The opioid crisis can be traced back to the 1990s. Amid rising demand for pain relief, pharmaceutical companies assured doctors that their drugs carried little risk of addiction, and doctors began prescribing them at a higher rate. Since then, prescription, production and misuse of opioids have risen dramatically. In 2017 alone, an estimated 1.7 million Americans suffered from substance abuse disorders involving prescription or illegal opioids, with some 47,000 deaths due to overdose. Clearly, more research is required to better understand and prevent opioid misuse. In this webinar sponsored by Data Sciences International, Dr. Ralph Lydic and Dr. Tally Largent-Milnes discuss the role of respiratory and neuroscience research in addressing the opioid epidemic. Dr. Ralph Lydic, professor of neuroscience and co-director of anesthesiology research at the University of Tennessee, presents data demonstrating the systemic and neurological effects of opioids on breathing and sleep in rodent models. Following, Dr. Tally Largent-Milnes, assistant professor of pharmacology at the University of Arizona, presents research investigating how cannabinoid receptors modulate opioid-induced respiratory depression in rodent models. Key discussion topics include: - How opiates induce changes in sleep and breathing - Animal model selection: which mouse lines are most suitable for preclinical respiratory research? - The scientific insight gained from simultaneously collecting plethysmography and telemetry signals in a single study - Brain chemistry as the basis of physiology and behavior - How opioids induce respiratory depression - Which cannabinoids, if any, induce respiratory depression in preclinical models - How the site of action determines how cannabinoid targeting agents interact with opioid-induced respiratory depression

1. Ralph Lydic, PhD
Professor, Psychology & Anesthesiology
Robert H. Cole Professor of Neuroscience
University of Tennessee
Joint Faculty Oak Ridge National Laboratory
Tally Largent-Milnes, PhD
Assistant Professor, Pharmacology
College of Medicine – Tucson
University of Arizona
Understanding Physiological Mechanisms of
Opioid Addiction: Advancements in CNS and
Respiratory Endpoint Measurement

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4. Opiate modulation of breathing, sleep, and
brain neurotransmitter concentrations
Ralph Lydic, PhD
Professor of Psychology & Anesthesiology
Robert H. Cole Professor of Neuroscience
University of Tennessee
Joint Faculty Oak Ridge National Laboratory
Copyright 2019 R. Lydic and InsideScientific. All Rights Reserved.

5. Prefrontal cortex
contains no respiratory
neurons but
contributes to opiate-
induced disruption of
sleep and breathing.
Trends Neurosci 18: 22; 1995
• Dysfunction of the prefrontal cortex in addiction
Nature Rev Neurosci 12: 652, 2011
• Prefrontal cortex modulates breathing
J Physiol 591: 6069, 2013
• Prefrontal cortex contains opiate receptorsOverarching
Hypothesis

6. National Drug Overdose Deaths: About 49,000 Due to Opiate Overdose in 2017
Centers for Disease Control and Prevention, National Center for Health Statistics. Multiple
Cause of Death 1999-2017 on CDC WONDER Online Database, released December 2018

7. Example Slide
Header
Weaponized use of
opiates is a potential
threat for civilians
and/or military
Figure from Science 360: 144, 2018

8. “The sine qua non of opioid intoxication is
respiratory depression”
Opiate-induced respiratory depression is
associated with increased risk among subjects who
are female and/or obese.
The opiate buprenorphine is an approved
medication for treating opiate use disorder.
Does buprenorphine causes weight-dependent and
sex-dependent respiratory depression in mice?
N Engl J Med 2012;
367:146-55
Anesthesiology 2018;
128:984-91
N Engl J Med 2017;
377:391–4
Question:

9. Advantages of Mouse Models

10. Mice for Studies of Buprenorphine-Induced Respiratory Depression

11. Whole Body Plethysmography + Telemetry of EEG and EMG
1 sec
Click here to learn
more about respiratory
solutions from DSI

12. Systemic buprenorphine
decreased respiratory
variability (dispersion of
points):
1. in normal weight male
(blue) and female (red)
B6 mice. (A. and B.)
2. in mice with diet-
induced obesity (DIO) (C.
and D.)
Anesthesiology 2018; 128:984-91
Results

13. Zhang et al., IARS Meeting, May 16–20, 2019
Dialysis Delivery of Fentanyl to Prefrontal Cortex Decreases Breathing

14. Systemic Opiates Increase Wakefulness and Decrease NREM and REM Sleep
Locklear et al., Soc Neurosci Abstract 4598 (in press) 2019
Saline (VEH)
Morphine (MSO4)
Fentanyl (FENT)
Buprenorphine (BUPE)

