Patricia Risède

Respiratory toxicity of buprenorphine results from the blockage of P-glycoprotein-mediated efflux of norbuprenorphine at the blood-brain barrier in mice.

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.

Effects of Various Combinations of Benzodiazepines with Buprenorphine on Arterial Blood Gases in Rats

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.

Characteristics and comparative severity of respiratory response to toxic doses of fentanyl, methadone, morphine, and buprenorphine in rats

Opioids are known to induce respiratory depression. We aimed to characterize in rats the effects of four opioids on arterial blood gases and plethysmography after intraperitoneal administration at 80% of their LD(50) in order to identify opioid molecule-specific patterns and classify response severity. Opioid-receptor (OR) antagonists, including intravenous 10 mg kg(-1)-naloxonazine at 5 min [mu-OR antagonist], subcutaneous 30 mg kg(-1)-naloxonazine at 24 h [mu1-OR antagonist], subcutaneous 3 mg kg(-1)-naltrindole at 45 min [delta-OR antagonist], and subcutaneous 5 mg kg(-1)-Nor-binaltorphimine at 6 h [kappa-OR antagonist] were pre-administered to test the role of each OR. Methadone, morphine, and fentanyl significantly decreased PaO(2) (P<0.001) and increased PaCO(2) (P<0.05), while buprenorphine only decreased PaO(2) (P<0.05). While all opioids significantly increased inspiratory time (T(I), P<0.001), methadone and fentanyl also increased expiratory time (T(E), P<0.05). Intravenous 10 mg kg(-1)-naloxonazine at 5 min completely reversed opioid-related effects on PaO(2) (P<0.05), PaCO(2) (P<0.001), T(I) (P<0.05), and T(E) (P<0.01) except in buprenorphine. Subcutaneous 30 mg kg(-1)-naloxonazine at 24 h completely reversed effects on PaCO(2) (P<0.01) and T(E) (P<0.001), partially reversed effects on T(I) (P<0.001), and did not reverse effects on PaO(2). Naltrindole reversed methadone-induced T(E) increases (P<0.01) but worsened fentanyls effect on PaCO(2) (P<0.05) and T(I) (P<0.05). Nor-binaltorphimine reversed morphine- and buprenorphine-induced T(I) increases (P<0.001) but worsened methadones effect on PaO(2) (P<0.05) and morphine (P<0.001) and buprenorphines (P<0.01) effects on pH. In conclusion, opioid-related respiratory patterns are not uniform. Opioid-induced hypoxemia as well as increases in T(I) and T(E) are caused by mu-OR, while delta and kappa-OR roles appear limited, depending on the specific opioid. Regarding severity of opioid-induced respiratory effects at 80% of their LD(50), all drugs increased T(I). Methadone and fentanyl induced hypoxemia, hypercapnia, and T(E) increases, morphine caused both hypoxemia and hypercapnia while buprenorphine caused only hypoxemia.

Mechanisms of respiratory insufficiency induced by methadone overdose in rats.

Methadone may cause respiratory depression. We aimed to understand methadone‐related effects on ventilation as well as each opioid‐receptor (OR) role. We studied the respiratory effects of intraperitoneal methadone at 1.5, 5, and 15 mg/kg (corresponding to 80% of the lethal dose‐50%) in rats using arterial blood gases and plethysmography. OR antagonists, including intravenous 10 mg/kg‐naloxonazine at 5 minutes (mu‐OR antagonist), subcutaneous 30 mg/kg‐naloxonazine at 24 hours (mu1‐OR antagonist), 3 mg/kg‐naltrindole at 45 minutes (delta‐OR antagonist) and 5 mg/kg‐Nor‐binaltorphimine at 6 hours (kappa‐OR antagonist) were pre‐administered. Plasma concentrations of methadone enantiomers were measured using high‐performance liquid chromatography coupled to mass‐spectrometry. Methadone dose‐dependent inspiratory time (TI) increase tended to be linear. Respiratory depression was observed only at 15 mg/kg and characterized by an increase in expiratory time (TE) resulting in hypoxemia and respiratory acidosis. Intravenous naloxonazine completely reversed all methadone‐related effects on ventilation, while subcutaneous naloxonazine reduced its effects on pH (P < 0.05), PaCO2 (P < 0.01) and TE (P < 0.001) but only partially on TI (P < 0.001). Naltrindole reduced methadone‐related effects on TE (P < 0.001). Nor‐binaltorphimine increased methadone‐related effects on pH and PaO2 (P < 0.05) Respiratory effects as a function of plasma R‐methadone concentrations showed a decrease in PaO2 (EC50: 1.14 µg/ml) at lower concentrations than those necessary for PaCO2 increase (EC50: 3.35 µg/ml). Similarly, increased TI (EC50: 0.501 µg/ml) was obtained at lower concentrations than those for TE (EC50: 4.83 µg/ml). Methadone‐induced hypoxemia is caused by mu‐ORs and modulated by kappa‐ORs. Additionally, methadone‐induced increase in TE is caused by mu1‐ and delta‐opioid receptors while increase in TI is caused by mu‐ORs.

