L-cysteine ethylester reverses the adverse effects of morphine on breathing and arterial blood-gas chemistry while minimally affecting antinociception in unanesthetized rats.

L-cysteine ethylester (L-CYSee) is a membrane-permeable analogue of L-cysteine with a variety of pharmacological effects. The purpose of this study was to determine the effects of L-CYSee on morphine-induced changes in ventilation, arterial-blood gas (ABG) chemistry, Alveolar-arterial (A-a) gradient (i.e., a measure of the index of alveolar gas-exchange), antinociception and sedation in male Sprague Dawley rats. An injection of morphine (10 mg/kg, IV) produced adverse effects on breathing, including sustained decreases in minute ventilation. L-CYSee (500 µmol/kg, IV) given 15 min later immediately reversed the actions of morphine. Another injection of L-CYSee (500 µmol/kg, IV) after 15 min elicited more pronounced excitatory ventilatory responses. L-CYSee (250 or 500 µmol/kg, IV) elicited a rapid and prolonged reversal of the actions of morphine (10 mg/kg, IV) on ABG chemistry (pH, pCO2, pO2, sO2) and A-a gradient. L-serine ethylester (an oxygen atom replaces the sulfur; 500 µmol/kg, IV), was ineffective in all studies. L-CYSee (500 µmol/kg, IV) did not alter morphine (10 mg/kg, IV)-induced sedation, but slightly reduced the overall duration of morphine (5 or 10 mg/kg, IV)-induced analgesia. In summary, L-CYSee rapidly overcame the effects of morphine on breathing and alveolar gas-exchange, while not affecting morphine sedation or early-stage analgesia. The mechanisms by which L-CYSee modulates morphine depression of breathing are unknown, but appear to require thiol-dependent processes.

The ventilatory depressant actions but not the antinociceptive effects of morphine are blunted in rats receiving intravenous infusion of L-cysteine ethyl ester.

This study demonstrates that intravenous infusion of the cell-penetrant thiol ester, L-cysteine ethyl ester (L-CYSee), to adult male Sprague-Dawley rats elicited (a) minor alterations in frequency of breathing, expiratory time, tidal volume, minute ventilation, or expiratory drive but pronounced changes in inspiratory time, end-inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50, relaxation time, apneic pause, inspiratory drive and non-eupneic breathing index, (b) minimal changes in arterial blood-gas (ABG) chemistry (pH, pCO2, pO2, SO2) and Alveolar-arterial (A-a) gradient (index of alveolar gas exchange), and (c) minimal changes in antinociception (tail-flick latency). Subsequent injection of morphine (10 mg/kg, IV) elicited markedly smaller effects on the above parameters, ABG chemistry, and A-a gradient in rats receiving L-CYSee, whereas morphine antinociception was not impaired. Infusions of L-cysteine or L-serine ethyl ester (oxygen rather than sulfur moiety), did not affect morphine actions on ABG chemistry or A-a gradient. L-CYSee (250 µmol/kg, IV) injection elicited dramatic changes in ventilatory parameters given 15 min after injection of morphine in rats receiving L-CYSee. Our findings suggest that (a) L-CYSee acts in neurons that drive ventilation, (b) L-CYSee reversal of the adverse actions of morphine on ventilation, ABG chemistry and A-a gradient may be via modulation of intracellular signaling pathways activated by morphine rather than by direct antagonism of opioid receptors since morphine antinociception was not diminished by L-CYSee, and (c) the thiol moiety of L-CYSee is vital to efficacy, (d) intracellular conversion of L-CYSee to an S-nitrosylated form may be part of its mechanism of action.

S-nitroso-L-cysteine stereoselectively blunts the adverse effects of morphine on breathing and arterial blood gas chemistry while promoting analgesia.

