Optimization of a Pyrimidinone Series for Selective Inhibition of Ca2+/Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain

Adenylyl cyclase type 1 is an emerging target for the treatment of chronic pain that is downstream on the analgesic pathway from the traditional µ-opioid receptor. AC1 is expressed in the central nervous system and critical for signaling in pain sensitization. Behavioral studies have revealed AC1 knockout mice exhibit reduced behavioral pain sensitization responses similar to morphine administration. AC1, and a closely related isoform AC8, are also implicated to have a role in learning and memory signaling processes. However, reports suggest selectively targeting AC1 over AC8 may be a viable strategy to eliminate potential deleterious effects on learning and memory. Our team has carried out cellular screening for inhibitors of AC1 that yielded a pyrazolyl-pyrimidinone scaffold with potency comparable to previously published AC1 inhibitors, selectivity versus AC8, and improved drug-like physicochemical properties. Structure-activity relationship (SAR) studies produced 36 analogs that balanced improvements in potency with cellular IC50 values as low as 0.25 µM and selectivity versus AC8. Prioritized analogs were selective for AC1 compared to other AC isoforms and other common neurological targets. A representative analog was assessed for efficacy in a mouse model of inflammatory pain and displayed modest anti-allodynic effects. This series of compounds represents the most potent and selective inhibitors of Ca2+/Calmodulin-stimulated AC1 activity to date with reduced off-target liabilities and improved drug-like physicochemical properties making them promising lead compounds for the treatment of inflammatory pain.

Alternative Splicing Mechanisms Underlying Opioid-Induced Hyperalgesia

Prolonged use of opioids can cause opioid-induced hyperalgesia (OIH). The impact of alternative splicing on OIH remains partially characterized. A study of the absolute and relative modes of action of alternative splicing further the understanding of the molecular mechanisms underlying OIH. Differential absolute and relative isoform profiles were detected in the trigeminal ganglia and nucleus accumbens of mice presenting OIH behaviors elicited by chronic morphine administration relative to control mice. Genes that participate in glutamatergic synapse (e.g., Grip1, Grin1, Wnk3), myelin protein processes (e.g., Mbp, Mpz), and axon guidance presented absolute and relative splicing associated with OIH. Splicing of genes in the gonadotropin-releasing hormone receptor pathway was detected in the nucleus accumbens while splicing in the vascular endothelial growth factor, endogenous cannabinoid signaling, circadian clock system, and metabotropic glutamate receptor pathways was detected in the trigeminal ganglia. A notable finding was the prevalence of alternatively spliced transcription factors and regulators (e.g., Ciart, Ablim2, Pbx1, Arntl2) in the trigeminal ganglia. Insights into the nociceptive and antinociceptive modulatory action of Hnrnpk were gained. The results from our study highlight the impact of alternative splicing and transcriptional regulators on OIH and expose the need for isoform-level research to advance the understanding of morphine-associated hyperalgesia.

Exercise Reduces Morphine-Induced Hyperalgesia and Antinociceptive Tolerance

Chronic morphine intake for treating various pain is frequently concomitant with morphine-induced hyperalgesia and tolerance. The mechanisms can be explained by the activation of p38-MAPK proteins in microglia in the spinal cord horn. Exercise has been shown to prevent the development of microglia overactivation. Thus, we designed to test whether exercise prevents the morphine-induced hyperalgesia and tolerance as well as suppression of p38 phosphorylation. A p38 inhibitor SB203580, exercise, and exercise preconditioning were used for treating morphine-induced hyperalgesia and tolerance development in the present study. The behavior tests for hyperalgesia and tolerance were performed in male Wistar rats before and after morphine administration. Western blotting and immunostaining for examining phosphorylated-p38 expression were performed after the behavior tests. Our results showed that SB203580 and exercise, but not exercise preconditioning, prevented the occurrence of morphine-induced hyperalgesia and tolerance. Meanwhile, exercise decreased morphine-induced phosphorylated-p38 overexpression. In summary, exercise prevented the development of morphine-induced hyperalgesia and tolerance. The mechanism may be related to inhibition of p38 phosphorylation.

