Tuomas O. Lilius

Modulation of Morphine-induced Antinociception in Acute and Chronic Opioid Treatment by Ibudilast

Background:Opioid analgesics are effective in relieving chronic pain, but they have serious adverse effects, including development of tolerance and dependence. Ibudilast, an inhibitor of glial activation and cyclic nucleotide phosphodiesterases, has shown potential in the treatment of neuropathic pain and opioid withdrawal. Because glial cell activation could also be involved in the development of opioid tolerance in rats, the authors studied the antinociceptive effects of ibudilast and morphine in different models of coadministration. Methods:Antinociception was assessed using male Sprague- Dawley rats in hot plate and tail-flick tests. The effects of ibudilast on acute morphine-induced antinociception, induction of morphine tolerance, and established morphine tolerance were studied. Results:Systemic ibudilast produced modest dose-related antinociception and decreased locomotor activity at the studied doses of 2.5–22.5 mg/kg. The highest tested dose of 22.5 mg/kg produced 52% of the maximum possible effect in the tail-flick test. It had an additive antinociceptive effect when combined with systemic morphine. Coadministration of ibudilast with morphine did not attenuate the development of morphine tolerance. However, in morphine-tolerant rats, ibudilast partly restored morphine-induced antinociception. Conclusions:Ibudilast produces modest antinociception, and it is effective in restoring but not in preventing morphine tolerance. The mechanisms of the effects of ibudilast should be better understood before it is considered for clinical use.

Ketamine coadministration attenuates morphine tolerance and leads to increased brain concentrations of both drugs in the rat

The effects of ketamine in attenuating morphine tolerance have been suggested to result from a pharmacodynamic interaction. We studied whether ketamine might increase brain morphine concentrations in acute coadministration, in morphine tolerance and morphine withdrawal.

Pregabalin enhances the antinociceptive effect of oxycodone and morphine in thermal models of nociception in the rat without any pharmacokinetic interactions.

Oxycodone is increasingly being used in combination with pregabalin. Pregabalin use is prevalent in opioid‐dependent individuals. A high number of deaths caused by the co‐use of gabapentinoids and opioids occur. It is not known whether pregabalin affects concentrations of oxycodone or morphine in the central nervous system.

Intrathecal atipamezole augments the antinociceptive effect of morphine in rats.

BACKGROUND: Opioid analgesics are effective in the treatment of chronic pain, but they have serious adverse effects such as development of tolerance and dependence. Adrenergic &agr;2 agonists and &mgr;-opioid receptor agonists show synergistic potentiation and cross-tolerance in spinal analgesia, whereas &agr;2-adrenergic antagonists have shown pronociceptive effects. However, at ultralow doses, spinal &agr;2-adrenergic antagonists have been reported to paradoxically enhance opioid antinociception. New data have suggested a functional &mgr;-opioid-&agr;2-adrenoceptor complex, which may help in interpreting the paradoxical effect of the &agr;2-adrenergic antagonists. In the present study we assessed the effects of low doses of atipamezole, a nonselective &agr;2-adrenergic antagonist, on both systemic and spinal morphine antinociception and tolerance. METHODS: Antinociception was assessed in male Sprague-Dawley rats using hotplate, tail-flick, and paw pressure tests. Spinal or systemic opioid tolerance was induced for 4 days. The effects of both intrathecal and subcutaneous atipamezole on acute morphine-induced antinociception and established morphine tolerance were studied. RESULTS: Systemic or spinal atipamezole itself did not produce antinociception at the doses studied (subcutaneous 0.03, 0.3, 3 &mgr;g/kg or intrathecal 0.1, 1, 10 ng). The combined administration of spinal morphine and 1 ng of atipamezole increased the antinociceptive effect of acute spinal morphine 30 minutes after the administration of test drugs in the tail-flick test. Furthermore, 10 ng of intrathecal atipamezole attenuated established morphine tolerance 30 minutes after the administration of test drugs in the tail-flick test. However, subcutaneous atipamezole had no significant effect on systemic morphine antinociception, and it did not attenuate morphine tolerance. CONCLUSIONS: Spinal coadministration of low doses of atipamezole augmented the antinociceptive effect of morphine in naïve and tolerant rats. Heterodimerization of &mgr;-opioid- and &agr;2A-adrenoceptors with consequent changes in function and interaction could explain these results. This also suggests an interesting explanation for the variability in opioid response and tolerance in patients experiencing stress or having an increased noradrenergic tone due to other causes, e.g., drugs.

