Lucie Chevillard
email: luciechevillard@gmail.com

Xavier Declèves

Frédéric J Baud

Patricia Risède

Bruno Mégarbane

Respiratory effects of diazepam/methadone combination in rats: a study based on concentration/effect relationships

Keywords: ANOVA, analysis of variance, AUC, area under the curve, BZD, benzodiazepine, CYP, cytochrome P450, DZP, diazepam, EDDP, 2-ethylidene-1 Diazepam, effect/concentration relationships, metabolism, methadone, plethysmography, respiratory effects

Methadone may cause respiratory depression and fatalities. Concomitant use of benzodiazepines in methadone-treated patients for chronic pain or as maintenance therapy for opiate abuse is common.

However, the exact contribution of benzodiazepines to methadone-induced respiratory toxicity remains debatable. We investigated the respiratory effects of the combination diazepam (20 mg/kg)/methadone (5 mg/kg) in the rat, focusing on methadone concentration/effect relationships. Respiratory effects were studied using arterial blood gases and whole-body plethysmography. Plasma concentrations of both Rand S-methadone enantiomers were measured using high-performance liquid chiral chromatography coupled to mass spectrometry. To clarify mechanisms of diazepam/methadone interaction, methadone metabolism was investigated in vitro using rat liver microsomes. Diazepam/methadone co-administration significantly increased methadone-related effects on inspiratory time (p < 0.001) but did not significantly alter the other respiratory parameters when compared with methadone alone, despite significant increase in the area under the curve of plasma R-methadone concentrations measured during 240 min (p < 0.05).

Diazepam/methadone co-incubation with microsomes in vitro resulted in a significant inhibition of methadone metabolism (p < 0.01), with 50%-inhibitory diazepam concentrations of 25.02 ± 0.18 µmol/L and 25.18 ± 0.23 µmol/L for R-and S-methadone, respectively. We concluded that co-administration of high-doses of diazepam and methadone in rats is not responsible for additional respiratory depression in comparison to methadone alone, despite significant metabolic interaction between the drugs. In humans, although our experimental data may suggest the relative safety of benzodiazepine/methadone coprescription, physicians should remain cautious as other underlying conditions may enhance this drugdrug interaction.

Introduction

Methadone, a full agonist opioid, is widely used as treatment for chronic pain and for maintenance therapy in heroin addicts [START_REF] Cicero | Diversion and abuse of methadone prescribed for pain management[END_REF][START_REF] Mattick | Buprenorphine maintenance versus placebo or methadone maintenance for opioid dependence[END_REF]. Marketed methadone is a racemic mixture, with R-methadone believed to account for most, if not all, of its therapeutic effects and S-methadone suspected of playing a significant role in its adverse reactions [START_REF] Mitchell | Subjective and physiological responses among racemic-methadone maintenance patients in relation to relative (S)-vs (R)-methadone exposure[END_REF]. In humans, methadone (6-dimethylamino-4,4-diphenyl-3-heptanone) is metabolized by multiple cytochrome P450 (CYP) isoforms including 3A4, 2B6, 2C19, 2C9, and 2D6 to 2-ethyl-1,5-dimethyl-3,3diphenylpyrrolidine (EDDP), an inactive primary metabolite [START_REF] Iribarne | Involvement of cythochrome P450 3A4 enzyme in the N-demethylation of methadone in human liver microsomes[END_REF], with the potential risk of significant drug-drug interactions.

A steep rise in recreational use of prescription opioids was observed during the past two decades, resulting in increased fatalities mainly in relation to methadone abuse [START_REF] Paulozzi | Opioid analgesic involvement in drug abuse deaths in American metropolitan areas[END_REF]. Methadone is increasingly prescribed and, as a result of its increased availability to patients, adjunctively diverted to recreational or habitual opioid abusers [START_REF] Cicero | Diversion and abuse of methadone prescribed for pain management[END_REF]. To date, although representing less than 5% of prescribed opioids in the U.S., methadone has been implicated in one-third of opioid-related poisonings [START_REF] Webster | An analysis of the root causes for opioid-related overdose deaths in the United States[END_REF].

Circumstances of methadone poisonings include overdoses and drug-drug interactions.

Benzodiazepines (BZDs) are commonly used by opioid abusers to reduce anxiety, reinforce opioid effects, and treat craving and withdrawal symptoms [START_REF] Lintzeris | Benzodiazepines, methadone and buprenorphine: interactions and clinical management[END_REF]. Prevalence of BZD use among methadone-maintained patients ranges from 50 to 70% [START_REF] Jones | Polydrug abuse: A review of opioid and benzodiazepine combination use[END_REF]. Screening for BZDs was found to be positive in more than 85% of methadone-intoxicated patients admitted to the intensive care unit [START_REF] Mégarbane | Prospective comparative assessment of buprenorphine overdose with heroin and methadone: clinical characteristics and response to antidotal treatment[END_REF]. Among BZDs, diazepam is one of the most preferred, prescribed, and thus abused molecules (Bramness and Kornor, 2007;[START_REF] Iguchi | Benzodiazepine and sedative use/abuse by methadone maintenance clients[END_REF].