Acute renal failure alters the kinetics of pralidoxime in rats.

There is a trend towards increasing doses of pralidoxime to treat human organophosphate poisonings that may have relevance in subpopulations. Indeed, pralidoxime is eliminated unchanged by the renal route. This study assesses the effect of renal failure on the kinetics of pralidoxime in a rat model of acute renal failure induced by potassium dichromate administration. On the first day, Sprague-Dawley rats received subcutaneously potassium dichromate (study) or saline (control). Forty-eight hours post-injection, animals received pralidoxime methylsulfate (50mg/kg of pralidoxime base) intramuscularly. Blood specimens were sampled during 180min after the injection. Urine was collected daily during the 3 days of the study. Plasma pralidoxime concentrations were measured by liquid chromatography with electrochemical detection. There was a 2-fold increase in mean elimination half-life and a 2.5-fold increase in mean area under the curve in the study compared to the control group. The mean total body clearance was halved in the study compared to the control group. Our study showed acute renal failure does not modify the distribution of pralidoxime but significantly alters its elimination from plasma. These results suggest that dosages of pralidoxime should be adjusted in organophosphate-poisoned humans with renal failure when using high dosage regimen of pralidoxime.

Comparison of tolerance to morphine-induced respiratory and analgesic effects in mice.

Morphine is responsible for severe poisonings in chronically treated patients. We hypothesize that toxicity could be related to the development of weaker tolerance for morphine-induced deleterious respiratory effects in comparison to analgesic effects. Our objectives were to compare tolerance to both effects in mice and investigate possible mechanisms for such possible differences. Tolerance to morphine-induced analgesia and respiratory effects was assessed using hot plate response latencies and plethysmography, respectively. Mechanisms of tolerance were investigated using binding studies to mu-opioid receptors (MOR) and adenylate cyclase (AC) activity measurement in homogenates of cell membranes from the periaqueductal gray region (PAG) and brainstem. Morphine (2.5 mg/kg) was responsible for analgesia with significant increase in inspiratory time. Acute tolerance to analgesia (p<0.01) and effects on respiratory frequency (p<0.05) was observed in mice pre-treated with 100 mg/kg morphine in comparison to saline. Following repetitive administration (2.5 mg/kg/day during 10 days), we observed a 13-fold increase in the effective dose-50% (ED₅₀) of morphine-induced analgesia in comparison to a 2- or 4-fold increase in the ED₅₀ of its related increase in inspiratory time determined in air and 4% CO₂, respectively. No significant alteration in MOR expression was observed in either PAG or brainstem following repeated morphine administration. However, in PAG, in contrast to brainstem, superactivation of AC was observed in morphine-treated mice in comparison to controls (p<0.05). In conclusion, tolerance to morphine-induced respiratory effects is much more limited than tolerance to its analgesic effects in repeatedly morphine-treated mice. The difference in morphine-induced AC activation between the brainstem and the PAG contributes to the observed difference in tolerance between both morphine effects.

Pharmacokinetics and Toxicodynamics of Pralidoxime Effects on Paraoxon-Induced Respiratory Toxicity

Empirical studies suggest that the antidotal effect of pralidoxime depends on plasma concentrations with therapeutic effects associated with concentrations above 4 mg/l. The purpose of this study was to determine the pharmacokinetic-toxicodynamic (PK-TD) relationships for the antidotal effect of pralidoxime on paraoxon-induced toxicity in rats. Diethylparaoxon inactivation of whole-blood cholinesterase activity was studied both in vitro and in male Sprague-Dawley rats. Toxin-induced respiratory effects were measured via whole-body plethysmography in control and pralidoxime-treated animals (50 mg/kg im injection). In the in vitro analysis, cholinesterase reactivation by pralidoxime in blood-poisoned diethylparaoxon (10nM) was proportional to the logarithm of drug concentrations. A mechanism-based TD model was developed, which well described the inhibition of cholinesterases by diethylparaoxon and reactivation with pralidoxime. The in vitro pralidoxime EC(50) was estimated to be 4.67 mg/l. Animals exposed to diethylparaoxon exhibited a decrease in respiratory rate and an increase in expiratory time, and pralidoxime treatment resulted in a rapid complete but transient (< 30 min) correction in respiratory toxicity. In contrast, there was a fast and total reactivation of blood cholinesterase activity over the 210-min study period. The in vitro TD model was extended to capture the time-course of in vivo pralidoxime antidotal effects, which explained the complex relationship between drug exposure and pharmacological response profile. This study provides insights into the role of oxime-rescue of paraoxon-induced toxicity, and the final PK-TD model might prove useful in optimizing the design and development of such therapy.

