L-NAC and L-NAC methyl ester prevent and overcome physical dependence to fentanyl in male rats.

N-acetyl-L-cysteine (L-NAC) is a proposed therapeutic for opioid use disorder. This study determined whether co-injections of L-NAC (500 µmol/kg, IV) or its highly cell-penetrant analogue, L-NAC methyl ester (L-NACme, 500 µmol/kg, IV), prevent acquisition of acute physical dependence induced by twice-daily injections of fentanyl (125 µg/kg, IV), and overcome acquired dependence to these injections in freely-moving male Sprague Dawley rats. The injection of the opioid receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IV), elicited a series of withdrawal phenomena (i.e. behavioral and cardiorespiratory responses, hypothermia and body weight loss) in rats that received 5 or 10 injections of fentanyl and similar numbers of vehicle co-injections. With respect to the development of dependence, the NLX-precipitated withdrawal phenomena were reduced in rats that received had co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme. In regard to overcoming established dependence, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) were reduced in rats that had received co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme beginning with injection 6 of fentanyl. This study provides compelling evidence that co-injections of L-NAC and L-NACme prevent the acquisition of physical dependence and overcome acquired dependence to fentanyl in male rats. The higher efficacy of L-NACme is likely due to its greater cell penetrability in brain regions mediating dependence to fentanyl and interaction with intracellular signaling cascades, including redox-dependent processes, responsible for the acquisition of physical dependence to fentanyl.

L-cysteine ethyl ester prevents and reverses acquired physical dependence on morphine in male Sprague Dawley rats.

The molecular mechanisms underlying the acquisition of addiction/dependence on morphine may result from the ability of the opioid to diminish the transport of L-cysteine into neurons via inhibition of excitatory amino acid transporter 3 (EAA3). The objective of this study was to determine whether the co-administration of the cell-penetrant L-thiol ester, L-cysteine ethyl ester (L-CYSee), would reduce physical dependence on morphine in male Sprague Dawley rats. Injection of the opioid-receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IP), elicited pronounced withdrawal phenomena in rats which received a subcutaneous depot of morphine (150 mg/kg) for 36 h and were receiving a continuous infusion of saline (20 µL/h, IV) via osmotic minipumps for the same 36 h period. The withdrawal phenomena included wet-dog shakes, jumping, rearing, fore-paw licking, 360° circling, writhing, apneas, cardiovascular (pressor and tachycardia) responses, hypothermia, and body weight loss. NLX elicited substantially reduced withdrawal syndrome in rats that received an infusion of L-CYSee (20.8 µmol/kg/h, IV) for 36 h. NLX precipitated a marked withdrawal syndrome in rats that had received subcutaneous depots of morphine (150 mg/kg) for 48 h) and a co-infusion of vehicle. However, the NLX-precipitated withdrawal signs were markedly reduced in morphine (150 mg/kg for 48 h)-treated rats that began receiving an infusion of L-CYSee (20.8 µmol/kg/h, IV) at 36 h. In similar studies to those described previously, neither L-cysteine nor L-serine ethyl ester (both at 20.8 µmol/kg/h, IV) mimicked the effects of L-CYSee. This study demonstrates that 1) L-CYSee attenuates the development of physical dependence on morphine in male rats and 2) prior administration of L-CYSee reverses morphine dependence, most likely by intracellular actions within the brain. The lack of the effect of L-serine ethyl ester (oxygen atom instead of sulfur atom) strongly implicates thiol biochemistry in the efficacy of L-CYSee. Accordingly, L-CYSee and analogs may be a novel class of therapeutics that ameliorate the development of physical dependence on opioids in humans.

Lipophilic analogues of D-cysteine prevent and reverse physical dependence to fentanyl in male rats.

