published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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Opioid-based agents represent the cornerstone of analgesia in modern clinical practice. Additionally however opioids produce a range of unwelcome side-effects including immunomodulation. It has been suggested that this immunomodulation may result either as a direct effect of opioids on circulating immune cells or via a central action. Meanwhile studies show that classical opioid receptors are up-regulated in peripheral inflammation, while endogenous opioids are released from circulating immune cells producing local analgesia. Expression of opioid receptors on immune cells however remains contentious. This thesis has made a significant contribution to understanding the interaction between opioids and a neurovascularimmune axis by employing radioligand binding, flow cytometry and polymerase chain reaction techniques to make a systematic and detailed examination of the expression of the classical opioid receptors (MOP, DOP and KOP) and the non-classical opioid receptor (NOP) and the precursor for its endogenous ligand N/OFQ (ppN/OFQ) in the peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Using these techniques we have shown (1) that naïve human PBMCs do not express classical opioid receptors, (2) that PCR techniques support the view that PBMCs do express gene transcripts for NOP and ppN/OFQ. In an additional clinical study during a profound vascular insult we have used quantitative PCR and radioimmunoassay techniques to follow the expression of the opioid receptors and native N/OFQ throughout a septic episode in patients admitted to the intensive care unit (ICU). Here we report for the first time an elevation in plasma N/OFQ concentration in non-survivors of sepsis requiring ICU admission, 3.0 [2.5 – 5.0]pg ml-1 in non-survivors vs. 1.0 [1.0 – 2.5]pg ml-1 in survivors (p=0.028). Similarly we are first in reporting an elevation in plasma N/OFQ following major abdominal surgery in septic patients. These findings lead us to suggest an amendment to the previously proposed neuroimmune axis to include the N/OFQ-NOP system.

Oxycodone is a semi-synthetic opioid originally developed as a safer alternative to morphine. It is commonly prescribed for its pain-relieving effects, but has recently been implicated as a major underlying cause of the current opioid epidemic due to its clinical limitations that include tolerance, dependence and a high abuse liability. Simultaneous consumption of opioids and ethanol has been shown to increase the risk of overdose and death from opioids in opioid-tolerant individuals. We hypothesized that ethanol reversed opioid tolerance and previous studies showed that ethanol reversed morphine tolerance. This dissertation investigated whether ethanol reversed tolerance to other opioids in mice, primarily oxycodone. We found that tolerance developed to the antinociceptive effects of both oxycodone and hydrocodone, and that the same dose of ethanol (1 g/kg i.p.) reversed that tolerance. Oral ethanol (2 g/kg) also effectively reversed oxycodone tolerance. Ethanol did not significantly alter either acute or chronic oxycodone brain concentrations, suggesting that the reversal effect was mediated by neuronal mechanisms. DRG neurons were isolated from adult mice and the effects of oxycodone were assessed using whole-cell patch clamp electrophysiology experiments. Oxycodone [3µM] acutely reduced neuronal excitability as measured by a shift in threshold potentials to a more positive value. DRG neurons incubated overnight with 10µM oxycodone did not respond to the 3µM oxycodone challenge, indicating tolerance developed within these neurons. To test if ethanol was reversing tolerance through neuronal mechanisms, we incubated DRG neurons overnight with 10µM oxycodone and applied 20mM ethanol to the media prior to recording. Tolerance was robustly reversed in these neurons, as indicated by a response to 3µM oxycodone. The PKC inhibitor, Bis XI, also reversed oxycodone tolerance. In these studies we have clearly shown that tolerance develops to oxycodone in both the whole animal in an isolated neuronal preparation. In addition we have shown that the tolerance produced in these two preparations was reversed by ethanol at blood levels similar to those seen in humans. Further we have also included preliminary data that suggest that this reversal of oxycodone tolerance by ethanol may well be due to its actions on PKC.