15. https://www.jove.com/video/60333/computer-based-multitaper-spectrogram-program-for (In Press) 2019
Systemic Opiates Cause Drug-Specific Changes in Mouse EEG Power and Frequency

16. Future Directions: Clarify Neurotransmitter Mechanisms Causing
Opiate-Induced Respiratory Depression
Zhang et al., (in review) 2019

17. Acknowledgements
Oak Ridge National Lab
Piet C Jones, MS
Benjamin J Garcia PhD
Jonathon Romero, BS
Ashley Cliff, BA
Daniel A Jacobson, PhD
Lawrence Berkeley Nat’l Lab
James B Brown, PhD
University of Texas HSC
Yandong Jiang, MD PhD
Chinese PLA General Hospital-301
Xiaoying Zhang, MD, PhD
Weidong Mi, MD, PhD
University of Tennessee Knoxville
Helen A Baghdoyan, PhD
Aaron G Baer, BA
Zach Glovak, BA
Joshua M Price, MS
Chris O’Brien, BS
Clarence Locklear, BA
Diana Unzaga, DVM
Support: NIH grant HL-65272 and University of Tennessee
Click Here to
Watch the
Webinar

18. Cannabinoids: The Solution for When
Opioids Take our Breath Away?
Tally Largent-Milnes, PhD
Assistant Professor
University of Arizona
Copyright 2019 T. Largent-Milnes and InsideScientific. All Rights Reserved.

19. Acknowledgements
Ph.D Students
Jared Wahl (Med Pharm)
Beth Wiese, M.Sc. (NGIDP)
Seph Palomino (Med Pharm)
Vani Verkovsky, J.D. (Med Pharm)
Aidan Levine (MD/PhD)
Undergraduate Students
Kiera Blawn (NSCS, MCB, Biochem)
Kathryn Kellohen (NSCS)
Anjali Vivek (NSCS)
Collaborators
Todd W. Vanderah (UA)
Ralph Fregosi (UA)
Alex Makriyannis (Northeastern
University)
Igor Spiegelman (UCLA)
Post-Docs
Erika Liktor-Busa, Ph.D.
M.Sc. Students
Alysha Swanson (Med Pharm,
Perfusion)
Nothing to disclose

20. Points to Consider
1. Circuits by which opioids induce respiratory
depression
2. Which cannabinoids, if any, induce respiratory
depression in preclinical models
3. How the site of action determines how
cannabinoid targeting agents interact with opioid-
induced respiratory depression

21. Opioid Epidemic
https://www.azdhs.gov/prevention/womens-childrens-health/injury-prevention/opioid-
prevention/opioids/index.php#dashboard
https://www.cdc.gov/drugoverdose/epidemic/index.html

22. Respiratory Control in the Central Nervous System
Nagappa et al., 2017

23. Morphine Reduces Respiratory Rate, Tidal Volume, and Minute
Ventilation after 5% CO2 Challenge

24. Cannabis in the US
http://www.ncsl.org/research/civil-and-criminal-justice/marijuana-
overview.aspx; Lachenmeier and Rehm 2015
Are Cannabinoids the solution?

25. CB1/CB2 Distribution
Hypothesis: Targeting CB Receptors in Central Respiratory Groups Will Attenuate
Morphine-Depressed Respiration Without Dampening Antinociception
Specific Aim 1: Determine opioid/cannabinoid
interactions during respiratory challenges
Specific Aim 2: Evaluate antinociceptive effects of
select CB targeting agents alone and in
combination with morphine
Specific Aim 3: Assess the expression of CB
receptor subtypes to in central inspiratory centers

26. Phyto Cannabinoids Do Not Suppress Respiration and Variably Attenuate
Opioid-induced Respiratory Depression

27. Phyto-cannabinoid Synthetic cannabinoid
CB1R Ki = 1.89 -123 nM, CB2R Ki = 0.28 – 16.2 nMCB1R Ki = 5.05-80.3 nM, CB2R Ki = 3.13-75.3 nM
Mixed CB1/CB2 Synthetic Agonists ≠ Phyto Cannabinoids

28. Systemic Activation
of CB2, but not CB1
Mitigates Morphine-
Induced Respiratory
Depression
CB1 agonist
CB1R Ki = 20 nM
CB2 Ki > 1,000 nM
Veh
M
SA
M
365
A
M
365
+
M
S
Veh
M
SA
M
365
A
M
365
+
M
S
Veh
M
SA
M
365
A
M
365
+
M
S
0
50
100
150
Frequency(BPM)±SEM
NormalizedtoBL
Brain Penatrible CB1 Activation Induces Respiratory Depres
Equivalent to Morphine
11 10
Baseline 0% CO2 5% CO2
10 10
11 118 8
8 8
8
8
†
y
y
y y
y
y
†
CB2 agonist
CB2 Ki = 1.3 nM
CB1 Ki = 172 nM
Veh
M
SA
M
2301
A
M
2301
+
M
S
Veh
M
SA
M
2301
A
M
2301
+
M
S
Veh
M
SA
M
2301
A
M
2301
+
M
S
0
50
100
150
Frequency(BPM)±SEM
NormalizedtoBL
Brain penatribile CB2 agonism prevents respiratory depres
11 10
Baseline 0% CO2 5% CO2
10
10
11 11
8 8 8
8
8 8y
†
††
†
y
Nevalainen 2014
Selwood 2009
AM compounds gift from
Makriyannis lab