We examined whether co-injections of the cell-permeant D-cysteine analogues, D-cysteine ethyl ester (D-CYSee) and D-cysteine ethyl amide (D-CYSea), prevent acquisition of physical dependence induced by twice-daily injections of fentanyl, and reverse acquired dependence to these injections in freely-moving male Sprague Dawley rats. Injection of the opioid receptor antagonist, naloxone HCl (NLX, 1.5 mg/kg, IV), elicited a series of withdrawal phenomena that included cardiorespiratory and behavioral responses, and falls in body weight and body temperature, in rats that received 5 or 10 injections of fentanyl (125 µg/kg, IV), and the same number of vehicle co-injections. Regarding the development of physical dependence, the NLX-precipitated withdrawal phenomena were markedly reduced in fentanyl-injected rats that had received co-injections of D-CYSee (250 µmol/kg, IV) or D-CYSea (100 µmol/kg, IV), but not D-cysteine (250 µmol/kg, IV). Regarding reversal of established dependence to fentanyl, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) was markedly reduced in rats that received co-injections of D-CYSee (250 µmol/kg, IV) or D-CYSea (100 µmol/kg, IV), but not D-cysteine (250 µmol/kg, IV), starting with injection 6 of fentanyl. This study provides evidence that co-injections of D-CYSee and D-CYSea prevent the acquisition of physical dependence, and reverse acquired dependence to fentanyl in male rats. The lack of effect of D-cysteine suggests that the enhanced cell-penetrability of D-CYSee and D-CYSea into cells, particularly within the brain, is key to their ability to interact with intracellular signaling events involved in acquisition to physical dependence to fentanyl.

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.

L-cysteine methyl ester overcomes the deleterious effects of morphine on ventilatory parameters and arterial blood-gas chemistry in unanesthetized rats.

We are developing a series of thiolesters that produce an immediate and sustained reversal of the deleterious effects of opioids, such as morphine and fentanyl, on ventilation without diminishing the antinociceptive effects of these opioids. We report here the effects of systemic injections of L-cysteine methyl ester (L-CYSme) on morphine-induced changes in ventilatory parameters, arterial-blood gas (ABG) chemistry (pH, pCO2, pO2, sO2), Alveolar-arterial (A-a) gradient (i.e., the index of alveolar gas-exchange within the lungs), and antinociception in unanesthetized Sprague Dawley rats. The administration of morphine (10 mg/kg, IV) produced a series of deleterious effects on ventilatory parameters, including sustained decreases in tidal volume, minute ventilation, inspiratory drive and peak inspiratory flow that were accompanied by a sustained increase in end inspiratory pause. A single injection of L-CYSme (500 µmol/kg, IV) produced a rapid and long-lasting reversal of the deleterious effects of morphine on ventilatory parameters, and a second injection of L-CYSme (500 µmol/kg, IV) elicited pronounced increases in ventilatory parameters, such as minute ventilation, to values well above pre-morphine levels. L-CYSme (250 or 500 µmol/kg, IV) also produced an immediate and sustained reversal of the deleterious effects of morphine (10 mg/kg, IV) on arterial blood pH, pCO2, pO2, sO2 and A-a gradient, whereas L-cysteine (500 µmol/kg, IV) itself was inactive. L-CYSme (500 µmol/kg, IV) did not appear to modulate the sedative effects of morphine as measured by righting reflex times, but did diminish the duration, however, not the magnitude of the antinociceptive actions of morphine (5 or 10 mg/kg, IV) as determined in tail-flick latency and hindpaw-withdrawal latency assays. These findings provide evidence that L-CYSme can powerfully overcome the deleterious effects of morphine on breathing and gas-exchange in Sprague Dawley rats while not affecting the sedative or early stage antinociceptive effects of the opioid. The mechanisms by which L-CYSme interferes with the OR-induced signaling pathways that mediate the deleterious effects of morphine on ventilatory performance, and by which L-CYSme diminishes the late stage antinociceptive action of morphine remain to be determined.

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.

S-Nitroso-L-Cysteine Stereoselectively Blunts the Deleterious Effects of Fentanyl on Breathing While Augmenting Antinociception in Freely-Moving Rats.