BACKGROUND: It is well known that those taking chronic opioid pain medications often become tolerant to the medications and require escalating doses over time (Drewes, 2017). No objective method to identify tolerance currently exists. OBJECTIVE: The objective of this study is to determine the usability and feasibility of a wearable biosensor technology to determine a transition point between opioid naivety and tolerance. METHODS: Participant’s were recruited in the Emergency Department setting and were being admitted with a treatment plan that includes opioid analgesics. Participants were instructed to wear the sensor at all times and to ‘tag’ the sensor when opioid pain medications were administered. This data was analyzed for trends and changes in sensor data before and after opioid administration. Research staff also conducted formative interviews during and after hospital admission to gather information on the participants’ perception of the wearable biosensor and of opioid tolerance. RESULTS: The sample included 17 participants who received, on average, 21.2 morphine equivalents per day during admission. Over 90% of participants stated that they would wear the sensor again, and 70% would even wear two. Data analysis from the E4 biosensor indicated a difference between baseline physiological signaling and post-opioid administration. CONCLUSIONS: In this study, feasibility of wearable mHealth technology was assured, and the preliminary findings of the biosensor data suggest that the features from activity data at different axes can predict opioid use. Future studies will evaluate the development of tolerance among these participants. / 2021-06-17T00:00:00Z

This report describes the synthesis, chemical derivation, and pharmacological and behavioral characterization of several unique classes of opioid receptor modulators. In chapter one, a general overview of opioid receptor history, signaling biology, and therapeutic applications is provided. Also reviewed are several topics of high current interest, including, biased signaling, opioid receptor splice variants/heteromers, and applications of opioid modulators in the treatment of mood disorders. This introduction aims to frame the work that follows, and emphasize to the reader the untapped potential of the opioid receptor system, particularly in the realm of therapeutics development. Chapter two discusses the development of several new C-H activation reactions to provide rapid access to the core molecular scaffold of alkaloids from Tabernanthe iboga. The methods described permit the expedient construction of structurally diverse ibogamine analogs via a modular approach. In chapter three, this work is extended by applying the new reaction methodologies to explore a novel class of oxaibogamine analogs, which act as opioid receptor agonists and antagonists. The thorough exploration of structure-activity relationships within this skeleton is described, along with the pharmacological characterization of several select analogs as biased agonists at both the kappa- and mu-opioid receptors. This section concludes with a discussion of potential therapeutic applications for the synthesized compounds as new analgesics and antidepressants, and future goals and plans for this structural class. In chapter four, the isolation and pharmacological study of several alkaloids of Mitragyna speciosa is presented. Mitragynine, the primary natural alkaloid in this plant, is isolated, along with several naturally occurring analogs, and the modulatory activity of these compounds at the opioid receptors is fully characterized. Further, preliminary results are presented suggesting activity of these alkaloids at several other classes of central nervous system targets, including serotonin and adrenergic receptors. Also discussed are the preparations of semi-synthetic and fully synthetic mitragynine derivatives, including a total synthesis of mitragynine itself. These novel analogs are applied to explore key structure-activity relationships in this unusual opioid-active scaffold. Again, potential applications of Mitragyna alkaloid analogs in the treatment of pain and depression are discussed. In the final chapter, I describe our discovery that tianeptine, a clinically used atypical antidepressant of previously unknown mechanism of action, acts as an agonist of both the mu- and delta-opioid receptors. Activation of the mu-opioid receptor is thus proposed as the initial molecular-level event responsible for eliciting the beneficial therapeutic effects of this agent. This hypothesis is integrated with the large body of literature describing this compound, and mechanistic theories connecting the opioid activity of tianeptine to previous observations are described, with a particular emphasis on indirect modulation of glutamate signaling. Behavioral studies in mice employing both genetic knockout and pharmacological inhibition are then used to confirm the involvement of the opioid receptors in tianeptine's mechanism of action. Also described are thorough explorations of opioid structure-activity relationships within the tianeptine scaffold, and the design and synthesis of novel analogs having improved pharmacokinetic properties. It is hoped that these derivatives may one day serve as new therapeutic options for patients suffering from treatment-resistant depression.

Class of 2007 Abstract / Objectives: To conduct a survey instrument on a pharmacists’ knowledge of and attitudes toward dispensing narcotic medications for the management of chronic pain. Methods: A focus group of 39 pharmacy managers for a local chain drug store reviewed 6 knowledge questions and 10 attitude statements for content validity, clarity and readability. The results of their responses to the survey and other comments were tabulated and analyzed. Results: The focus group sample was small and results were not statistically significant. Pharmacists were highly confident about their training, yet most did not score well on the test, especially those questions designed to distinguish between addiction, pseudo-addiction and tolerance. This limited knowledge may have been related to age since many of the wrong answers selected were based on older definitions. Several questions and statements were identified as ambiguous, plus having unclear directions or incorrect information. Focus group discussions confirmed the limited knowledge found in the survey and clarified pharmacist's responses to the attitude statements. Conclusions: What is clear from the literature and our study is that pharmacists' knowledge about chronic pain and the uses of opioids strongly influences their attitdues. Therefore, the survey questions and statements need to be reworded and restructured to specifically evaluate the relationship between pharmacists' knowledge and their attitudes.