Differential Spinal and Supraspinal Activation of Glia in a Rat Model of Morphine Tolerance

Development of tolerance is a well known pharmacological characteristic of opioids and a major clinical problem. In addition to the known neuronal mechanisms of opioid tolerance, activation of glia has emerged as a potentially significant new mechanism. We studied activation of microglia and astrocytes in morphine tolerance and opioid-induced hyperalgesia in rats using immunohistochemistry, flow cytometry and RNA sequencing in spinal- and supraspinal regions. Chronic morphine treatment that induced tolerance and hyperalgesia also increased immunoreactivity of spinal microglia in the dorsal and ventral horns. Flow cytometry demonstrated that morphine treatment increased the proportion of M2-polarized spinal microglia, but failed to impact the number or the proportion of M1-polarized microglia. In the transcriptome of microglial cells isolated from the spinal cord (SC), morphine treatment increased transcripts related to cell activation and defense response. In the studied brain regions, no activation of microglia or astrocytes was detected by immunohistochemistry, except for a decrease in the number of microglial cells in the substantia nigra. In flow cytometry, morphine caused a decrease in the number of microglial cells in the medulla, but otherwise no change was detected for the count or the proportion of M1- and M2-polarized microglia in the medulla or sensory cortex. No evidence for the activation of glia in the brain was seen. Our results suggest that glial activation associated with opioid tolerance and opioid-induced hyperalgesia occurs mainly at the spinal level. The transcriptome data suggest that the microglial activation pattern after chronic morphine treatment has similarities with that of neuropathic pain.

Interactions of (2S,6S;2R,6R)‐Hydroxynorketamine, a Secondary Metabolite of (R,S)‐Ketamine, with Morphine

Ketamine and its primary metabolite norketamine attenuate morphine tolerance by antagonising N‐methyl‐d‐aspartate (NMDA) receptors. Ketamine is extensively metabolized to several other metabolites. The major secondary metabolite (2S,6S;2R,6R)‐hydroxynorketamine (6‐hydroxynorketamine) is not an NMDA antagonist. However, it may modulate nociception through negative allosteric modulation of α7 nicotinic acetylcholine receptors. We studied whether 6‐hydroxynorketamine could affect nociception or the effects of morphine in acute or chronic administration settings. Male Sprague Dawley rats received subcutaneous 6‐hydroxynorketamine or ketamine alone or in combination with morphine, as a cotreatment during induction of morphine tolerance, and after the development of tolerance induced by subcutaneous minipumps administering 9.6 mg morphine daily. Tail flick, hot plate, paw pressure and rotarod tests were used. Brain and serum drug concentrations were quantified with high‐performance liquid chromatography–tandem mass spectrometry. Ketamine (10 mg/kg), but not 6‐hydroxynorketamine (10 and 30 mg/kg), enhanced antinociception and decreased rotarod performance following acute administration either alone or combined with morphine. Ketamine efficiently attenuated morphine tolerance. Acutely administered 6‐hydroxynorketamine increased the brain concentration of morphine (by 60%), and brain and serum concentrations of 6‐hydroxynorketamine were doubled by morphine pre‐treatment. This pharmacokinetic interaction did not, however, lead to altered morphine tolerance. Co‐administration of 6‐hydroxynorketamine 20 mg/kg twice daily did not influence development of morphine tolerance. Even though morphine and 6‐hydroxynorketamine brain concentrations were increased after co‐administration, the pharmacokinetic interaction had no effect on acute morphine nociception or tolerance. These results indicate that 6‐hydroxynorketamine does not have antinociceptive properties or attenuate opioid tolerance in a similar way as ketamine.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

Ketamine and norketamine attenuate oxycodone tolerance markedly less than that of morphine: from behaviour to drug availability