Endogenous and exogenously administered S-nitrosothiols modulate the activities of central and peripheral systems that control breathing. We have unpublished data showing that the deleterious effects of morphine on arterial blood-gas chemistry (i.e., pH, pCO2, pO2, and sO2) and Alveolar-arterial gradient (i.e., index of gas exchange) were markedly diminished in anesthetized Sprague Dawley rats that received a continuous intravenous infusion of the endogenous S-nitrosothiol, S-nitroso-L-cysteine. The present study extends these findings by showing that unanesthetized adult male Sprague Dawley rats receiving an intravenous infusion of S-nitroso-L-cysteine (100 or 200 nmol/kg/min) markedly diminished the ability of intravenous injections of the potent synthetic opioid, fentanyl (10, 25, and 50 µg/kg), to depress the frequency of breathing, tidal volume, and minute ventilation. Our study also found that the ability of intravenously injected fentanyl (10, 25, and 50 µg/kg) to disturb eupneic breathing, which was measured as a marked increase of the non-eupneic breathing index, was substantially reduced in unanesthetized rats receiving intravenous infusions of S-nitroso-L-cysteine (100 or 200 nmol/kg/min). In contrast, the deleterious effects of fentanyl (10, 25, and 50 µg/kg) on frequency of breathing, tidal volume, minute ventilation and non-eupneic breathing index were fully expressed in rats receiving continuous infusions (200 nmol/kg/min) of the parent amino acid, L-cysteine, or the D-isomer, namely, S-nitroso-D-cysteine. In addition, the antinociceptive actions of the above doses of fentanyl as monitored by the tail-flick latency assay, were enhanced by S-nitroso-L-cysteine, but not L-cysteine or S-nitroso-D-cysteine. Taken together, these findings add to existing knowledge that S-nitroso-L-cysteine stereoselectively modulates the detrimental effects of opioids on breathing, and opens the door for mechanistic studies designed to establish whether the pharmacological actions of S-nitroso-L-cysteine involve signaling processes that include 1) the activation of plasma membrane ion channels and receptors, 2) selective intracellular entry of S-nitroso-L-cysteine, and/or 3) S-nitrosylation events. Whether alterations in the bioavailability and bioactivity of endogenous S-nitroso-L-cysteine is a key factor in determining the potency/efficacy of fentanyl on breathing is an intriguing question.

L-NAC reverses of the adverse effects of fentanyl infusion on ventilation and blood-gas chemistry.

There is an urgent need for development of drugs that are able to reverse the adverse effects of opioids on breathing and arterial blood-gas (ABG) chemistry while preserving opioid analgesia. The present study describes the effects of bolus injections of N-acetyl-L-cysteine (L-NAC, 500 µmol/kg, IV) on ventilatory parameters, ABG chemistry, Alveolar-arterial (A-a) gradient, sedation (righting reflex) and analgesia status (tail-flick latency assay) in unanesthetized adult male Sprague Dawley rats receiving a continuous infusion of fentanyl (1 µg/kg/min, IV). Fentanyl infusion elicited pronounced disturbances in (1) ventilatory parameters (e.g., decreases in frequency of breathing, tidal volume and minute ventilation), (2) ABG chemistry (decreases in pH, pO2, sO2 with increases in pCO2), (3) A-a gradient (increases that were consistent with reduced alveolar gas exchange), and (4) sedation and analgesia. Bolus injections of L-NAC given 60 and 90 min after start of fentanyl infusion elicited rapid and sustained reversal of the deleterious effects of fentanyl infusion on ventilatory parameters and ABG chemistry, whereas they did not affect the sedative or analgesic effects of fentanyl. Systemic L-NAC is approved for human use, and thus our findings raise the possibility that this biologically active thiol may be an effective compound to combat opioid-induced respiratory depression in human subjects.

Inducible re-expression of HEXIM1 causes physiological cardiac hypertrophy in the adult mouse.

AIMS: The transcription factor hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) regulates myocardial vascularization and growth during cardiogenesis. Our aim was to determine whether HEXIM1 also has a beneficial role in modulating vascularization, myocardial growth, and function within the adult heart. METHODS AND RESULTS: To achieve our objective, we created and investigated a mouse line wherein HEXIM1 was re-expressed in adult cardiomyocytes to levels found in the foetal heart. Our findings support a beneficial role for HEXIM1 through increased vascularization, myocardial growth, and increased ejection fraction within the adult heart. HEXIM1 re-expression induces angiogenesis, that is, essential for physiological hypertrophy and maintenance of cardiac function. The ability of HEXIM1 to co-ordinate processes associated with physiological hypertrophy may be attributed to HEXIM1 regulation of other transcription factors (HIF-1-α, c-Myc, GATA4, and PPAR-α) that, in turn, control many genes involved in myocardial vascularization, growth, and metabolism. Moreover, the mechanism for HEXIM1-induced physiological hypertrophy appears to be distinct from that involving the PI3K/AKT pathway. CONCLUSION: HEXIM1 re-expression results in the induction of angiogenesis that allows for the co-ordination of tissue growth and angiogenesis during physiological hypertrophy.