Study of Blood and Brain Lithium Pharmacokinetics in the Rat According to Three Different Modalities of Poisoning

Lithium-induced neurotoxicity may be life threatening. Three patterns have been described, including acute, acute-on-chronic, and chronic poisoning, with unexplained discrepancies in the relationship between clinical features and plasma lithium concentrations. Our objective was to investigate differences in plasma, erythrocyte, cerebrospinal fluid, and brain lithium pharmacokinetics using a multicompartmental approach in rat models mimicking the three human intoxication patterns. We developed acute (intraperitoneal administration of 185 mg/kg Li₂CO₃ in naive rats), acute-on-chronic (intraperitoneal administration of 185 mg/kg Li₂CO₃ in rats receiving 800 mg/l Li₂CO₃ in water during 28 days), and chronic poisoning models (intraperitoneal administration of 74 mg/kg Li₂CO₃ during 5 days in rats with 15 mg/kg K₂Cr₂O₇-induced renal failure). Delayed absorption (4.03 vs 0.31 h), increased plasma elimination (0.65 vs 0.37 l/kg/h) and shorter half-life (1.75 vs 2.68 h) were observed in acute-on-chronically compared with acutely poisoned rats. Erythrocyte and cerebrospinal fluid kinetics paralleled plasma kinetics in both models. Brain lithium distribution was rapid (as early as 15 min), inhomogeneous and with delayed elimination (over 78 h). However, brain lithium accumulation was more marked in acute-on-chronically than acutely poisoned rats [area-under-the-curve of brain concentrations (379 ± 41 vs 295 ± 26, P < .05) and brain-to-plasma ratio (45 ± 10 vs 8 ± 2, P < .0001) at 54 h]. Moreover, brain lithium distribution was increased in chronically compared with acute-on-chronically poisoned rats (brain-to-plasma ratio: 9 ± 1 vs 3 ± 0, P < .01). In conclusion, prolonged rat exposure results in brain lithium accumulation, which is more marked in the presence of renal failure. Our data suggest that differences in plasma and brain kinetics may at least partially explain the observed variability between human intoxication patterns.

Respiratory effects of buprenorphine/naloxone alone and in combination with diazepam in naive and tolerant rats

Respiratory depression has been attributed to buprenorphine (BUP) misuse or combination with benzodiazepines. BUP/naloxone (NLX) has been marketed as maintenance treatment, aiming at preventing opiate addicts from self-injecting crushed pills. However, to date, BUP/NLX benefits in comparison to BUP alone remain debated. We investigated the plethysmography effects of BUP/NLX in comparison to BUP/solvent administered by intravenous route in naive and BUP-tolerant Sprague-Dawley rats, and in combination with diazepam (DZP) or its solvent. In naive rats, BUP/NLX in comparison to BUP significantly increased respiratory frequency (f, P<0.05) without altering minute volume (VE). In combination to DZP, BUP/NLX significantly increased expiratory time (P<0.01) and decreased f (P<0.01), tidal volume (VT, P<0.001), and VE (P<0.001) while BUP only decreased VT (P<0.5). In BUP-tolerant rats, no significant differences in respiratory effects were observed between BUP/NLX and BUP. In contrast, in combination to DZP, BUP/NLX did not significantly alter the plethysmography parameters, while BUP increased inspiratory time (P<0.001) and decreased f (P<0.01) and VE (P<0.001). In conclusion, differences in respiratory effects between BUP/NLX and BUP are only significant in combination with DZP, with increased depression in naive rats but reduced depression in BUP-tolerant rats. However, BUP/NLX benefits in humans remain to be determined.

Influence of various combinations of specific antibody dose and affinity on tissue imipramine redistribution

1 This study was designed to evaluate the distribution kinetics of imipramine (Imip) in the brain and the main peripheral organs (heart, kidney, liver and lung) of rats, and to establish the relationship between the redistribution of Imip from these tissues and the immunoreactive capacity (dose and affinity) of anti‐TCA IgG. 2 [3H]‐Imip (1 nmol kg−1 body weight) was injected intravenously 6 min before the i.v. injection of antibodies. At this time, the concentrations of Imip and its main metabolites in plasma were determined. The radioactivity measured corresponded to 91.7% Imip, indicating that the pharmacokinetics reflected essentially Imip. Plasma and tissue Imip contents were measured over the interval 1 to 90 min in control and in treated rats. The antibodies used were a murine monoclonal IgG1 (Ka=3.8 107 M−1) at an IgG1/Imip molar ratio of 1000 (IgG1 1000), and a sheep polyclonal IgG (TAb, Ka=1.3 1010 M−1) at IgG/Imip molar ratios of 1, 10 and 100 (TAb1, TAb10 and TAb100). 3 The anti‐TCA IgG increased the plasma [3H]‐Imip concentrations: the AUC1→60 min for [3H]‐Imip were 4 (IgG1 1000), 9 (TAb1), 33.9 (TAb10) and 41.4 (TAb100) times higher in the treated groups than in the controls. The opposite effect occurred in the brain, heart and lungs, with large, rapid decreases in Imip. The increase in plasma Imip and the decrease in tissue Imip depended on the immunoreactive capacity (NKa) of the antibody, where N=molar concentration of IgG binding sites and Ka=IgG affinity constant. Maximal plasma and tissue redistribution occurred when NKa=33.8×104. 4 Imip redistribution can be controlled using various doses or affinities of specific antibodies, and the resulting rapid, extensive Imip redistribution from the main target organs could be very promising for TCA detoxification.