S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 µmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO2 and a decrease in pCO2, (d) a decrease in mean arterial blood pressure (MAP), and (e) an increase in tail-flick (TF) latency (antinociception). Infusion of S-nitroso-D-cysteine (SNO-D-CYS, 1 µmol/kg/min, IV), did not elicit similar responses, except for a sustained decrease in MAP equivalent to that elicited by SNO-L-CYS. A bolus injection of morphine (2 mg/kg, IV) in rats receiving an infusion of vehicle elicited (a) sustained decreases in frequency of breathing tidal volume, and therefore minute ventilation, (b) a sustained decrease in MAP, (c) sustained decreases in pH, pO2 and maximal sO2 with sustained increases in pCO2 and A-a gradient, and (d) a sustained increase in TF latency. In rats receiving SNO-L-CYS infusion, morphine elicited markedly smaller changes in minute ventilation, arterial blood gas chemistry, A-a gradient and MAP. In contrast, the antinociceptive effects of morphine were enhanced in rats receiving the infusion of SNO-L-CYS. The morphine-induced responses in rats receiving SNO-D-CYS infusion were similar to vehicle-infused rats. These data are the first to demonstrate that infusion of an S-nitrosothiol, such as SNO-L-CYS, can stereoselectively ameliorate the adverse effects of morphine on breathing and alveolar gas exchange while promoting antinociception.

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

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.

D-cysteine ethyl ester and D-cystine dimethyl ester reverse the deleterious effects of morphine on arterial blood-gas chemistry and Alveolar-arterial gradient in anesthetized rats.

We determined whether intravenous injections of the membrane-permeable ventilatory stimulants, D-cysteine ethyl ester (ethyl (2 S)- 2-amino-3-sulfanylpropanoate) (D-CYSee) and D-cystine dimethyl ester (methyl (2 S)- 2-amino-3-[[(2 S)- 2-amino-3-methoxy-3-oxopropyl]disulfanyl] propanoate) (D-CYSdime), could overcome the deleterious actions of intravenous morphine on arterial blood pH, pCO2, pO2 and sO2, and Alveolar-arterial (A-a) gradient (i.e., the measure of exchange of gases in the lungs) in Sprague Dawley rats anesthetized with isoflurane. Injection of morphine (2 mg/kg, IV) caused pronounced reductions in pH, pO2 and sO2 accompanied by elevations in pCO2, all which are suggestive of diminished ventilation, and elevations in A-a gradient, which suggests a mismatch of ventilation-perfusion. Subsequent boluses of D-cysteine ethyl ester (2 ×100 µmol/kg, IV) or D-cystine dimethyl ester (2 ×50 µmol/kg, IV) rapidly reversed of the negative actions of morphine on pH, pCO2, pO2 and sO2, and A-a gradient. Similar injections of D-cysteine (2 ×100 µmol/kg, IV) were without effect, whereas injections of D-cystine (2 ×50 µmol/kg, IV) produced a modest reversal. Our data show that D-cysteine ethyl ester and D-cystine dimethyl ester readily overcome the deleterious effects of morphine on arterial blood gas (ABG) chemistry and A-a gradient by mechanisms that may depend upon their ability to rapidly enter cells. As a result of their known ability to enter the brain, lungs, muscles of the chest wall, and most likely the major peripheral chemoreceptors (i.e., carotid bodies), the effects of the thiolesters on changes in ABG chemistry and A-a gradient elicited by morphine likely involve central and peripheral mechanisms. We are employing target prediction methods to identify an array of in vitro and in vivo methods to test potential functional proteins by which D-CYSee and D-CYSdime modulate the effects of morphine on breathing.

D-Cystine di(m)ethyl ester reverses the deleterious effects of morphine on ventilation and arterial blood gas chemistry while promoting antinociception.