The real contribution of BZDs to methadone-related respiratory toxicity and fatalities remains under debate [START_REF] Häkkinen | Comparison of fatal poisonings by prescription opioids[END_REF][START_REF] Jones | Polydrug abuse: A review of opioid and benzodiazepine combination use[END_REF][START_REF] Paulozzi | A comparison of drug overdose deaths involving methadone and other opioid analgesics in West Virginia[END_REF]. In rats, methadone has been shown to induce a dose-dependent increase in inspiratory time (T I ) with an additional increase in expiratory time (T E ) at elevated doses, resulting in respiratory acidosis and hypoxemia [START_REF] Chevillard | Mechanisms of respiratory insufficiency induced by methadone overdose in rats[END_REF][START_REF] Mccormick | Effects of diazepam on the arterial blood gas concentrations and pH of adults rats acutely and chronically exposed to methadone[END_REF][START_REF] White | Acute and chronic methadone exposure in adult rats: studies on arterial blood gas concentrations and pH[END_REF]. In contrast, BZDs were shown to weakly alter arterial blood gases, even at elevated doses [START_REF] Mccormick | Effects of diazepam on the arterial blood gas concentrations and pH of adults rats acutely and chronically exposed to methadone[END_REF][START_REF] Mégarbane | Cerebral and plasma kinetics of a high dose of midazolam and correlations with its respiratory effects in rats[END_REF][START_REF] Nielsen | The effect of buprenorphine and benzodiazepines on respiration in the rat[END_REF], while BZD pre-treatment reduced methadone's median lethal dose [START_REF] Borron | Flunitrazepam variably alters morphine, buprenorphine, and methadone lethality in the rat[END_REF] and potentiated methadone-related respiratory depression [START_REF] Mccormick | Effects of diazepam on the arterial blood gas concentrations and pH of adults rats acutely and chronically exposed to methadone[END_REF][START_REF] Nielsen | The effect of buprenorphine and benzodiazepines on respiration in the rat[END_REF]. A pharmacokinetic interaction between methadone and diazepam was suggested in rats, in relation to a non-competitive inhibition of methadone metabolism by diazepam [START_REF] Liu | Correlation of urinary excretion of methadone metabolites with methadone metabolism and analgesia in the rat[END_REF][START_REF] Spaulding | The effect of diazepam on the metabolism of methadone by the liver of methadone-dependent rats[END_REF]. However, using human microsomes, diazepam appeared to be a weak competitive CYP3A4 inhibitor in vitro, questioning the clinical relevance of such metabolic interaction [START_REF] Iribarne | Interaction of methadone with substrates of human hepatic cytochrome P450 3A4[END_REF].

Consistently, clinical investigations in methadone-treated patients ruled out significant pharmacokinetic interactions between diazepam and methadone [START_REF] Pond | The lack of effect of diazepam on methadone metabolism in methadone-maintained addicts[END_REF][START_REF] Preston | Diazepam and methadone blood levels following concurrent administration of diazepam and methadone[END_REF].

Given the possible role of BZDs in methadone-related toxicity and concerns regarding the in vivo relevance of pharmacokinetic interaction between these drugs, we designed an experimental study to better quantify the respiratory effects resulting from diazepam/methadone interaction, aiming to analyze its mechanism and clarify the exact role of each methadone enantiomer.

Experimental procedures

Animals

Male Sprague-Dawley rats (Janvier, France) weighing between 250 and 350 g at the time of experimentation were used. Animals were housed for 5 days before experimentation in an environment maintained at 21  0.5°C with controlled humidity and a light-dark cycle (lights on between 08:00 and 20:00 h). Food and tap water were provided ad libitum. All experimental protocols used in this study were carried out within the ethical guidelines established by the National Institutes of Health, the French Minister of Agriculture, and the European Union Legislation. Protocols were approved by the local ethics committee for animal experimentation of Paris-Descartes University.

Chemicals and drugs

Methadone hydrochloride and diazepam were purchased from Francopia, France. Pooled male rat liver microsomes (20 mg/mL in a 0.25 mol/L sucrose solution) and nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system were purchased from Becton Dickinson Gentest, Le Pont de Claix, France. All other chemicals were purchased from Sigma, Saint-Quentin Fallavier, France.

Methadone was diluted in Tween-20 ® and sterile water to obtain solutions of 16 and 0.1 mmol/L, respectively. Diazepam was diluted in Tween-20 ® and methanol to obtain solutions of 70 and 10 mmol/L, respectively.

Femoral artery catheterization

The day before the study, animals were anesthetized with 70 mg/kg ketamine (Ketalar  ) and 10 mg/kg xylazine (Rompum  ) intraperitoneally (i.p.) then placed on a warming blanket with regulating thermostat.. The femoral artery was catheterized using 30 cm-silastic tubing with external and internal diameters of 0.94 and 0.51 mm, respectively (Dow Corning Co., Midland, MI). Arterial catheters were tunneled subcutaneously (s.c.) and fixed at the back of the neck. Heparinized saline was injected into the catheter to avoid thrombosis and catheter obstruction. Rats were then returned to their individual cages for a minimum recovery period of 24 h, to allow complete anesthesia washout. Moreover, ketamine/xylazine combination, routinely used for short surgical procedures, was chosen based on drug's short elimination half-lives to avoid any further significant interaction on ventilation with the investigated drugs tested 24h later.

