Reduced Opioid Analgesic Requirements Via Improved Endogenous Opioid Function
Phase: NA
Conditions: Chronic Pain
Sponsor: Vanderbilt University Medical Center

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Reduced Opioid Analgesic Requirements Via 
Improved Endogenous Opioid Function 
 
 
Stephen Bruehl, Ph.D. (Co-Principal Investigator, Contact PI) 
Professor of Anesthesiology 
Vanderbilt University School of Medicine 
701 Medical Arts Building 
1211 21st Avenue South 
Nashville, TN  37212 
 
 
Vanderbilt University School of Medicine Co-Investigator 
Rajnish Gupta, M.D., Assistant Professor 
Department of Anesthesiology 
Vanderbilt University School of Medicine 
 
Rush University Site Co-Principal Investigator 
John Burns, Ph.D., Professor 
Department of Behavioral Science  
Rush University School of Medicine 
 
Rush University Site Co-Investigator 
Asokumar Bunavendran, M.D., Associate Professor 
Department of Anesthesiology 
Rush University School of Medicine  
 
University of Wisconsin Co-Investigator (Exercise Protocol Expert) 
Kelli Koltyn, Ph.D., Professor  
Department of Kinesiology  
University of Wisconsin 

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Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
13.0 Literature Cited 

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1.0 
Background 
 
Chronic pain (CP) affects 100 million individuals in the U.S.  CP management has 
increasingly utilized long-term opioid analgesic therapy, a change associated with 
increased opioid abuse (via greater exposure in vulnerable individuals), non-pain health 
consequences (hormone changes, falls), and a dramatic rise in opioid-related overdoses 
and deaths.  Treatment strategies that minimize the need for chronic high-dose opioids 
are sorely needed.  This project will test the novel hypothesis that effective pain relief 
can be achieved at lower opioid analgesic doses by increasing levels of endogenous 
opioids (EOs).  This hypothesis follows from our prior work demonstrating that analgesic 
responses to opioid medications are significantly influenced by individual differences in 
EO activity18,19.  Results indicated that for individuals exhibiting higher natural EO levels, 
morphine produced little incremental analgesic benefit.  In contrast, for those displaying 
less natural EO analgesia, morphine produced significant analgesia.  Findings suggested 
that morphine in effect supplemented lower levels of EO analgesia, making pain 
responses of individuals lower in EO function who had received morphine look 
statistically similar to individuals higher in EO function not receiving morphine18.  An 
Opioid Supplement Model was suggested, in which opioid analgesics supplement EO 
function, making up the difference necessary to maximize opioid receptor-mediated pain 
inhibition.  Our previous work looked only at natural variations in EO function, and only 
at a single opioid analgesic dosage.  Therefore, the key clinical implication of the Opioid 
Supplement Model has not been tested: can effective pain relief be achieved at lower 
opioid analgesic dosages by enhancing EO function?  This is one primary aim of the 
current proposal.  While several controlled trials indicate that aerobic exercise is 
effective for reducing CP intensity, and it is widely assumed these benefits are related to 
enhanced EO analgesia, this assumption has received surprisingly little empirical testing.  
Therefore, a second aim of this project is for the first time to directly test whether 
aerobic exercise training enhances EO function in individuals with chronic low back pain 
(CLBP), and whether resulting reductions in CP intensity are attributable to these EO 
changes.  Results will provide important information on mechanisms underlying a 
common CP therapy and have potentially important implications for public health, as 
results would highlight the potential for strategic combination of lower dose opioid 
analgesic therapy with nonpharmacological EO-enhancing interventions, potentially 
reducing negative side effects and mitigating opioid therapy risks.   
 
2.0 
Rationale and Specific Aims 
 
Daily use of high-dose opioid analgesics for CP management has increased dramatically, 
and is associated with increasing numbers of patients experiencing opioid-related 
negative health effects, abuse, overdose, and even death. Ways of providing effective 
chronic pain relief with less reliance on high-dose opioid analgesics are sorely needed.  
Based on recent work indicating that responses to opioid analgesics are influenced by 
functioning in endogenous opioid systems18,19, this project will determine whether 
enhancing endogenous opioids (via aerobic exercise training) permits achieving desired 
levels of analgesia with lower dosages of opioid analgesics, and fewer side effects and 
abuse-relevant drug effects.  This 4 year project will test study hypotheses in a sample 
of 116 chronic low back pain patients.  The study will have two key elements: 1) a 
randomized, controlled aerobic exercise manipulation in CP patients completing daily 

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electronic pain diaries and 2) laboratory evoked thermal pain protocols pre- and post-
exercise permitting direct examination of changes in both opioid analgesic effects (in 
response to a series of incremental morphine doses) and EO activity (indexed by 
comparing pain responses after placebo vs. opioid blockade).  This design will permit 
testing the novel hypothesis that aerobic exercise can facilitate desired analgesia levels 
at lower dosages of opioid analgesics via enhanced EO function.   
 
