* INSERM U26 et Université Paris VII, Hôpital Fernand Widal, 200, rue du Faubourg Saint-Denis, 75475 Paris Cedex 10, France
Department of Emergency Medicine, George Washington University, Washington, DC 20037
International Toxicology Consultants, LLC, 2000 L Street NW, Suite 200, Washington, DC 200364924
Laboratoire de Biochimie-Toxicologie, Hôpital Fernand Widal, 75475 Paris, France and
¶ Laboratoire de Biomathématiques, Université Paris V, UFR Pharmacie, 75006 Paris, France
Received March 30, 2001; accepted September 4, 2001
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ABSTRACT |
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Key Words: buprenorphine; midazolam; acute toxicity; respiratory depression; safety; rats; LD50; arterial blood gas; opioids; heroin substitution; drug abuse.
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INTRODUCTION |
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The approach to treatment of heroin addiction has undergone a profound evolution with the development of substitution treatments such as methadone, levomethadyl acetate, and buprenorphine. Products of substitution have been introduced in a number of countries with the goal of reducing mortality and morbidity associated with intravenous drug abuse, improving the addict's chances of reintegration into society. High dose (816 mg/day) buprenorphine has been available in France since 1996 and a recent report suggests that high-dose buprenorphine, like high dose methadone and levomethadyl acetate, substantially reduces the use of illicit opioids (Johnson et al., 2000).
Dose-effect relationships of buprenorphine both in animals and humans suggest a plateau of respiratory effects (Cowan et al., 1977; Walsh et al., 1994
) or no effect at all (Ohtani et al., 1997
). The plateau effect of buprenorphine appears of utmost importance regarding its safety for use in substitution treatment (Cowan et al., 1977
; Walsh et al., 1994
). In a previous study, we assessed the LD50 of intravenous buprenorphine in adult rats and measured the arterial blood gases in rats acutely exposed to high doses of buprenorphine (Gueye et al., 2001
). We did not observe any significant effect of the 3, 30, or 90 mg/kg doses of buprenorphine distinct from the effect of the aqueous solvent on arterial blood gases. It should be noted that 90 mg/kg approaches the minimum lethal dose (120 mg/kg) found in our LD50 study (Gueye et al., 2001
). In contrast with methadone, which induces potent respiratory depressant effects in rats (McCormick et al., 1984
), our data are consistent with a limited effect on respiration of a single intravenous dose of up to 90 mg/kg buprenorphine alone, as assessed by arterial blood gases.
However, deaths have been reported during substitution with buprenorphine in humans (Brenet et al., 1998; Reynaud et al., 1998
; Tracqui et al., 1998
). Deaths may result from either misuse or overdose with substitution treatment (Robinson et al., 1993
; Tracqui et al., 1998
). In legitimate substitution treatment, buprenorphine is prescribed sublingually. However, there is now considerable evidence that buprenorphine is misused and administered by the intravenous route (Robinson et al., 1993
).
Furthermore, recent reports have emphasized the combination of substitution products (methadone or buprenorphine) with psychotropic drugs as a major factor in fatalities among heroin addicts. More especially, the combination of buprenorphine and benzodiazepines is widely used by heroin addicts, and this combination is considered as a risk factor of lethal overdose (Drummer et al., 1993; Hammersley et al., 1995
; Reynaud et al., 1998
; Tracqui et al., 1998
). However, benzodiazepines are considered relatively safe drugs and deaths caused by benzodiazepines alone in the absence of other pathology are uncommon (Drummer et al., 1993
; Finkle et al., 1979
; Serfaty and Masterton, 1993
). In rats, diazepam alone did not cause any significant changes in arterial pH or PaCO2. In contrast, there is a real potential for severe respiratory depression, as assessed by arterial blood gas measurement, when the combination of methadone and diazepam is given acutely to drug-naive rats (McCormick et al., 1984
).
To address the acute toxicity of buprenorphine in combination with midazolam, we performed the following study. We first determined the maximum nonlethal dose of intravenous midazolam in adult rats. Then, we studied the effects of buprenorphine and midazolam alone and in combination on respiratory rate and arterial blood gases in adult rats.