29. Veh
M
SPrN
M
1
PrN
M
1
+
M
S
Veh
M
SPrN
M
1
PrN
M
1
+
M
S
Veh
M
SPrN
M
1
PrN
M
1
+
M
S
0
50
100
150
Frequency(BPM)±SEM
NormalizedtoBL
Peripherally Restricted CB1 Activation Prevents Respiratory De
11 10
Baseline 0% CO2 5% CO2
8 8 8 8
8 8
10 10
11 11
y
y
† † †
†
PRNMI gifted from Speigelman lab
Seltzman, Shiner et al. 2016
AM compound gifted from
Makriyannis lab
Nevalainen 2014
CB1 agonist
CB1R Ki = 1.18 nM,
%Emax: 108
CB2R Ki = 1 nM,
% Emax: 69
CB2 agonist
CB2 Ki = 17 nM
Veh
M
SA
M
1710
A
M
1710
+
M
S
Veh
M
SA
M
1710
A
M
1710
+
M
S
Veh
M
SA
M
1710
A
M
1710
+
M
S
0
50
100
150
Frequency(BPM)±SEM
NormalizedtoBL
Peripherally restricted CB2 agonism does not prevent respir
depression compared to morphine
11 10
Baseline 0% CO2 5% CO2
10
10
11 11
6
66
6
6 6
†
y
yy y
Peripherally
Restricted CB1, but
not CB2 Agonists
Limit Morphine-
Induced Respiratory
Depression

30. Hypothesis: Targeting CB Receptors in Central Respiratory Groups Will Attenuate Morphine
Depressed Respiration Without Dampening Antinociception
Specific Aim 1: Determine opioid/cannabinoid
interactions during respiratory challenges
Specific Aim 2: Evaluate antinociceptive effects of
select CB compounds alone and in combination
with morphine
Specific Aim 3: Assess the expression of CB
receptor subtypes to in central inspiratory centers
B
L
7
m
in
1
4
m
in
7 0
8 0
9 0
1 0 0
1 1 0
Frequency(BPM)SEM
NormalizedtoBL
V e h ic le
J W H 0 1 5
*
JWH015
Grenald et al., 2017
Click Here to
Watch the
Webinar

31. Painful
Normal
Post-Surgical Pain
Painful
Antinociceptive
Acute Pain
52°C
Morphine
Antinociception is
Retained in the
Presence of AM2301

32. Hypothesis: Targeting CB Receptors in Central Respiratory Groups Will Attenuate Morphine
Depressed Respiration Without Dampening Antinociception
CB1/CB2 Distribution
Specific Aim 1: Determine opioid/cannabinoid
interactions during respiratory challenges
Specific Aim 2: Evaluate antinociceptive effects of
select CB targeting agents alone and in
combination with morphine
Specific Aim 3: Assess the expression of CB
receptor subtypes to in central inspiratory centers

33. A
Wiese et al., 2019 under review
Gray et al., 1999
Herkenham et al., 1990
Opioid and Cannabinoid Receptors are in Mouse Prebötzinger Complex (pBC)

34. Summary/ Next Steps
Phyto Cannabinoids Do Not Suppress Respiration and Variably Attenuate Opioid-induced Respiratory Depression
Opioid and Cannabinoid Receptors are in Mouse Prebötzinger Complex (pBC)
Mixed CB1/CB2 Synthetic Agonists ≠ Phyto Cannabinoids
Systemic activation of CB2, but not CB1 mitigates morphine-induced respiratory depression
Peripherally restricted CB1, but not CB2 agonists limit morphine-induced respiratory depression
CB2 agonism with BBB penetrant AM2301 does not ablate morphine antinociception
Hua et al., 2017, Ramos et al., 2011
Are Cannabinoids the solution? MAYBE

35. Ralph Lydic, PhD
Professor, Psychology & Anesthesiology
Robert H. Cole Professor of Neuroscience
University of Tennessee
Joint Faculty Oak Ridge National Laboratory
Tally Largent-Milnes, PhD
Assistant Professor, Pharmacology
College of Medicine – Tucson
University of Arizona
Thank You