While there are a variety of therapeutics that interact with the opioid receptor system, they are not without side effects; including constipation, dysphoria and respiratory depression. A better understanding of the opioid receptor system may yield therapeutic agents with a limited side effect profile. The neoclerodane diterpene, salvinorin A, appears to interact at opioid receptors through a unique mode of action. A better understanding of its interactions with opioid receptors will yield valuable information about the opioid system. In order to probe further how salvinorin A interacts at opioid receptors, a series of novel analogues modified at the C-2 and furan ring were synthesized and evaluated for their ability to interact at opioid receptors. Synthetic methods were identified to modulate the furan ring, including the synthesis of Diels-Alder cycloadducts and phenyl rings derived from a reductive elimination. The cycloadducts are one of the first reported examples of Diels-Alder chemistry being applied to modify a neoclerodane while the phenyl ring analogues are the first to have aromatic rings directly off the salvinorin A core. C-2 sulfonate analogues were found to interact differently then their ester counterparts at opioid receptors while several of the cycloadduct analogues maintained affinity and efficacy demonstrating the furan is not required for opioid receptor activity. These findings demonstrate that salvinorin A is amenable for chemical modification, illustrating its potential as a novel scaffold for the development of opioid ligands.

Animal research suggests that exposure to inescapable stressors can lead to an endogenous opioid-mediated form of pain inhibition, known as stress-induced analgesia (SIA). Similar results have been found with humans, although the literature is much less extensive and at times contradictory where uncontrollable stressors have led to an increase, rather than a decrease in pain. More recently, there has been some suggestion that emotions play an important role in pain modulation, and that particular negative moods are associated with opioid-mediated hypoalgesia. This research aimed to clarify the psychological (cognitive and affective) factors underlying endogenous opioid-mediated pain inhibition in humans. The purpose of Study 1 was to examine the effects of stressor controllability and predictability on pain intensity (PI) and unpleasantness (UP) ratings during a cold pressor task (CPT) in 56 male and female subjects. The stressor involved a timed mental arithmetic task during which three moderately noxious electrical shocks were delivered. Although subjects were informed that shock delivery was contingent on math performance, the shock schedule was preset and identical across conditions. Perceived control over the shocks was manipulated between subjects by altering the difficulty of the math task. Shock predictability was manipulated by changing the colour of the computer screen to warn of an impending shock. Subjects were randomly allocated to four experimental conditions (controllable-predictable, controllable-unpredictable, uncontrollable-predictable, and uncontrollable-unpredictable shocks). Visual analogue ratings of ‘perceived self-efficacy’ (to avoid the shocks) and mood (anxiety, confusion, discouragement, anger, sluggishness, liveliness) were completed before, during and after the math task. Significantly greater discouragement and lower self-efficacy was reported in ‘uncontrollable’ conditions indicating that ‘controllability’ was manipulated effectively. Results indicated that a perceived lack of control over shocks during the math task led to significantly greater decreases in PI, but not UP, ratings during the last stages of a 4-minute fixed interval CPT after the math task. Shock predictability failed to influence subjective pain ratings alone; however, unpredictability interacted with lack of control to initially increase pain, followed by analgesia. Stress-induced increases in negative affect (anxiety, discouragement, anger) were associated with decreases in cold pressor PI, but with increased shock PI and UP during the math task. It was concluded that lack of control over an aversive event and negative affect led to SIA during a prolonged pain stimulus, whereas shock predictability had little influence on pain. In Study 2, 70 male and female subjects received either an opioid antagonist (naltrexone) or a placebo before the math task (using a double-blind, counterbalanced design), in order to determine the role of endogenous opioids in SIA. Subjects were randomly assigned to one of three experimental conditions to investigate whether the shocks themselves may have contributed to analgesia observed after the math task: (1) easy task-few shocks, (2) hard task-few shocks, (3) hard task-many shocks. Increases in systolic blood pressure (SBP), diastolic blood pressure (DBP), anxiety, anger and discouragement indicated that negative affect and sympathetic arousal were induced during the math task. Endogenous opioids inhibited the rise in anger, but not discouragement or anxiety, during the math task. There was some evidence that perceived lack of control over shocks, and not the shocks themselves, led to opioid-mediated decreases in cold pressor UP after the math task. In correlational analyses, discouraged subjects under opioid blockade reported more cold pressor UP after the math task than their placebo counterparts. However, this effect was not strong enough to reach statistical significance in regression analyses. Anxiety, anger, discouragement and lack of control over shocks increased shock PI and UP during the math task. A growing body of research with normotensive subjects has linked increased cardiovascular activity with insensitivity to pain, but the role of endogenous opioids remains contentious. In