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MATERIALS AND METHODS |
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Animals
Animals employed were Sprague-Dawley male rats (Iffa-Credo, France) weighing between 250 and 300 g at the time of experimentation. They were housed for 8 days before experimentation in a temperature- and light-controlled animal-care unit. They were allowed food and water ad libitum until 1 day prior to experimentation.
Drugs
Buprenorphine hydrochloride was generously supplied by Schering-Plough, SA. It was subsequently diluted in a mixture of sterile water, ethanol, and hydrochloric acid 0.1 N at a pH of 4.0, at a concentration of 18.5 mg/ml, the greatest concentration achievable without further lowering the pH. This mixture of water, ethanol, and hydrochloric acid is referred to throughout this paper as "aqueous solvent."
Midazolam was generously supplied by Hoffman-La Roche, Inc. It was subsequently diluted in a mixture of sterile water and hydrochloric acid 0.1 N at a pH of 4.0.
Study 1: Determination of the Maximum Nonlethal Dose of Midazolam
To determine a toxic but nonlethal dose of midazolam, we first determine the LD50 of intravenous midazolam. Approximately 18 h prior to experimentation, the animals were fasted but allowed free access to water. The rats were placed individually in horizontal, Plexiglas cylinders (internal diameter: 6.5 cm, adjustable length: up to 20 cm, Harvard Apparatus, Inc., Massachusetts, U.S.). Midazolam was administered in awake, restrained animals via the tail vein. Animals were examined repeatedly during the first 4 h after injection, then daily, for evidence of drug-related side effects or other illness. Following drug administration, animals were placed in individual cages and allowed to eat and drink, and were maintained in the laboratory, which was temperature-controlled with day lighting.
Animals were observed for a period of 7 days after midazolam injection. At the end of the study period, animals were euthanized using a CO2 chamber.
The up-and-down method, as proposed by Dixon (Dixon 1991; Dixon and Mood 1948
) and refined by Bruce (Bruce, 1985
, 1987
), was employed to determine the LD50 in triplicate, calculated on the basis of final dose, outcome/dose pattern, and dose interval.
Study 2: Effects of a High Dose of Buprenorphine or Midazolam Alone and in Combination on Respiratory Rate and Arterial Blood Gases
Catheterization.
The day before the study, the animals were anesthetized with ketamine (Ketalar®), 70 mg/kg, and xylazine (Rompum®), 10 mg/kg, intraperitoneally, then placed on a warming blanket with a regulating thermostat. A rectal probe permitted feedback control of the temperature. The femoral vein and artery were catheterized with Silastic® tubing with external and internal diameters of 0.94 and 0.51 mm, respectively; length 30 cm (Dow Corning Co, Michigan). The technique of catheterization was described elsewhere (Gueye et al., 2001).
The day of experimentation, rats were placed individually in horizontal Plexiglas cylinders (internal diameter: 6.5 cm, adjustable length up to 20 cm, Harvard Apparatus, Inc., Massachusetts). The Plexiglas cylinders are provided with several openings on the cranial end in order to prevent CO2 rebreathing.
Drug administration and collection of arterial blood gases.
The venous catheter permitted the administration of the study drug. The arterial catheter permitted blood collection for arterial blood gases. Midazolam (160 mg/kg) was administered by the intraperitoneal route. Buprenorphine (30 mg/kg) or aqueous solvent, in a volume of 1.2 ml, was administered by the femoral vein over 3 min by an infusion pump at a constant rate (Harvard InstrumentsPHD 2000, USA). In combination, midazolam was first administered, ip, followed 30 min later by the iv injection of buprenorphine. For the measurement of arterial blood gases, blood samples of 300 µl were collected in a heparinized syringe from the arterial catheter. Arterial blood samples were collected before and at 5, 20, 60, 90, 120, and 180 min after the administration of the drug, and immediately measured by means of a blood gas analyzer (Radiometer ABL 300, Copenhagen, Denmark). The first blood sample collected after injection in the midazolam group was 35 min after intraperitoneal injection to allow for direct comparison between groups. This delay permitted the onset of coma in animals having received midazolam.
Respiratory rate.
At each sampling time, the respiratory rate was counted for 1 min, the count being based on the up-and-down movement of the abdomen caused by the animal's breathing.
At the end of experiments, rats were euthanized by the injection of a lethal dose of sodium pentobarbital.
