D-Cysteine Ethyl Ester Reverses the Deleterious Effects of Morphine on Breathing and Arterial Blood-Gas Chemistry in Freely-Moving Rats

Abstract

Cell-penetrant thiol esters including the disulfides, D-cystine diethyl ester and D-cystine dimethyl ester, and the monosulfide, L-glutathione ethyl ester, prevent and/or reverse the deleterious effects of opioids, such as morphine and fentanyl, on breathing and gas exchange within the lungs of unanesthetized/unrestrained rats without diminishing the antinociceptive or sedative effects of opioids. We describe here the effects of the monosulfide thiol ester, D-cysteine ethyl ester (D-CYSee), on intravenous morphine-induced changes in ventilatory parameters, arterial blood-gas chemistry, alveolar-arterial (A-a) gradient (i.e., index of gas exchange in the lungs), and sedation and antinociception in freely-moving rats. The bolus injection of morphine (10 mg/kg, IV) elicited deleterious effects on breathing, including depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive. Subsequent injections of D-CYSee (2 × 500 μmol/kg, IV, given 15 min apart) elicited an immediate and sustained reversal of these effects of morphine. Morphine (10 mg/kg, IV) also A-a gradient, which caused a mismatch in ventilation perfusion within the lungs, and elicited pronounced changes in arterial blood-gas chemistry, including pronounced decreases in arterial blood pH, pO₂ and sO₂, and equally pronounced increases in pCO₂ (all responses indicative of decreased ventilatory drive). These deleterious effects of morphine were immediately reversed by the injection of a single dose of D-CYSee (500 μmol/kg, IV). Importantly, the sedation and antinociception elicited by morphine (10 mg/kg, IV) were minimally affected by D-CYSee (500 μmol/kg, IV). In contrast, none of the effects of morphine were affected by administration of the parent thiol, D-cysteine (1 or 2 doses of 500 μmol/kg, IV). Taken together, these data suggest that D-CYSee may exert its beneficial effects via entry into cells that mediate the deleterious effects of opioids on breathing and gas exchange. Whether D-CYSee acts as a respiratory stimulant or counteracts the inhibitory actions of µ-opioid receptor activation remains to be determined. In conclusion, D-CYSee and related thiol esters may have clinical potential for the reversal of the adverse effects of opioids on breathing and gas exchange, while largely sparing antinociception and sedation.

Keywords: D-cysteine ethyl ester; Sprague Dawley rats; antinociception; gas exchange; morphine; respiratory depression; sedation; thiol esters.

FIGURE 1

Values of frequency of breathing (Panel (A)), tidal volume (Panel (B)), and minute ventilation (Panel (C)) in freely-moving rats prior to (pre) and following injection of morphine (10 mg/kg, IV) and subsequent injections of vehicle (saline, IV) or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). Data are presented as mean ± SEM. There were 5 rats in the vehicle group and 6 rats in the D-CYSee group.

FIGURE 2

Values of inspiratory time (Panel (A)), expiratory time (Panel (B)), and expiratory time/inspiratory time (Te/Ti) (Panel (C)) in freely-moving rats prior to (pre) and following injection of morphine (10 mg/kg, IV) and subsequent injections of vehicle (saline, IV) or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). Data are presented as mean ± SEM. There were 5 rats in the vehicle group and 6 rats in the D-CYSee group.

FIGURE 3

Values of peak inspiratory flow (Panel (A)), peak expiratory flow (Panel (B)), and peak expiratory flow/peak inspiratory flow (PEF/PIF) (Panel (C)) in freely-moving rats prior to (pre) and following injection of morphine (10 mg/kg, IV) and subsequent injections of vehicle (saline, IV) or D-cysteine ethyl ester (D-CYSee, 2 × 500 μmol/kg, IV). The data are presented as mean ± SEM. There were 5 rats in the vehicle group and 6 rats in the D-CYSee group.