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, USA), modified by the addition of several holes at the cephalic end to avoid CO 2 rebreathing [START_REF] Chevillard | Mechanisms of respiratory insufficiency induced by methadone overdose in rats[END_REF]. Before drug administration, the catheter was exteriorized, purged, and its permeability verified.

Clinical findings

Sedation level based on a 4-stage scale from 0 (awake) to 3 (coma) was assessed. 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 muscular 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. Temperature was measured using i.p. implanted temperature transmitters for the purpose of plethysmography study.

Arterial blood gas study

In catheterized rats, 100 µL-arterial blood samples were collected before (T0), and at 5, 10, 15, 30, 60, 120, 180, and 240 min after i.p. injection of methadone or its solvent. Samples were immediately analyzed using Rapidlab 248 ® (Siemens Healthcare Diagnostics, Saint Denis, France). Every hour, 1000 µL heparinized 0.9% NaCl was administered to the rat via the arterial catheter to reduce the risk of catheter clotting and to compensate for volume losses. Rats were subsequently euthanized using a carbon dioxide chamber.

Whole body plethysmography

Four days before the study, temperature transmitters (DSI, Chatillon, France) were implanted in the peritoneal cavity. Rats were then returned to their individual cages for a 72 h-recovery period. Ventilatory parameters were recorded in a whole-body plethysmograph by the barometric method described and validated in the rat [START_REF] Bartlett | Control of breathing in experimental anemia[END_REF]. On the day of experimentation, animals were placed in a rectangular Plexiglas chamber with a 3 L-volume connected to a reference chamber of the same size by a high-resistance leak to minimize the effect of pressure changes in the experimentation room. The animal chamber was flushed continuously with humidified air at a rate of 5 L/min. During the recording periods, the inlet and outlet tubes were temporarily clamped and pressure changes associated with each breath were recorded using a differential pressure transducer (Validyne MP, 45 ± 3 cm H 2 O, Northridge, CA), connected to the animal and reference chambers. During each measurement, calibration was performed by three injections of 1 mL of air into the chamber and the ambient temperature was noted. The spirogram was recorded and stored on a computer with an acquisition data card (PCI-DAS 1000, Dipsi, Chatillon, France) using respiratory acquisition software (Acquis1 Software, CNRS, Gif-sur-Yvette, France) for offline analysis.

This technique was daily validated with a series of leak tests (leak was signaled if diminution of the signal amplitude exceeded 33% in 5 s) [START_REF] Bonora | Ventilatory responses to hypercapnia and hypoxia in conscious cystic fibrosis knockout mice Cftr[END_REF]. The quantification threshold corresponded to a minimum air volume injection of 30 µl. Within the range of tested volumes (0.1 to 3 mL), measurement was linear. The mean coefficient of intra-day variability (four series of five measurements carried out the same day) was 1.32 ± 0.18%. The mean coefficient of inter-day variability (25 measurements carried out on three different days) was 1.69 ± 0.11%. We checked that the mean CO 2 measured using an Ohmeda 5250 RGM capnograph (rebreathing test) during clamping periods did not exceed 0.6% of the air contained in the chamber.

Before each measurement, the temperature of the enclosure, the temperature of the animal, the volume of calibration and the atmospheric pressure were noted to permit calculation of plethysmography parameters. The first measurement was performed after a 30-60 min-period of accommodation of the animal in the plethysmography unit. During this period, the rat was quiet and not in deep or rapid eye movement sleep which could be roughly estimated from its behavior, response to noise, and breathing pattern. Then, the rat was gently removed from the chamber for i.p. injection, and replaced in the chamber for the remaining measurements. Ventilation was recorded at 5, 10, 15, 30, and then every 60 min during four hours, each record lasting about 60 s. The following parameters were determined using a specific formula [START_REF] Bartlett | Control of breathing in experimental anemia[END_REF]: tidal volume (V T ), T I , and T E . Additional parameters were calculated: the respiratory cycle duration (T TOT = T I + T E ), the respiratory frequency (f), and the minute volume (V E = V T x f). At the end of experimentation, rats were euthanized using a carbon dioxide chamber.

In vitro study of methadone metabolism using liver microsomes

Methadone metabolism by rat liver microsomes was studied in vitro as previously described [START_REF] Cho | Stereochemical differences in the metabolism of 3,4-methylenedioxymethamphetamine in vivo and in vitro: a pharmacokinetic analysis[END_REF]. The incubation mixture consisted in 0.98 mL of 0.1 mol/L HEPES buffer, pH 7.6 containing NADPH regenerating system (1.3 mmol/L NADP, 3.3 mmol/L glucose-6-phosphate, 3.3 mmol/L MgCl 2 , 0.4 IU/mL glucose-6-phosphate dehydrogenase) and 1 mg/ml rat microsomal protein. This mixture was pre-incubated for 15 min at 37°C, and reactions were initiated by adding methadone at 1 µmol/L, the minimal concentration we previously obtained in plasma between 5 and 240 min after i.p. administration of 5 mg/kg methadone to rats [START_REF] Chevillard | Mechanisms of respiratory insufficiency induced by methadone overdose in rats[END_REF]. To study the inhibition of methadone metabolism by diazepam, increasing concentrations of diazepam were added. For the in vitro time-course of methadone metabolism, aliquots (100 μL) were removed from this mixture at 0, 15, 30, 60, and 120 min.

