12%) and three of 35 death cases (9%), and their participation in the lethal process is strongly plausible in eight (buprenorphine) and 11 (methadone) additional deaths., Conclusions: Analysis of causes of death reveals the difficulties in determining the role of substitution drugs in the death process, as many other factors may be involved, including circumstances surrounding death, past history, differential selection of subjects into either substitution modality and concomitant intake of other drugs (especially benzodiazepines and neuroleptics). The potential for synergistic or additive actions by other isolated molecules-particularly opioids, benzodiazepines, other psychotropes and alcohol-must be also considered.

High dose buprenorphine is used as substitution treatment in human heroin addiction. Deaths have been reported in addicts using buprenorphine, frequently in association with benzodiazepines. In the current study, we observed the effects of buprenorphine and midazolam alone and in combination on arterial blood gases. Four groups of 10 male Sprague-Dawley rats received a parenteral injection of aqueous solvent, buprenorphine (30 mg/kg, iv), midazolam (160 mg/kg, ip), or buprenorphine (30 mg/kg, iv) plus midazolam (160 mg/kg, ip). Serial blood gases were obtained over 3 hours. There was a mild but significant effect of buprenorphine alone in comparison with the aqueous solvent on PaCO2 at 60 min (6.24 vs. 5.65 kPa, p< 0.01). There was also a mild but significant effect of midazolam alone in comparison with aqueous solvent on arterial pH at 90 min (7.33 vs. 7.41,p< 0.001) and PaCO2 at 60 min (6.52 vs. 5.65 kPa,p< 0.01). The combination of midazolam and buprenorphine produces a rapid, profound, and prolonged respiratory depression, as demonstrated by an increase in PaCO2 at 7.65 +/- 0.12 kPa at 20 min and a decrease in arterial pH at 7.25 +/- 0.02 at 20 min, with appearance of delayed hypoxia with a decrease in PaO2 at 8.74 +/- 0.20 kPa at 120 min. These data show that high doses of midazolam and buprenorphine alone have limited effects on arterial blood gases in rats while midazolam and buprenorphine appear to act in an additive or synergistic fashion to depress ventilation in rats.

High dose buprenorphine, a potent semisynthetic agonist-antagonist for opiate receptors, is now used in substitution treatment of human heroin addiction. Deaths have been reported in addicts misusing buprenorphine. We determined the median lethal dose (LD(50)) and studied the effects of high doses of intravenous buprenorphine on arterial blood gases in rats. Male Sprague-Dawley rats were administered buprenorphine intravenously to determine the LD(50) using the up-and-down method. Subsequently, catheterized groups of 10 restrained rats received no drug, saline, acid-alcohol aqueous solvent (required to dissolve buprenorphine at a high concentration), or 3, 30, or 90 mg/kg of buprenorphine intravenously. Serial arterial blood gases were obtained over 3 h. The LD(50) determined in triplicate was 146.5 mg/kg (median of 3 series, range: 142.6-176.5). The mean dose received by surviving animals was 96.9 +/- 46.7 mg/kg. There was a significant effect of the acid-alcohol aqueous solvent on arterial blood gases. Excluding the solvent effect, 3, 30, and 90-mg/kg buprenorphine doses had no significant effects on arterial blood gases. The toxicity of intravenous buprenorphine in adult rats, assessed by the LD(50), is low. These data are consistent with a wide margin of safety of buprenorphine. The mechanism of death after the intravenous administration of a lethal dose of buprenorphine remains to be determined.