FIGURE 4

Calculated values of inspiratory drive (TV/Ti) (Panel (A)) and expiratory drive (TV/Te) (Panel (B)) in freely moving rats prior to (pre) and following injection of morphine (10 mg/kg, IV) and subsequent injections of vehicle (saline) or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). The data are presented as mean ± SEM. There were 5 rats in the vehicle group and 6 rats in the D-CYSee group.

FIGURE 5

Total changes (expressed as % change from pre-values) in Freq, TV, MV, Ti, Te and Te/Ti (A), EIP, EEP, PIF, PEF, PEF/PIF and EF50 (B), and RT, ApP, TV/Ti, TV/Te, NEBI, and NEBI/Freq (C) that occurred over the 15-min period following the first injection of vehicle (VEH, IV) or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). The vehicle (VEH) group received two injections of vehicle. The D-CYSee (2x) group received two injections of D-CYSee, whereas the D-CYSee (1x) group received one injection of D-CYSee and then an injection of vehicle. There were 5 rats in the vehicle group and 6 rats in the D-CYSee groups. *p < 0.05, significant change from pre. p < 0.05, D-CYSee versus vehicle.

FIGURE 6

Total changes (expressed as % change from pre-values) in ventilatory parameters that occurred over the 60-min period following the second set of injections of either vehicle (VEH) or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). The vehicle (VEH) group received two injections of vehicle. The D-CYSee (2x) group received two injections of D-CYSee, whereas the D-CYSee (1x) group received one injection of D-CYSee and then an injection of vehicle. There were 5 rats in the vehicle group and 6 rats in the D-CYSee group. *p < 0.05, significant change from pre-values. p < 0.05, D-CYSee versus vehicle.

FIGURE 7

Values of pH (Panel (A)), pCO₂ (Panel (B)), pO₂ (Panel (C)), and sO₂ (Panel (D)) before (Pre) and after injection of morphine (10 mg/kg, IV) in three separate groups of freely-moving rats followed by injection of vehicle (VEH (saline), IV), D-cysteine (500 μmol/kg, IV), or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). The terms M15, M20, M30, M45, and M60 denote 15, 30, 45, and 60 min after injection of morphine. The terms D5, D15, D30, and D45 denote 5, 15, 30, and 45 min after injection of the drug (vehicle, D-cysteine, or D-CYSee). The data are mean ± SEM. There were 8 rats in the vehicle- or D-CYSee–treated groups and 6 rats in the D-cysteine–treated group. *p < 0.05, significant change from pre-values. p < 0.05, D-CYSee versus vehicle.

FIGURE 8

Alveolar–arterial (A-a) gradient values before (Pre) and after injection of morphine (10 mg/kg, IV) in three groups of freely-moving rats followed by an injection of vehicle (VEH (saline), IV), D-cysteine (500 μmol/kg, IV), or D-cysteine ethyl ester (D-CYSee, 500 μmol/kg, IV). The terms M15, M20, M30, M45, and M60 denote 15, 30, 45, and 60 min after injection of morphine. The terms D5, D15, D30, and D45 denote 5, 15, 30, and 45 min after injection of drug (vehicle, D-cysteine, or D-CYSee). The data are mean ± SEM. There were 8 rats in the vehicle- or D-CYSee-treated groups and 6 rats in the D-cysteine–treated group. *p < 0.05, significant change from pre-values. p < 0.05, D-CYSee versus vehicle.

FIGURE 9

Tail-flick latencies elicited by intravenous injection of 5 mg/kg (Panel (A)) or 10 mg/kg (Panel (B)) of morphine in freely-moving rats that received injections of vehicle (VEH (saline), IV) or D-CYSee (500 μmol/kg, IV) 20 min after the injection of morphine. Panels (C,D) display the data as percentage maximum possible effect (%MPE). The data are shown as mean ± SEM. There were 6 rats in each group. *p < 0.05, significant change from pre-values. There were no between group comparisons at any time-point (p > 0.05, for all comparisons).