Pharmacological blockade of neurokinin1 receptor restricts morphine-induced tolerance and hyperalgesia in the rat

Objectives The most notable adverse side effects of chronic morphine administration include tolerance and hyperalgesia. This study investigated the involvement of dorsal root ganglion (DRG) protein kinase Cɛ (PKCɛ) expression during chronic morphine administration and also considered the relationship between DRG PKCɛ expression and the substance P- neurokinin1 receptor (SP- NK1R) activity. Methods Thirty-six animals were divided into six groups (n=6) in this study. In the morphine and sham groups, rats received 10 µg intrathecal (i.t.) morphine or saline for eight consecutive days, respectively. Behavioral tests were performed on days 1 and 8 before and after the first injections and then 48 h after the last injection (day 10). In the treatment groups, rats received NK1R antagonist (L-732,138, 25 µg) daily, either alone or 10 min before a morphine injection, Sham groups received DMSO alone or 10 min before a morphine injection. Animals were sacrificed on days 8 and 10, and DRG PKCɛ and SP expression were analyzed by western blot and immunohistochemistry techniques, respectively. Results Behavioral tests indicated that tolerance developed following eight days of chronic morphine injection. Hyperalgesia was induced 48 h after the last morphine injection. Expression of SP and PKCɛ in DRG significantly increased in rats that developed morphine tolerance on day 8 and hyperalgesia on day 10, respectively. NK1R antagonist (L-732,138) not only blocked the development of hyperalgesia and the increase of PKCɛ expression but also alleviated morphine tolerance. Conclusions Our results provide evidence that DRG PKCɛ and SP-NK1R most likely participated in the generation of morphine tolerance and hyperalgesia. Pharmacological inhibition of SP-NK1R activity in the spinal cord suggests a role for NK1R and in restricting some side effects of chronic morphine. All experiments were performed by the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised1996) and were approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1396.130).

Enhanced negative response to stress following morphine administration increases wanting of social reward

Animal research suggests a central role of the mu-opioid receptor (MOR) system in mediating contact seeking and the stress-buffering function of social touch. However, the human neurochemistry of social motivation in aversive situations is still poorly understood. In a randomized, double-blind, between-subject design, healthy female volunteers (N = 80) received either 10 mg of the mu-opioid agonist morphine sulfate or a placebo. Following psychosocial stress induction, participants engaged in a social reward task, in which the motivation (subjective ratings of wanting and physical effort) to obtain skin-to-skin social touch and the hedonic reactions (subjective ratings of liking and facial electromyography) elicited by it were assessed. Morphine administration prevented the increase of salivary cortisol, usually observed in response to acute stress exposure. The dampened physiological reaction to the psychosocial stress was associated with increased negative mood and subsequent higher subjective wanting of the most pleasurable touch. Furthermore, participants administered with morphine displayed greater activity of the corrugator muscle during reward anticipation, possibly tracking enhanced attention toward the social stimuli. Overall, the results provide novel evidence on the effect of exogenous opioids administration on the reactions to psychosocial stress and point to a state-dependent regulation of social motivation.

Paroxetine effects on morphine analgesic tolerance in rats

Objectives To alleviate different pain intensities, morphine administration has been extensively used. However, prolonged administration of morphine leads to a progressive decline of its analgesic effect which limits their overall utility. Morphine tolerance is considered as a challenging issue for the treatment of both acute and chronic pain. We conducted this study in rats to investigate the effect of paroxetine on morphine tolerance when used preemptively or after morphine tolerance had developed. Methods Male Wistar rats (weight 250–300 g, n=10) were used to evaluate the effects of paroxetine on tolerance to morphine. In order to induce tolerance, daily intraperitoneal injection of morphine (7 mg/kg) was done. After tolerance induction, a group of animals received intraperitoneal injection of 10 mg/kg paroxetine 30 min prior to each morphine dose. In another trial, to investigate the potential of paroxetine to prevent tolerance to morphine, animals were pretreated with 10 mg/kg paroxetine 30 min before morphine administration. In the control groups, 10 mL/kg of saline was injected. The behavioral test (tail-flick test) was done for all groups. Results Our data showed that paroxetine significantly reversed tolerance to morphine when used after tolerance induction (p<0.001). However, administration of paroxetine before occurrence of tolerance had no effect. Conclusions We conclude that paroxetine could decrease tolerance to morphine when used after the occurrence of morphine tolerance, while it was not able to prevent morphine tolerance when administered preemptively. Ethical committee number IRIB.SBMU.MSP.REC.1394.098.