To study the inhibition of methadone metabolism by diazepam, aliquots were removed at 0 and 120 min.

The reaction was stopped by adding an equal volume of cold methanol. The reaction mixture was centrifuged at 10,000 g for 10 min, and supernatants stored at -80 °C until analyzed. Concentrations of methadone enantiomers and EDDP were measured in supernatants using liquid chromatography-mass spectrometry (HPLC/MS), without liquid phase extraction (see below). An aliquot of each supernatant (100 μL) was mixed with 20 μL internal standards (5 µmol/l [(R,S)-methadone-d 3 + EDDP-d 3 ]) and directly injected into the system. Triplicate samples were used in all the inhibition experiments.

HPLC/MS determination of R-and S-methadone and EDDP

Samples were collected in catheterized rats concomitantly to those dedicated for arterial blood gas analysis. Three hundred microliters of blood were obtained from the arterial catheter before (T0), and at 5, 10, 15, 30, 60, 120, 180, and 240 min after i.p. administration of methadone or its solvent. Samples were placed in Eppendorf tubes and centrifuged at 3,500 g for 10 min. Subsequently, plasma (about 200 µl) was separated and refrigerated at -20°C, until liquid-liquid extraction in Toxitubes A ® (Varian, USA) and quantification using a specific HPLC/MS assay.

Analyses were performed on a ThermoFinnigan ® liquid chromatograph/ion trap mass spectrometer LCQ Advantage ® , including a Surveyor ® HPLC system which consisted of a degasser, a binary pump, and an autosampler. Chromatographic separation was carried out on a Cyclobond ® I-2000 HP-RSP chiral column (250 mm×4.6 mm I.D., 5 μm, Thermo Electron Corporation, Waltham, MA). Single ion storage detection was performed (m/z ((R and S)-methadone) = 310.1, m/z (EDDP) = 278.2). R-methadone, Smethadone, and racemic EDDP concentrations were calculated using the peak area ratios between the compound and its corresponding internal standard, i.e. deuterated racemic methadone [(R,S)-methadoned 3 ] or deuterated EDDP [EDDP-d 3 ], which was added before extraction at 5 µmol/l (20 μL). Limits of quantification were 3 nmol/L for both R-and S-methadone enantiomers and 36 nmol/L for EDDP, with coefficients of variation of 15.8%, 13.5%, and 10.8%, respectively.

Study designs:

In vivo study of diazepam/methadone combination

Rats were randomized into four groups (N=8) and treated consecutively with s.c. 20 mg/kg diazepam + i.p. 5 mg/kg methadone (diazepam/methadone group), s.c. Tween-20 ® + i.p. 5 mg/kg methadone (methadone group), s.c. 20 mg/kg diazepam + i.p. Tween-20 ® (diazepam group), and s.c. Tween-20 ® + i.p. Tween-20 ® (solvent group). Subsequently, ventilation at rest was studied using arterial blood gas analysis and plethysmography. Plasma concentrations of both methadone enantiomers were measured at each sampling time. We investigated methadone/effects relationships regarding T I (the hallmark of opioid respiratory effects) and PaCO 2 (the hallmark of respiratory depression) [START_REF] Chevillard | Characteristics and comparative severity of respiratory response to toxic doses of fentanyl, methadone, morphine, and buprenorphine in rats[END_REF].

Methadone (5 mg/kg) and diazepam (20 mg/kg) doses and routes of administration were those previously used in rats to assess methadone/diazepam interactions [START_REF] Mccormick | Effects of diazepam on the arterial blood gas concentrations and pH of adults rats acutely and chronically exposed to methadone[END_REF][START_REF] Nielsen | The effect of buprenorphine and benzodiazepines on respiration in the rat[END_REF]. Both drug doses can be considered as elevated doses, taking into account the lethal doses-50% (LD 50 ), i.e. 18 mg/kg for i.p. methadone [START_REF] Lynch | A study of the selective antagonism by N-allylnormorphine against meperidine, methadone, and morphine[END_REF] and 32 mg/kg for intravenous diazepam [START_REF] Pfizer | Material safety data sheet[END_REF].

In vitro study of diazepam-related effects on methadone metabolism

We studied the effects of 0 (control group), 25, 50, and 75 µmol/L diazepam on methadone metabolism (1 µmol/L). The dose range of diazepam was used in accordance with the value of the inhibition constant reported with human microsomes (K I = 50 µmol/L; [START_REF] Iribarne | Interaction of methadone with substrates of human hepatic cytochrome P450 3A4[END_REF].

Statistical analysis

Results were expressed as mean ± S.E.M. For plethysmography, T0 was the mean of the three baseline measurements. To permit simultaneous analysis of the effect of time and various treatments, we calculated for each animal and for each studied parameter, the area under the curve (AUC) from T0 to the completion of the measurement (240 min) using the trapezoid method (Tallarida and Murray, 1984). For each measurement, we compared the baseline values, the values at each sampling time, and the AUCs.