Statistical Analysis
Four groups (aqueous solvent, midazolam, buprenorphine, and combination groups) of 10 restrained animals were used. The results are expressed as mean ± SEM. Baseline values were compared using one-way analysis of variance, followed by multiple comparison tests using Bonferroni's correction. In each group, comparisons of drug effects to baseline values were performed using repeated measures ANOVA and Dunnett's multiple comparison tests. The effects of the acute administration of buprenorphine or midazolam alone were compared to those of the aqueous solvent. Then, the effects of buprenorphine alone or midazolam alone were compared to those induced by the two in combination. Then, for each sampling time and each drug we calculated the difference between the value at that time and its corresponding baseline value. These differences were compared using one-way analysis of variance followed by multiple-comparison tests using Bonferroni's correction. All tests were performed using Prism version 2.0 (GraphPad Software, Inc., San Diego, CA), were 2-tailed, and p values of less than 0.05 were considered significant.
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RESULTS |
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Respiratory rate: Baseline values before treatment.
There were no significant differences of the baseline values of the respiratory rate in the 4 treatment groups.
Comparison with baseline values
In the aqueous solvent and buprenorphine groups, there were no significant differences in the respiratory rate at any time in comparison with the baseline value. In the midazolam group, the respiratory rate was significantly lower than the baseline value at only 5 min post-injection (84 ± 7/min vs. 107 ± 2/min: p < 0.01). In the combination group, the respiratory rate was significantly lower than the baseline value at all times (p < 0.01); the lowest mean respiratory rate was recorded at 20 min post-injection (76 ± 5/min vs. 112 ± 5/min, respectively).
Effects of treatment at each sampling time.
The respiratory rate in the combination group was significantly lower than that in the buprenorphine group at 20 min (76 ± 5/min vs. 101±3/min, respectively: p < 0.05) and 60 min (77 ± 4/min vs. 104 ± 4/min, respectively: p < 0.01). The respiratory rate in the combination group was significantly lower than that in the midazolam group at 120 min (86 ± 6/min vs. 104 ± 4/min, respectively: p < 0.05; Fig. 2).
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Comparison with Baseline Values
The aqueous solvent group.
There were no significant effects in this treatment group on the arterial pH, PaCO2, and blood bicarbonate concentrations in comparison with the baseline values. The PaO2 value at 5 min was slightly but significantly lower than the baseline value (10.74 ± 0.54 kPa vs. 11.95 ± 0.15 kPa, p < 0.05).
The midazolam group (160 mg/kg, ip).
When compared with baseline values, this group showed significant differences on the arterial pH from 90 min post-injection, on PaCO2 at 60 and 90 min, on blood bicarbonate concentrations from 60 min, and on PaO2 at 90 and 180 min.
The buprenorphine group (30 mg/kg, iv).
In comparison with the baseline values, this group had significant differences in the arterial pH at 20 and 60 min, and in the PaO2 at 5 and 20 min. There was no significant effect on the PaCO2 or blood bicarbonate concentrations compared with the baseline values.
The combination group.
In comparison with the baseline values, there were significant differences on the arterial pH and the PaCO2 at all times in this group. The blood bicarbonate concentrations were significantly lower in comparison with the baseline value at 5, 120, and 180 min. The PaO2 values were significantly lower in comparison with the baseline value at 5, 90, 120, and 180 min.
Effects of Treatment at Each Sampling Time
pH.
There were no significant differences of arterial pH between the aqueous solvent and midazolam or buprenorphine groups, except at 90 min when the arterial pH in the midazolam group was significantly lower than that of the aqueous solvent (p < 0.001; Fig. 3). The lowest pH values in the aqueous solvent, midazolam, and buprenorphine groups were 7.39 ± 0.02 at 120 min, 7.33 ± 0.01 at 90 min, and 7.38 ± 0.01 at 120 min, respectively.
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PaCO2.
The PaCO2 in both midazolam and buprenorphine groups was significantly greater than that in the aqueous solvent group at only 60 min (p < 0.01) (Fig. 4). The greatest PaCO2 values in the aqueous solvent, midazolam, and buprenorphine groups were 6.10 ± 0.33 kPa at 5 min, 6.52 ± 0.17 kPa at 60 min, and 6.43 ± 0.31 kPa at 20 min, respectively.