Background: Ketamine attenuates morphine tolerance by antagonising N‐methyl‐d‐aspartate receptors. However, a pharmacokinetic interaction between morphine and ketamine has also been suggested. The interaction between oxycodone and ketamine is unclear. We studied the effects of ketamine and norketamine on the attenuation of morphine and oxycodone tolerance focusing on both the pharmacodynamic and pharmacokinetic interactions. Methods: Morphine 9.6 mg day−1 or oxycodone 3.6 mg day−1 was delivered to Sprague–Dawley rats by subcutaneous pumps. Once tolerance had developed, the rats received subcutaneous injections of ketamine or norketamine. Tail‐flick, hot‐plate, and rotarod tests were performed. Drug concentrations were measured with high‐performance liquid chromatography–tandem mass spectrometry. Results: Anti‐nociceptive tolerance to morphine and oxycodone developed similarly by Day 6. Acute ketamine 10 mg kg−1 and norketamine 30 mg kg−1 attenuated morphine tolerance for 120 and 150 min, respectively, whereas in oxycodone‐tolerant rats the effect lasted only 60 min. Both ketamine and norketamine increased the brain and serum concentrations of morphine, and inhibited its metabolism to morphine‐3‐glucuronide, whereas oxycodone concentrations were not changed. Morphine, but not oxycodone, pretreatment increased the brain and serum concentrations of ketamine and norketamine. Ketamine, but not norketamine, significantly impaired the motor coordination. Conclusions: Ketamine and norketamine attenuated morphine tolerance more effectively than oxycodone tolerance. Ketamine and norketamine increased morphine, but not oxycodone brain concentrations, which may partly explain this difference. Norketamine is effective in attenuating morphine tolerance with minor effects on motor coordination. These results warrant pharmacokinetic studies in patients who are co‐treated with ketamine and opioids.

Simultaneous electrochemical detection of tramadol and O-desmethyltramadol with Nafion-coated tetrahedral amorphous carbon electrode

Abstract Tramadol (TR) is a member of the opioid family and is widely used for pain treatment in clinical patient care. The analgesic effect of tramadol is induced primarily by its main metabolite O-desmethyltramadol (ODMT). Due to inter-individual differences in the TR metabolism to ODMT, the responses to TR vary highly between patients. Thus, a fast and selective method for simultaneous detection of TR and ODMT would increase the patient safety and pain treatment efficacy. In this study, a tetrahedral amorphous carbon (ta-C) electrode coated with a thin dip-coated recast Nafion membrane was fabricated for selective electrochemical determination of TR and ODMT. With this Nafion/ta-C electrode, simultaneous detection of TR and ODMT was achieved with linear ranges of 1–12.5 μM and 1–15 μM, respectively. The limits of detection were 131 nM for TR and 209 nM for ODMT. Both analytes were also measured in the presence of several common interferents, demonstrating the high selectivity of the fabricated electrode. In addition, the effect of pH on the peak potential was studied to observe the electrochemical behavior of the analytes at the electrode. Finally, clinically relevant concentrations of TR and ODMT were simultaneously detected from diluted human plasma to assess the applicability of the electrode in real samples. The fabricated Nafion/ta-C electrode was found successful in the simultaneous electrochemical detection of TR and ODMT in both buffer solution and in human plasma.

Do Diuretics have Antinociceptive Actions: Studies of Spironolactone, Eplerenone, Furosemide and Chlorothiazide, Individually and with Oxycodone and Morphine

Spironolactone, eplerenone, chlorothiazide and furosemide are diuretics that have been suggested to have antinociceptive properties, for example via mineralocorticoid receptor antagonism. In co‐administration, diuretics might enhance the antinociceptive effect of opioids via pharmacodynamic and pharmacokinetic mechanisms. Effects of spironolactone (100 mg/kg, i.p.), eplerenone (100 mg/kg, i.p.), chlorothiazide (50 mg/kg, i.p.) and furosemide (100 mg/kg, i.p.) were studied on acute oxycodone (0.75 mg/kg, s.c.)‐ and morphine (3 mg/kg, s.c.)‐induced antinociception using tail‐flick and hot plate tests in male Sprague Dawley rats. The diuretics were administered 30 min. before the opioids, and behavioural tests were performed 30 and 90 min. after the opioids. Concentrations of oxycodone, morphine and their major metabolites in plasma and brain were quantified by mass spectrometry. In the hot plate test at 30 and 90 min., spironolactone significantly enhanced the antinociceptive effect (% of maximum possible effect) of oxycodone from 10% to 78% and from 0% to 50%, respectively, and that of morphine from 12% to 73% and from 4% to 83%, respectively. The brain oxycodone and morphine concentrations were significantly increased at 30 min. (oxycodone, 46%) and at 90 min. (morphine, 190%). We did not detect any independent antinociceptive effects with the diuretics. Eplerenone and chlorothiazide did not enhance the antinociceptive effect of either opioid. The results suggest that spironolactone enhances the antinociceptive effect of both oxycodone and morphine by increasing their concentrations in the central nervous system.