Noncompartmental analysis (WinNonlin version 5.1, Pharsight Corp, Mountain View, CA) was used to calculate methadone terminal elimination half-life (T 1/2 ) as well as the AUC from 0 to 240 (AUC 0→240 ) and the percentage of extrapolation of the AUC from 0 to infinity (AUC 0→∞ ) regarding methadone concentrations. We used Mann-Whitney U-tests for comparisons between two groups and two-way analyses of variance (ANOVA) followed by multiple comparison tests using Bonferroni's correction for comparisons between more than two groups. For the in vitro inhibition study, the 50%-inhibitory diazepam concentration (IC 50 ) was determined for each enantiomer based on the metabolism inhibition curves using nonlinear regression. Inhibition of methadone metabolism was determined for each enantiomer as the relative difference in its concentrations between 0 and 120 min in the reaction aliquot.

EDDP formation was determined as the relative difference in its concentrations between 0 and 120 min in the reaction aliquot. These variables were compared using one-way ANOVA followed by multiple comparison tests with Dunnett's correction. The concentration-effect relationships were analyzed using linear regression. All tests were performed using Prism version 4.0 (GraphPad Software, Inc., San Diego, CA), and p-values of less than 0.05 were considered significant.

Results

In vivo study of diazepam/methadone combination's effects on ventilation

Rats were free of symptoms in the diazepam group in comparison to the solvent group. In contrast, they were significantly sedated in the methadone and diazepam/methadone groups in comparison to the solvent and diazepam groups (p < 0.001, Fig. 1). Interestingly, no significant differences were observed in sedation between the methadone and diazepam/methadone groups, while these groups significantly differed regarding body temperature (p < 0.01).

Regarding blood gases and plethysmography, no significant differences between baseline values were observed for each parameter between the groups. Methadone administration (p < 0.05) and diazepam/methadone co-administration (p < 0.01) were responsible for a significant decrease in PaO 2 when compared to the solvent and diazepam groups. Similarly, diazepam/methadone co-administration was responsible for a significant decrease in arterial pH when compared to the solvent (p < 0.05) and diazepam groups (p < 0.001). Interestingly, none of the blood gas parameters significantly differed between the methadone and diazepam/methadone groups (Fig. 2).

Using AUC measurements, only T I was significantly increased in the diazepam/methadone group in comparison to the solvent, diazepam, and methadone groups (p < 0.001, Fig. 3A). No significant differences regarding f, T E , and V T were observed between the methadone and diazepam/methadone groups. However, in the diazepam/methadone group, T I was significantly increased at 120 and 180 min (p < 0.05) and T E at 30 (p < 0.05), 60 (p < 0.01), 120 (p < 0.05), and 180 min (p < 0.01), in comparison to the methadone group (Fig. 3B). Regarding f, a significant difference was measured at 120 min between the diazepam/methadone and the methadone group (p < 0.05).

In vivo study of diazepam/methadone combination's effects on methadone concentrations

Diazepam/methadone co-administration resulted in a significant increase in plasma R-methadone concentrations at 120 (p < 0.01), 180 (p < 0.01), and 240 min (p < 0.001), without any significant modification in S-methadone concentrations (Fig. 4). Using the AUC method, there was a 2.0-fold increase in plasma R-methadone concentrations in relation to diazepam co-administration (p < 0.01), whereas plasma S-methadone concentrations were not significantly altered (1.5-fold increase). Regarding T max and C max of R-methadone, no significant differences were observed between the two groups. In rats treated with diazepam/methadone combination, neither R-methadone T 1/2 (155.1 versus 155.5 min) nor S-methadone T 1/2 (131.3 versus 158.3 min) was altered in comparison to rats treated with methadone alone.

Analysis of methadone-related concentration/effect relationships

We studied the relationships between methadone-induced respiratory effects (i.e. on PaCO 2 and T I ) and plasma concentrations of the active R-methadone enantiomer, when 5 mg/kg methadone was administered alone or in combination with 20 mg/kg diazepam (Fig. 5). A clockwise hysteresis was observed for both parameters, whether diazepam was co-administered or not.

In vitro study of diazepam-related effects on methadone metabolism

No significant differences between the kinetics of R-and S-methadone disappearance were observed.

Diazepam co-incubation resulted in a dose-dependent decrease in R-and S-methadone concentrations in comparison to the control, however this was only significant with the two higher diazepam doses (p < 0.01) (Fig. 6A). As a consequence of the inhibitory effect of diazepam on methadone metabolism, EDDP formation was significantly decreased for all diazepam doses (Fig. 6B). IC 50 values of diazepam on Rand S-methadone metabolism were 25.02 ± 0.18 µmol/L and 25.18 ± 0.23 µmol/L, respectively.

Discussion and conclusions

In rats, the diazepam/methadone combination does not significantly worsen methadone-induced respiratory effects despite a significant increase in plasma R-methadone attributed to diazepam-mediated inhibition of methadone metabolism, as assessed in vitro.

Like any full mu-opioid receptor agonist, methadone-induced respiratory effects are characterized by a dose-related, naloxone-reversible depression of the resting total volume with a proportional reduction in V T , decrease in PaO 2 and arterial pH, increase in PaCO 2 , and reduction in the ventilatory drive stimulated by hypercapnia [START_REF] Shook | Differential roles of opioid receptors in respiration, respiratory disease, and opiate-induced respiratory depression[END_REF]. Previously, we showed that methadone increases T I in a dosedependent manner but only increases T E at elevated doses (~15 mg/kg) resulting in respiratory depression [START_REF] Chevillard | Mechanisms of respiratory insufficiency induced by methadone overdose in rats[END_REF].