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Blood bicarbonate concentrations.
The blood bicarbonate concentrations in the midazolam group were significantly lower than in the aqueous solvent group at only 180 min (p < 0.05). There were no significant differences of the blood bicarbonate concentrations between the aqueous solvent and buprenorphine groups at any time (Fig. 5). The lowest blood bicarbonate concentration values in the aqueous solvent, midazolam, and buprenorphine groups were 26.8 ± 0.3 mmol/l at 120 min, 24.2 ± 0.9 mmol/l at 180 min, and 27.2 ± 0.6 mmol/l at 90 min, respectively.
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PaO2.
There were no significant differences of the PaO2 between the aqueous solvent and midazolam or buprenorphine group at any time (Fig. 6). The lowest PaO2 values in the aqueous solvent, midazolam, and buprenorphine groups were 10.74 ± 0.54 kPa at 5 min, 11.40 ± 0.22 kPa at 20 min, and 10.37 ± 0.36 kPa at 20 min, respectively.
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DISCUSSION |
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Benzodiazepines have been shown to be relatively free of severe respiratory effects when used alone (McCormick et al., 1984; Bahar et al., 1997
; Verborgh et al., 1998
). Midazolam is a benzodiazepine commonly used as an induction agent or to provide sedation. In an initial phase of our study, we assessed the acute toxicity of midazolam, finding an intravenous LD50 of 357 mg/kg, far greater than that previously reported (Schläppi 1983
). Our LD50 study demonstrated a low toxicity of intravenous midazolam alone in rat. In the subsequent blood-gas study, we used a dose of midazolam (160 mg/kg, ip), which alone reproducibly induced a deep coma with zero mortality and moderate respiratory depression. Indeed, the highest PaCO2 was 6.52 ± 0.17 kPa while the lowest arterial pH was 7.33 ± 0.01 after 90 min. An important finding was that in spite of respiratory acidosis, the PaO2 did not change following midazolam administration while the animals were breathing room air. In fact, an increase in PaO2 has been reported in rats following subcutaneous administration of 10 mg/kg chlordiazepoxide (Verborgh et al., 1998
), or ip administration of 20 mg/kg diazepam (McCormick et al., 1984
). A decrease in oxygen consumption may explain the lack of effect on PaO2. Indeed, a decrease in oxygen consumption has been shown in man following the administration of anesthetic doses of various benzodiazepines (Marty et al., 1986
; Nitenberg et al., 1983
). The greatest decrease in oxygen consumption in man was observed with midazolam (Marty et al., 1986
; Mohan et al., 1988
).
The doses used in this study were far greater (on a mg/kg basis) than those used in humans. Indeed, the doses of buprenorphine and midazolam used in this animal study were in the toxic range. Rats received 160 mg/kg midazolam, ip, while sedative doses used in humans range between 0.05 to 0.2 mg/kg (Forster et al., 1983; Morel et al., 1984
). Our rats received 30-mg/kg buprenorphine, iv. In adult humans, buprenorphine is used as an analgesic at the recommended parenteral dose of 0.3 to 0.6 mg (Watson et al., 1982
). In substitution treatment of opioid addiction, the doses typically range between 2 and 16 mg sublingually, daily, which corresponds to approximately 0.03 to 0.23 mg/kg (Johnson et al., 1995
). However, Walsh showed in healthy adult male volunteers a plateau of respiratory effect by sublingual buprenorphine at doses up to 32 mg (Walsh et al., 1994
). Our study showed that large doses of buprenorphine and midazolam alone induced limited effects on respiratory rate and arterial blood gases in rats. However, the two in combination induced a severe respiratory depression as assessed by frank respiratory acidosis and hypoxemia. The lowest arterial pH, 7.25 ± 0.02, occurred within 5 min after the combination of midazolam + buprenorphine treatment, while the greatest PaCO2, 7.65 ± 0.12 kPa, was recorded 20 min later. Both the arterial pH and the PaCO2 improved over time but did not return to control values, even at the end of the study. Similarly, McCormick et al reported severe and long-lasting respiratory acidosis following the administration of 20-mg/kg diazepam subcutaneously in association with 5 mg/kg of methadone intraperitoneally. Furthermore, Verborgh et al. (1998) showed that the combination of chlordiazepoxide with either morphine or sufentanyl significantly increased the PaCO2. However, in contrast to buprenorphine, both morphine and methadone are known to significantly depress respiration when administered alone (McCormick et al., 1984
; Verborgh et al., 1998
). Our results and those of others (Cowan et al., 1977
; Ohtani et al., 1997
) showed limited or no effect of buprenorphine alone on arterial blood gases in rats. Thus, we conclude that the limited and transient effect on respiration of high-dose buprenorphine alone does not hold when used in combination with midazolam. In fact, buprenorphine and midazolam appear to act in at least an additive fashion to depress respiration in rats.