addition to the investigations outlined above, Study 2 aimed to examine the contribution of endogenous opioids in the cardiovascular-pain relationship. However, there was no evidence of an interaction between pain and cardiovascular activity in this study. Study 3 was carried out to investigate opioid involvement in the effects of an uncontrollable stressor and stress-induced negative mood on cold pressor PI, UP and pain tolerance, and onset/thresholds of the nociceptive flexion reflex (RIII). Forty-three male and female subjects were administered either naltrexone or a placebo using a double-blind, counterbalanced design before completing a timed mental arithmetic stressor (identical to the ‘hard task-many shocks’ condition in Study 2). Increases in physiological (SBP, DBP) and affective measures (anxiety, anger and discouragement) indicated that the math task induced a marked state of stress. Negative affect increased shock PI and UP during the task, whereas self-efficacious subjects taking the placebo experienced less shock pain. However, uncontrollable stress led to an opioid-antagonised increase in cold pressor UP. Stressor controllability had a similar, but marginal, effect on cold pressor PI, but not pain tolerance. Tolerance of cold pressor pain was not associated with subjective PI and UP ratings, but was positively associated with endurance to non-painful, but unpleasant tasks (Valsalva Manoeuvre, Letter-Symbol Matching Task), indicating that pain tolerance was measuring the ability to tolerate discomfort, in addition to pain. Results of hierarchical multiple regressions demonstrated that increases in discouragement were positively related to increases in cold pressor UP after the math task, for naltrexone recipients only. These findings suggest that discouragement inhibits the UP of a prolonged pain stimulus via opioid mechanisms. RIII latencies and thresholds were not affected by the math task or by opioid blockade; however, these null effects may be due to methodological limitations. Unlike Study 2, higher blood pressure was associated with shock and cold pressor pain inhibition in normotensive subjects, and this relationship appeared to be mediated by opioids. The strong association between chronic pain and depression has led to speculation that the endogenous opioid system and pain modulatory mechanisms may be impaired in depression. At the time that this research was carried out, no studies had examined whether this was the case. In Study 4, the effect of a cognitive stressor (math task used in Study 3) on foot cold pressor PI, UP and pain tolerance and the nociceptive, or R2 component, of the blink reflex was investigated in 61 participants with or without major depression (as met by DSM-IV diagnostic criteria and confirmed by psychometric testing). Naltrexone or placebo was administered to subjects an hour before the math task using a double-blind, counterbalanced design. Increases in physiological (SBP, DBP) and affective measures (anxiety, anger and discouragement) confirmed that the math task induced the targeted emotional state. An opioid-mediated reduction in anxiety occurred mid-way through the math task. Opioid-mediated decreases in foot cold pressor PI and UP were observed in depressed and non-depressed subjects after the math task. R2 onset to 10 mA was facilitated after the task regardless of opioid blockade, suggesting that endogenous opioids are not involved in the modulation of the BR. Increased anxiety and discouragement led to opioid-mediated inhibition of shock PI and UP during the task and, to a lesser extent, foot cold pressor PI and UP after the math task. Anger increased shock pain without being influenced by opioid blockade. Pain tolerance was not influenced by depression, opioid blockade or mood. These findings failed to support the idea that SIA is impaired in major depression, suggesting instead that uncontrollable aversive events and negative mood (anxiety, discouragement) lead to opioid activation and insensitivity to acute pain. Multiple regression analyses revealed that the inverse relationship between resting blood pressure and foot cold pressor PI and UP was opioid-mediated in controls only, suggesting that opioid dysregulation in depression might influence regulatory functions other than SIA. In Study 4, opioid involvement in hetero-segmental pain inhibitory phenomena termed diffuse noxious inhibitory controls (DNIC) was examined separately, before psychological stress. Specifically, the effect of a heterotopic noxious conditioning stimulus (CS i.e., hand CPT) on R2 onset latency was compared before and after drug absorption (before the math task). An inhibitory effect of the first CS was detected at each electrical stimulus intensity consistent with a DNIC effect. However, this effect was not detected during the second CS, suggesting that some other process masked the DNIC effect. In summary, the findings indicate that uncontrollable aversive events and negative emotion (primarily discouragement) activates endogenous opioids and inhibits pain in human subjects, whether depressed or not. Notably, opioids inhibited the affective component of pain perception, or pain UP, more consistently than PI, suggesting that the antinociceptive function of opioids may be secondary to an important emotional-modulatory role. Endogenous opioids also appeared to mediate the cardiovascular-pain relationship in normotensive non-depressed subjects, suggesting an important stress-regulatory role for these peptides. Opioid-mediated masking of this relationship in major depression suggests that functioning of the endogenous opioid system may be impaired in baroreceptor-mediated analgesia. This finding provides preliminary support for the notion that opioid antinociceptive system dysfunction may contribute to cardiovascular disease in depression.