Mechanisms and consequences of BZD/methadone interaction are debated in the literature [START_REF] Jones | Polydrug abuse: A review of opioid and benzodiazepine combination use[END_REF]. In mice, neither locomotor activity nor analgesia was altered when administering the diazepam/methadone combination in comparison to methadone alone [START_REF] Shannon | Interactions between narcotic analgesics and benzodiazepine derivatives on behavior in the mouse[END_REF]. In contrast, diazepam was reported to markedly potentiate methadone respiratory depressant effects when given in combination with methadone in rats [START_REF] Mccormick | Effects of diazepam on the arterial blood gas concentrations and pH of adults rats acutely and chronically exposed to methadone[END_REF]. The most severe respiratory depression occurred when both drugs were used together acutely, suggesting that the maximum deleterious effect occurred when concurrently used for the first time. Here, surprisingly, and although using the same doses and administration routes, only a significant increase in T I with a trend to a decrease in PaO 2 was observed, suggesting limited deleterious effects on rat ventilation of the diazepam/methadone combination in comparison to methadone alone. It is noteworthy that in McCormick's study, diazepam was diluted in a solution containing 10% ethyl alcohol and 1.5% benzyl alcohol, and the interaction with methadone could have been responsible for the observed significant respiratory effects. Interestingly, the second animal study attributing deleterious respiratory effects using arterial blood gases to the diazepam/methadone combination, compared rats treated with a diazepam/methadone combination to rats treated with diazepam or solvent but not to rats treated with methadone alone [START_REF] Nielsen | The effect of buprenorphine and benzodiazepines on respiration in the rat[END_REF].

These concerns question the exact respiratory consequences of the diazepam/methadone combination, as shown in our study.

As previously assessed with human microsomes [START_REF] Iribarne | Interaction of methadone with substrates of human hepatic cytochrome P450 3A4[END_REF], we confirmed the diazepammediated inhibition of methadone metabolism, using rat microsomes in vitro. Interestingly, our calculated diazepam IC 50 (25.02 µmol/L) was in the same range as the value reported by Iribarne et al. (50 µmol/L, Iribarne et al., 1997). Moreover, no differences in either the time-course of methadone disappearance or diazepam IC 50 values for both methadone enantiomers were observed. Surprisingly, we found marked diazepam-induced inhibition of methadone metabolism into EDDP in vivo: AUCs of R-and S-methadone were altered in rats treated with the diazepam/methadone combination but this was only significant for Rmethadone, which suggested possible involvement in vivo of additional pathways in S-methadone clearance (i.e. transport or metabolic enzymes) that were not inhibited by diazepam. There are no published data regarding the stereoselectivity of metabolism enzymes for methadone in rats. In studies using human microsomes, CYP2B6 preferentially metabolizes S-methadone [START_REF] Totah | Role of CYP2B6 in stereoselective human methadone metabolism[END_REF] and CYP2C19 R-methadone [START_REF] Gerber | Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19[END_REF], while CYP1A2 was not stereoselective [START_REF] Eap | Fluvoxamine and fluoxetine do not interact in the same way with the metabolism of the enantiomers of methadone[END_REF].

Interestingly, CYP3A4 affinity for R-methadone was reported to be higher than for S-methadone [START_REF] Foster | Methadone N-demethylation in human liver microsomes: lack of stereselectivity and involvement of CYP3A4[END_REF]; however stereoselectivity of CYP3A4-mediated methadone metabolism is still debated [START_REF] Gerber | Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19[END_REF][START_REF] Totah | Enantiomeric Metabolic Interactions and Stereoselective Human Methadone Metabolism[END_REF][START_REF] Wang | Involvement of CYP3A4, CYP2C8, and CYP2D6 in the metabolism of (R)-and (S)-methadone in vitro[END_REF].

Increased AUC of methadone concentrations without alterations in its T 1/2 when pre-treating rats with diazepam supported a diazepam-mediated increase in methadone i.p. bioavailability (Fig. 4). Based on the peak diazepam concentration (1.76 µmol/L) measured after intravenous administration of 1.2 mg/kg diazepam in rats [START_REF] Igari | Tissue distribution of 14 C-diazepam and its metabolites in rats[END_REF] and assuming that diazepam pharmacokinetics are linear up to 20 mg/kg with a rapid and complete absorption by the s.c. route, we hypothesize that plasma diazepam concentrations may have reached ~29.4 µmol/L in our study. Compared to diazepam IC 50 (25.02 µmol/L), such expected concentrations supported the occurrence of significant CYP inhibition in vivo as reported.

Additionally, the diazepam elimination half-life of 24.8 min [START_REF] Igari | Tissue distribution of 14 C-diazepam and its metabolites in rats[END_REF] clearly explained why diazepam inhibitory effects on methadone metabolism occurred during its absorption and distribution phases (liver first-pass) rather than its elimination phase.