We have identified thiolesters that reverse the negative effects of opioids on breathing without compromising antinociception. Here we report the effects of D-cystine diethyl ester (D-cystine diEE) or D-cystine dimethyl ester (D-cystine diME) on morphine-induced changes in ventilation, arterial-blood gas chemistry, A-a gradient (index of gas-exchange in the lungs) and antinociception in freely moving rats. Injection of morphine (10 mg/kg, IV) elicited negative effects on breathing (e.g., depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive). Subsequent injection of D-cystine diEE (500 µmol/kg, IV) elicited an immediate and sustained reversal of these effects of morphine. Injection of morphine (10 mg/kg, IV) also elicited pronounced decreases in arterial blood pH, pO2 and sO2 accompanied by pronounced increases in pCO2 (all indicative of a decrease in ventilatory drive) and A-a gradient (mismatch in ventilation-perfusion in the lungs). These effects of morphine were reversed in an immediate and sustained fashion by D-cystine diME (500 µmol/kg, IV). Finally, the duration of morphine (5 and 10 mg/kg, IV) antinociception was augmented by D-cystine diEE. D-cystine diEE and D-cystine diME may be clinically useful agents that can effectively reverse the negative effects of morphine on breathing and gas-exchange in the lungs while promoting antinociception. Our study suggests that the D-cystine thiolesters are able to differentially modulate the intracellular signaling cascades that mediate morphine-induced ventilatory depression as opposed to those that mediate morphine-induced antinociception and sedation.

Glutathione ethyl ester reverses the deleterious effects of fentanyl on ventilation and arterial blood-gas chemistry while prolonging fentanyl-induced analgesia.

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 µg/kg, IV) in male adult Sprague-Dawley rats that had received a bolus injection of GSHee (100 µmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO2 and sO2 and increases in pCO2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

Role of central and peripheral opiate receptors in the effects of fentanyl on analgesia, ventilation and arterial blood-gas chemistry in conscious rats.

This study determined the effects of the peripherally restricted µ-opiate receptor (µ-OR) antagonist, naloxone methiodide (NLXmi) on fentanyl (25µg/kg, i.v.)-induced changes in (1) analgesia, (2) arterial blood gas chemistry (ABG) and alveolar-arterial gradient (A-a gradient), and (3) ventilatory parameters, in conscious rats. The fentanyl-induced increase in analgesia was minimally affected by a 1.5mg/kg of NLXmi but was attenuated by a 5.0mg/kg dose. Fentanyl decreased arterial blood pH, pO2 and sO2 and increased pCO2 and A-a gradient. These responses were markedly diminished in NLXmi (1.5mg/kg)-pretreated rats. Fentanyl caused ventilatory depression (e.g., decreases in tidal volume and peak inspiratory flow). Pretreatment with NLXmi (1.5mg/kg, i.v.) antagonized the fentanyl decrease in tidal volume but minimally affected the other responses. These findings suggest that (1) the analgesia and ventilatory depression caused by fentanyl involve peripheral µ-ORs and (2) NLXmi prevents the fentanyl effects on ABG by blocking the negative actions of the opioid on tidal volume and A-a gradient.

L-Cysteine ethyl ester reverses the deleterious effects of morphine on, arterial blood-gas chemistry in tracheotomized rats.

This study determined whether the membrane-permeable ventilatory stimulant, L-cysteine ethylester (L-CYSee), reversed the deleterious actions of morphine on arterial blood-gas chemistry in isoflurane-anesthetized rats. Morphine (2 mg/kg, i.v.) elicited sustained decreases in arterial blood pH, pO2 and sO2, and increases in pCO2 (all responses indicative of hypoventilation) and alveolar-arterial gradient (indicative of ventilation-perfusion mismatch). Injections of L-CYSee (100 µmol/kg, i.v.) reversed the effects of morphine in tracheotomized rats but were minimally active in non-tracheotomized rats. L-cysteine or L-serine ethylester (100 µmol/kg, i.v.) were without effect. It is evident that L-CYSee can reverse the negative effects of morphine on arterial blood-gas chemistry and alveolar-arterial gradient but that this positive activity is negated by increases in upper-airway resistance. Since L-cysteine and L-serine ethylester were ineffective, it is evident that cell penetrability and the sulfur moiety of L-CYSee are essential for activity. Due to its ready penetrability into the lungs, chest wall muscle and brain, the effects of L-CYSee on morphine-induced changes in arterial blood-gas chemistry are likely to involve both central and peripheral sites of action.