Numerous reports have shown evidence of significant interactions between benzodiazepines and opioids with regard to analgesia (Dosaka-Akita et al., 1992; Paakkari et al., 1993
). These interactions seem rather complex, as some reports indicated potentiation of the effects, while others suggested antagonism (Bradshaw et al., 1973
; Brady et al., 1984
; Shannon et al., 1976
). It has long been recognized that the respiratory depression induced by opiates may be aggravated by the addition of sedative-hypnotic drugs such as benzodiazepines or barbiturates. In spite of the awareness of this phenomenon, little is known about the quantitative aspects of these relationships. Our data show that 2 drugs with limited and transient effects on respiration can induce a severe, prolonged respiratory depression when used in combination.
A number of mechanisms have been suggested to explain the interaction of opioids and benzodiazepines. An interaction may result from a pharmacokinetic or a pharmacodynamic process. The major metabolite of buprenorphine, norbuprenorphine, was shown to have a severe respiratory depressant effect (Ohtani et al., 1997). However, we are not aware of any study dealing with the metabolic interactions of buprenorphine or norbuprenorphine with midazolam. At pharmacological doses, midazolam is metabolized in the rat by cytochrome P450 3A1 and 3A2 (Greenblatt and Abernethy, 1985
) while buprenorphine is metabolized by cytochrome P450 3A4 (Kobayashi et al., 1998
; Kilicarslan, and Sellers, 2000
). Furthermore, it seems unlikely that metabolic interactions may explain the rapid onset, 5 min after buprenorphine injection, of the respiratory depression observed in our study.
The opioids appear to depress respiration through their agonist activity at µ and receptors (Shook et al., 1990
) while the
receptor agonists appear to be protective (Dosaka-Akita et al., 1992
; Shook et al., 1990
). The benzodiazepines act at the GABAA receptors (Gravish and Snyder, 1980
; Haefeley, 1990
). The potential for effects of certain benzodiazepines at the opiate receptors and vice versa has been demonstrated (Brady et al., 1984
; Rodgers et al., 1985
). Indeed, the anti-nociceptive effects of morphine may be diminished by the administration of flumazenil, a specific benzodiazepine receptor antagonist (Brady et al., 1984
). Conversely, the diminished activity noted after administration of the benzodiazepine chlordiazepoxide, when administered in high doses (20 mg/kg) could be further diminished by the administration of the specific µ-antagonist naloxone (Rodgers et al., 1985
). However, receptor specificity is typically studied at pharmacological doses. We cannot assume that these relationships hold at the high doses employed in our study.
Nonspecific factors may also explain our findings. Stress may antagonize or mask the respiratory effects of opiates (Hanks and Twycross, 1984). Several authors have suggested that benzodiazepines may reduce the stress, increasing the magnitude of opiate-induced respiratory depression (Van den Hoogen et al., 1989
; Verborgh et al., 1998
).
Finally, it is worth noting that delayed significant hypoxia was observed in the combination group, even after other respiratory parameters began to improve. In the absence of respiratory physiological studies, we cannot be sure of the mechanisms of this effect.
Conclusion
Buprenorphine and midazolam alone, in the doses tested, induce a mild and transient respiratory depression in comparison with the aqueous solvent. In contrast, the combination of midazolam and buprenorphine produces a rapid, profound, and prolonged respiratory depression as demonstrated by the early and sustained increase in PaCO2, decrease in arterial pH, and delayed decrease in PaO2. These data suggest that midazolam and buprenorphine act in an at least additive fashion to depress ventilation in rats.
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ACKNOWLEDGMENTS |
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NOTES |
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