Here, the diazepam/methadone combination increased the mean peak R-methadone concentrations in plasma from 3.4 to 4.9 µmol/L. We previously studied R-methadone concentrations associated with the half-maximum effects (EC 50 ) on T I (1.6 µmol/L), PaO 2 (3.7 µmol/L), PaCO 2 (10.8 µmol/L), and T E (15.6 µmol/L) [START_REF] Chevillard | Mechanisms of respiratory insufficiency induced by methadone overdose in rats[END_REF]. These data clearly explain why alterations in ventilation resulting from the combination of diazepam with 5 mg/kg methadone were limited to the observed significant increase in T I . Moreover, diazepam-mediated inhibition of methadone metabolism appears to explain the observed effects on ventilation induced by the diazepam/methadone combination, even though additional mechanisms could not be definitively ruled out, including a pharmacodynamic interaction or the alternative pharmacokinetic interaction on protein binding previously suggested to explain the significant increase in brain and liver diazepam concentrations observed when administering diazepam one hour before intravenous methadone [START_REF] Liu | Correlation of urinary excretion of methadone metabolites with methadone metabolism and analgesia in the rat[END_REF].

In humans, inter-individual variability makes it impossible to anticipate, with acceptable approximation, the relationship between the dose, blood concentrations, and clinical effects of methadone [START_REF] Ferrari | Methadone-metabolism, pharmacokinetics and interaction[END_REF]. Several drugs, including BZDs, may influence CYP3A4, 2B6, 2C19, and 2D6mediated metabolism of methadone, resulting in potential clinical respiratory toxicity [START_REF] Eap | Cytochrome P450 2D6 genotype and methadone steady-state concentrations[END_REF][START_REF] Foster | Methadone N-demethylation in human liver microsomes: lack of stereselectivity and involvement of CYP3A4[END_REF][START_REF] Kharasch | Mechanism of ritonavir changes in methadone pharmacokinetics and pharmacodynamics: I. Evidence against CYP3A mediation of methadone clearance[END_REF][START_REF] Totah | Role of CYP2B6 in stereoselective human methadone metabolism[END_REF]. Methadone and diazepam are two competitive CYP3A4 substrates [START_REF] Ferrari | Methadone-metabolism, pharmacokinetics and interaction[END_REF], leading to diazepam-mediated reduction in methadone in vitro metabolism [START_REF] Iribarne | Interaction of methadone with substrates of human hepatic cytochrome P450 3A4[END_REF] attributed to diazepam's higher affinity for CYP3A4 [START_REF] Weschules | Actual and potential drug interactions associated with methadone[END_REF]. However, comparison of diazepam IC 50 values (25 µmol/L) with the range of maximum diazepam plasma concentrations (7.02-8.78 µmol/L) that could be achieved seriously questions the clinical pertinence of this inhibition mechanism in vivo [START_REF] Walker | Comparison of single-and repeated-dose pharmacokinetics of diazepam[END_REF]. Interestingly, when combining methadone with various CYP3A4-metabolised BZDs, higher R-methadone concentrations were achieved, supporting a mechanism of BZD-mediated competitive inhibition of methadone metabolism in vivo [START_REF] Hallinan | Cannabis and benzodiazepines as determinants of methadone trough plasma concentration variability in maintenance treatment: a transnational study[END_REF]. However, in contrast to these data as well as to our findings in rats, Preston et al. found that concurrent methadone and diazepam administration did not significantly change the time-course or AUC of methadone, diazepam or N-desmethyldiazepam concentrations in plasma in comparison to concentrations measured by the administration of either drug alone [START_REF] Preston | Diazepam and methadone blood levels following concurrent administration of diazepam and methadone[END_REF].

In humans, several studies supported potentiating clinical effects with the BZD/methadone combination. BZD users exhibited higher levels of psychopathology and social dysfunction among methadone maintenance clients [START_REF] Drake | Drug use, HIV risk-taking and psychosocial correlates of benzodiazepine use among methadone maintenance clients[END_REF]. Diazepam significantly altered subjective drug responses and psychological performance in methadone-maintained subjects [START_REF] Farré | Abuse liability of flunitrazepam among methadone-maintained patients[END_REF][START_REF] Lintzeris | Pharmacodynamics of diazepam coadministered with methadone or buprenorphine under high dose conditions in opioid dependent patients[END_REF]. Interestingly, diazepam significantly increased reports of good, like, strong, and high when using methadone, resulting in a decrease in the amount of methadone consumed [START_REF] Spiga | Human methadone self-administration: effects of diazepam pretreatment[END_REF]. The diazepam/methadone combination elicited greater alterations in subjective drug effects, psychological performance, and physiologic parameters including pupil diameter and sedation in comparison to those induced by either drug alone [START_REF] Lintzeris | Pharmacodynamics of diazepam coadministered with methadone or buprenorphine under high dose conditions in opioid dependent patients[END_REF][START_REF] Preston | Diazepam and methadone interactions in methadone maintenance[END_REF]. However, none of the potentiated effects implicated respiration.