The Paradoxical Effect Hypothesis of Abused Drugs in a Rat Model of Chronic Morphine Administration

A growing body of studies has recently shown that abused drugs could simultaneously induce the paradoxical effect in reward and aversion to influence drug addiction. However, whether morphine induces reward and aversion, and which neural substrates are involved in morphine’s reward and aversion remains unclear. The present study first examined which doses of morphine can simultaneously produce reward in conditioned place preference (CPP) and aversion in conditioned taste aversion (CTA) in rats. Furthermore, the aversive dose of morphine was determined. Moreover, using the aversive dose of 10 mg/kg morphine tested plasma corticosterone (CORT) levels and examined which neural substrates were involved in the aversive morphine-induced CTA on conditioning, extinction, and reinstatement. Further, we analyzed c-Fos and p-ERK expression to demonstrate the paradoxical effect—reward and aversion and nonhomeostasis or disturbance by morphine-induced CTA. The results showed that a dose of more than 20 mg/kg morphine simultaneously induced reward in CPP and aversion in CTA. A dose of 10 mg/kg morphine only induced the aversive CTA, and it produced higher plasma CORT levels in conditioning and reacquisition but not extinction. High plasma CORT secretions by 10 mg/kg morphine-induced CTA most likely resulted from stress-related aversion but were not a rewarding property of morphine. For assessments of c-Fos and p-ERK expression, the cingulate cortex 1 (Cg1), prelimbic cortex (PrL), infralimbic cortex (IL), basolateral amygdala (BLA), nucleus accumbens (NAc), and dentate gyrus (DG) were involved in the morphine-induced CTA, and resulted from the aversive effect of morphine on conditioning and reinstatement. The c-Fos data showed fewer neural substrates (e.g., PrL, IL, and LH) on extinction to be hyperactive. In the context of previous drug addiction data, the evidence suggests that morphine injections may induce hyperactivity in many neural substrates, which mediate reward and/or aversion due to disturbance and nonhomeostasis in the brain. The results support the paradoxical effect hypothesis of abused drugs. Insight from the findings could be used in the clinical treatment of drug addiction.

Profiling the Effects of Repetitive Morphine Administration on Motor Behavior in Rats

Efficient repetitive clinical use of morphine is limited by its numerous side effects, whereas analgesic tolerance necessitates subsequent increases in morphine dose to achieve adequate levels of analgesia. While many studies focused on analgesic tolerance, the effect of morphine dosing on non-analgesic effects has been overlooked. This study aimed to characterize morphine-induced behavior and the development and progression of morphine-induced behavioral tolerance. Adult male Sprague–Dawley rats were repetitively treated with subcutaneous morphine for 14 days in two dose groups (A: 5 mg/kg/day (b.i.d.) → 10 mg/kg/day; B: 10 mg/kg/day (b.i.d.) → 20 mg/kg/day). Motor behavior was assessed daily (distance traveled, speed, moving time, rearing, rotation) in an open-field arena, before and 30 min post-injections. Antinociception was measured using tail-flick and hot-plate assays. All measured parameters were highly suppressed in both dosing groups on the first treatment day, followed by a gradual manifestation of behavioral tolerance as the treatment progressed. Animals in the high-dose group showed increased locomotor activity after 10 days of morphine treatment. This excitatory phase converted to an inhibition of behavior when a higher morphine dose was introduced. We suggest that the excitatory locomotor effects of repetitive high-dose morphine exposure represent a signature of its behavioral and antinociceptive tolerance.

Effects of Preoperative Low-Dose Gabapentin on Postoperative Pain and Sedation of Patients Undergoing General Anesthesia

Objective: To study the drug interaction between gabapentin and morphine in surgical patients under general anesthesia. Materials and Methods: Two hundred sixty patients undergoing surgery under general anesthesia were randomized into two groups, A with 130 patients receiving gabapentin 2.0 to 3.5 mg/kg orally for premedication add-on, and B with 130 patients getting morphine 0.1 to 0.2 mg/kg intravenously. After surgery, a co-researcher assessed patients using Ramsay sedation scale (RSS) and pain numeric rating scale (NRS) at 2, 4, 8, 12, 16, and 24-hour intervals. Results: Two hundred thirty-two patients were included the present study without procedural adverse events. There were 120 and 112 patients in gabapentin and morphine group, respectively. The administration dosage of gabapentin and morphine between the two groups showed statistically significant differences (p=0.031). During the emergence, the RSS on the sedation, agitation, drowsiness, and pain scores of gabapentin (1.8±0.4) and morphine (1.7±0.5) appeared statistically significant differences (p=0.032); however, the RSS on that in the post-anesthetic care unit (PACU) were 2.0±0.1and 2.0±0.2, respectively, which showed insignificant differences (p-value 0.283). Conclusion: A small, single oral dose of gabapentin as premedication showed a synergistic effect on intraoperative morphine administration. However, this additive effect was not long lasting through the PACU and might not be suitable for an extended surgery. Keywords: Drug interaction; Gabapentin; Morphine; Anesthesia