GluN2B-Selective NMDA Receptor Negative Allosteric Modulation as a Treatment for Chronic Pain and Opioid Tolerance Pubblico
Harris, Lynnea (Spring 2023)
Abstract
Drug overdose is the leading cause of accidental death in the US. From April 2021 to April 2022, there were over 100,000 new cases — an increase of nearly 30% compared to the previous year. The majority of these cases involved use of an opioid. Opioids are sought after for their profound analgesic properties, however their utility is significantly hindered by adverse off-target effects including addiction, physical dependence, and tolerance. Analgesic tolerance to opioids is characterized by a decrease in the efficacy of an opioid over time with repeated use. Tolerance requires increasingly higher doses of the opioid to maintain suitable analgesia and can lead to overdose. Persistent activation of NMDA receptors is a key mechanism in the development of tolerance. Additionally, the NMDA receptors which contain the GluN2B subunit are of particular interest in this mechanism. Here, we have introduced a novel GluN2B-selective negative allosteric modulator of the NMDA receptor, EU93-108, and evaluated its effects on pain and tolerance in mice. EU93-108 is potent and brain penetrant, and possesses analgesic properties in allodynia and thermal nociception rodent pain models. The compound also produces a significant enhancement effect whereby morphine, when combined with EU93-108, produces stronger thermal antinociception compared to that of morphine alone. These results suggest that GluN2B negative modulation has utility in the treatment of chronic pain and tolerance. Further structure-activity relationship work around this compound could give rise to compounds that can function as analgesic adjuvant therapeutics to diminish the onset of tolerance due to chronic opioid use.
Table of Contents
Table of Contents
List of Illustrations
Figures
Tables
Schemes
List of Abbreviations
Chapter 1: Background and Overview of Pathological Pain, Opioids, Analgesic Tolerance, and NMDAR Allosteric Modulation
1.1 Overview of Beneficial and Pathological Pain 1
1.1.1 Neurocircuitry Underlying Pain 2
1.1.2 Types of Pain 8
1.1.3 Interventions for Pain Management 9
1.2 Overview of Opioids and Opioid Receptors 11
1.2.1 Opioid Receptors 12
1.2.2 Opioids and the Dopamine Reward System 16
1.3 The United States Opioid Epidemic 19
1.4 Strategies to Develop Safer Analgesics 22
1.5 Behavioral Paradigms to Assess Efficacy of Analgesic Therapeutics 24
1.6 Analgesic Tolerance to Opioids 28
1.7 The NMDA Receptor 32
1.7.1 NMDAR Uncompetitive Antagonists 37
1.7.2 GluN2B-Selective Antagonists 39
1.7.3 The Role of NMDARs in Tolerance Development 42
1.7.4 Connecting the GluN2B Subunit to Tolerance Development 45
1.7.5 The 93 Series 47
Chapter 2: Novel GluN2B-Selective NMDA Receptor Negative Allosteric Modulator Possesses Intrinsic Analgesic Properties and Enhances Analgesia of Morphine in Rodent Pain Models
2.1 Abstract 49
2.2 Introduction 50
2.3 Results 53
2.3.1 EU93-108 is a potent, GluN2B-selective NMDAR NAM 53
2.3.2 EU93-108 concentration-inhibition curves on diheteromeric and triheteromeric NMDARs 54
2.3.3 Crystal structure of GluN1-GluN2B amino terminal domain in complex with EU93-108 57
2.3.4 Determination of intrinsic antinociceptive properties of GluN2B-selective NAMs 60
2.3.5 EU93-108 decreases mechanical allodynia and has sustained brain and plasma concentrations 63
2.3.6 Acute morphine and EU93-108 co-administration produces enhanced tail flick latency 65
2.3.7 EU93-108 has sedative effects at high doses 67
2.3.8 Chronic co-administration of morphine with EU93-108 did not inhibit development of tolerance 69
2.3.9 Co-administration of EU93-108 and morphine slows worsening of pre-established tolerance 72
2.3.10 Off-Target Effects of EU93-108 76
2.4 Discussion 77
2.5 Materials and Methods 81
2.6 Supplemental Data 91
Chapter 3: Considerations, Future Directions, and Broader Implications
3.1 Summary 98
3.2 Experimental Considerations 99
3.2.1 Analgesia vs Sedation 99
3.2.2 Differences Between EU93-108 and Previous GluN2B-selective NAMs
101
3.2.3 Off-Target Effects 102
3.2.4 Sex Differences 104
3.3 Future Directions 106
3.4 Broader Implications for GluN2B Negative Modulation and Chronic Pain 108
3.5 References 109
List of Illustrations Page
List of Figures
Chapter 1:
Figure 1. Brain regions involved in processing nociceptive input. 7
Figure 2. Structure of the mu-opioid receptor and G protein complex . 12
Figure 3. The dopamine reward pathway. 17
Figure 4. The NMDA receptor. 33
Figure 5. Uncompetitive NMDAR antagonists. 38
Figure 6. GluN2B-selective NMDAR negative allosteric modulators (NAMs). 41
Figure 7. Analgesic tolerance development. 44
Chapter 2:
Figure 8. Previously published inhibitors of the NMDAR. 53
Figure 9. Inhibition of NMDA receptors by EU93-108. 55
Figure 10. Structure of GluN1-GluN2B ATD in complex with EU93-108. 59
Figure 11. Intrinsic antinociceptive properties of EU93-4, EU93-31, EU93-108, ifenprodil, and Ro25-6981 in male C57BL/6J mice. 61
Figure 12. Intrinsic antinociceptive properties of EU93-4, EU93-31, EU93-108, ifenprodil, and Ro25-6981 in female C57BL/6J mice. 62
Figure 13. EU93-108 is efficacious in the Chung model of allodynia and has sustained plasma and brain concentrations over 4 hours. 64
Figure 14. Acute co-administration of EU93-108 and 5 mg/kg of morphine in male and female mice. 66
Figure 15. Locomotor rest time data for EU93-108 in male and female mice. 68
Figure 16. Morphine analgesic tolerance “stair stepping” dosing regimen. 70
Figure 17. Effects of co-administration of EU93-108 and morphine on tolerance development in male and female mice. 71
Figure 18. Dosing regimen to assess effects of EU93-108 on tolerant mice. 73
Figure 19. Effects of EU93-108 on tolerant male and female mice. 74
Supplemental Figure S1. The mean raw current ± SEM in nanoamperes (nA) in response to exposure of Xenopus oocytes recorded under two electrode voltage clamp to 100 µM glutamate and 100 µM glycine. 92
Supplemental Figure S2. Morphine dose-response curve in male C57BL/6J mice. 94
Supplemental Figure S3. Locomotor activity of EU93-108. 95
Chapter 3:
Figure 20. Structural considerations for improving off-target effects of EU93-108 and similar GluN2B-selective NAMs 103
List of Tables
Chapter 2:
Table 1. EU93-108 is a GluN2B-selective NMDAR NAM. 54
Table 2. P values for tolerance development experiments. 72
Table 3. P values for tolerant mice experiments. 76
Table 4. Secondary Off-Target Screen of EU93-108. 77
Supplemental Table S1. Table of EU93-108 concentration-inhibition results at NMDA receptors expressed in Xenopus oocytes. 91
Supplemental Table S2. X-ray crystallographic data collection and model refinement statistics. 93
Supplemental Table S3. Off-target actions of EU93-108 at ligand-gated ion channels expressed in Xenopus oocytes. 96
Supplemental Table S4. Off-Target Actions of EU93-108: Primary GPCR Screen. 97
List of Schemes
Chapter 2:
Scheme 1. 93 Series synthesis 82
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