Consistently, although BZDs were hypothesized to play a significant role in methadone-related deaths by increasing upper airway obstruction [START_REF] Caplehorn | Fatal methadone toxicity: signs and circumstances, and the role of benzodiazepines[END_REF], no definitive demonstration has been obtained to date. The rate of BZD detection in methadone-attributed deaths, although variable from one series to another (32% to 83%), was not significantly different from that rate in deaths not attributed to methadone. Nevertheless, in these deaths, detectable levels of methadone were found in contrast to the other prescription opioids like tramadol, codeine, and buprenorphine [START_REF] Häkkinen | Comparison of fatal poisonings by prescription opioids[END_REF]Schields et al., 2007). Fatal poisonings seldom seem to be caused by methadone, although absence of significant differences in methadone concentrations between both groups of patients as reported in forensic studies could be attributed to a multitude of factors, including tolerance, route of intake, associated central sleep apnea, and circumstantial and environmental factors [START_REF] Corkery | The effects of methadone and its role in fatalities[END_REF]. Therefore, the role of BZD co-ingestion in methadone-induced toxicity appears less obvious than with buprenophine, where poisonings and deaths were clearly related to BZD co-ingestion [START_REF] Mégarbane | Prospective comparative assessment of buprenorphine overdose with heroin and methadone: clinical characteristics and response to antidotal treatment[END_REF][START_REF] Pirnay | A critical review of the causes of death among post-mortem toxicological investigations: analysis of 34 buprenorphineassociated and 35 methadone-associated deaths[END_REF]. However, in methadone-related fatalities, the median concentrations of methadone decreased as the number of co-ingested drugs increased [START_REF] Häkkinen | Comparison of fatal poisonings by prescription opioids[END_REF], suggesting toxicodynamic interactions with the concomitantly used central nervous system depressant drugs.

Our study suffers from a number of limitations: 1)-We studied the diazepam combination with a moderately toxic dose of methadone (30% of its LD 50 ). Thus, our study does not rule out deleterious interactions with the higher doses that may be encountered in severe overdoses and thus lead to life-threatening conditions. Based on the methadone EC 50 of PaCO 2 increase, we can hypothesize that combining diazepam with higher methadone doses would result in significant respiratory depression in comparison to methadone alone. 2)-From a technical point of view, the reliability of plethysmography in measuring ventilation in awake animals may be questionable for the measurement of V T which may be underestimated; however, this technique has been validated against pneumotachography and direct plethysmography with a systemic error on V T of < 7% [START_REF] Bonora | Ventilatory responses to hypercapnia and hypoxia in conscious cystic fibrosis knockout mice Cftr[END_REF]. The more important variability we observed in V T measurement in comparison to the other parameters could be also related to various factors that were not taken into account using Bartlett's conventional equation to determine the V T [START_REF] Bartlett | Control of breathing in experimental anemia[END_REF][START_REF] Chaui-Berlinck | The signal in total-body plethysmography: errors due to adiabatic-isothermic difference[END_REF]. In addition, as significant modification of rat temperature was observed, the results of arterial blood gases were corrected using the actual rat temperatures. We chose not to correct the rat temperatures using heating blankets as this might alter methadone pharmacodynamic effects on related respiratory responses. 3)-Due to the limited sampling time, we were unable to measure methadone pharmacokinetic parameters since the extrapolated AUC represented more than 20% (27 to 39%) of the total AUC of the methadone time-course. 4)-We observed a possible enantioselectivity of methadone pharmacokinetics when diazepam was co-administered in rats; however, our experiments could not allow us to definitively conclude about its mechanism.

In conclusion, respiratory effects resulting from the diazepam/methadone combination at elevated doses are limited in comparison to those related to methadone alone despite significant pharmacokinetic interaction in vivo attributed to diazepam-mediated inhibition of methadone metabolism. Although our experimental findings may suggest the relative safety of BZD/methadone co-prescription, physicians should remain cautious when prescribing BZD in methadone-treated patients like drug addicts, as other underlying conditions may enhance this drug-drug interaction.

Figure legends

Fig. 1. Effects of Tween20 ® (solvent, black), Tween20 ® + 5 mg/kg methadone (MTD, vertical line), 20 mg/kg diazepam + 5 mg/kg methadone (DZP+MTD, white), and 20 mg/kg diazepam + Tween20 ® (DZP, horizontal line) on rat body temperature (A) and sedation (B). Each group consisted of eight rats. Values represent mean ± S.E.M of the areas under the curve (AUC) for each parameter between the time before injection and 240 min postinjection. Comparisons were performed using ANOVA followed by multiple tests using Bonferroni's correction. Comparisons versus solvent: ### p < 0.001; versus diazepam: ***p < 0.001; versus methadone: § § § p < 0.001.

Fig. 2 .

 2 Fig. 2.Effects of Tween20 ® (solvent, black), Tween20 ® + 5 mg/kg methadone (MTD, vertical line), 20 mg/kg diazepam + 5 mg/kg methadone (DZP+MTD, white), and 20 mg/kg diazepam + Tween20 ® (DZP,

Fig. 3 .

 3 Fig. 3. (A)Effects of Tween20 ® (solvent, black), Tween20 ® + 5 mg/kg methadone (MTD, vertical line), 20 mg/kg diazepam + 5 mg/kg methadone (DZP+MTD, white), and 20 mg/kg diazepam + Tween20 ®

Fig. 4 .

 4 Fig. 4. Plasma R-and S-methadone concentrations in rats treated with Tween20 ® + 5 mg/kg methadone

Fig. 5 .

 5 Fig. 5. Concentration/effect relationships regarding PaCO 2 and inspiratory time in rats treated with 5 mg/kg methadone (A) and 20 mg/kg diazepam + 5 mg/kg methadone (B).

Fig. 6 .

 6 Fig. 6. (A) Disappearance of R-and S-methadone enantiomers after 120 min of incubation with different