1) To determine whether an aerobic exercise manipulation increases EO activity in CLBP 
patients, and whether these EO changes mediate manipulation-induced reductions in 
evoked pain responsiveness and daily CP intensity.  If hypotheses are supported, a) 
participants randomized to exercise will exhibit greater decreases in CP intensity and 
evoked pain responsiveness and greater increases in EO function (indexed by laboratory 
responses to opioid blockade) than those assigned to the control condition, and b) the 
indirect path linking the exercise condition to reduced CP intensity via increased EO 
function will be significant. 
 
2) To determine whether the exercise manipulation is related to achieving analgesic 
targets at lower opioid  analgesic dosages, and whether these changes in analgesic 
requirements are mediated by pre-post EO changes.  If hypotheses are supported, a) 
participants assigned to exercise will exhibit a 25% reduction in acute pain responses 
(relative to pre-manipulation placebo responses) at a significantly lower total morphine 
dose (based on incremental dosing) than controls, and b) the indirect path linking the 
exercise condition to reduced morphine requirements via increased EO function will be 
significant. 
 
3) To determine whether the exercise manipulation is related to achieving a 25% 
morphine-induced reduction in evoked pain responsiveness with fewer negative side 
effects and abuse-relevant effects, and whether these changes are mediated by pre- to 
post-manipulation EO changes. If hypotheses are supported, a) participants assigned to 
the exercise condition will exhibit a 25% reduction in acute pain responses (relative to 
pre-manipulation placebo responses) during morphine administration with significantly 
fewer negative side effects (e.g., nausea, sedation) and less abuse-relevant subjective 
drug effects (e.g., drug liking, euphoria) than those in the control condition, and b) the 
indirect path linking the exercise condition to reduced side effects and abuse-relevant 
effects via increased EO function will be significant. 
 
3.0 
Animal Studies and Previous Human Studies 
 
Both EOs and opioid medications produce analgesia through activating the same opioid 
receptors.  This common mechanism of action between EO analgesia and responses to 
opioid-based medications suggests that the former may be related to the latter.  
Surprisingly, until recently this question had never been directly addressed in humans, 
although indirect evidence did support these hypothesized associations.  For example, a 
systematic review of randomized acupuncture trials found that acupuncture not only 
significantly reduces postoperative acute pain, but also reduces opioid analgesic 
consumption and opioid side effects112. To the extent that the effects of acupuncture are 
EO-mediated, as suggested by several studies indicating its analgesic effects can be 
blocked by opioid antagonists57,58,113, these findings imply that enhancing EO function 

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might supplement analgesia gained from opioid-based medications and thus permit 
reduced dosages. 
The Co-PIs recently reported the first human work directly examining 
associations between EO function and opioid analgesic responses. Using controlled 
laboratory methods, associations were examined between spontaneous EO function 
(indexed by differences in acute pain responses between opioid blockade [naloxone] and 
placebo conditions) and analgesic responses to morphine, the prototypic mu opioid 
agonist18.  In a sample of 45 chronic low back pain patients and 31 pain-free healthy 
controls (combined for analyses given no significant main or interaction effects of 
subject type), significant associations were observed between EO function and morphine 
analgesic responses on all 7 measures of pain responsiveness examined across two 
different evoked pain tasks (ischemic pain, thermal pain)18.  We recently extended these 
findings to clinical chronic pain19.  Individuals exhibiting less EO inhibition of their 
chronic back pain (less effect of opioid blockade on back pain intensity) experienced 
greater relief of their back pain with morphine administration relative to higher EO 
individuals19.  To isolate the source of the effects noted in Bruehl et al.18 and provide a 
clear means of portraying them, median splits were conducted on EO function variables 
derived for each pain measure, with placebo condition and morphine condition 
responses for each measure then compared across the resulting groups.  The pattern of 
results indicated an intriguing source of these observed associations.   First, as 
expected, when EO systems were intact (placebo condition), individuals exhibiting 
relatively lower spontaneous EO activity were significantly more responsive to both 
evoked pain tasks on all measures.  Second, individuals with lower EO function exhibited 
substantial morphine analgesic responses whereas those with higher EO function did 
not.   
A key finding in Bruehl et al.18 was that morphine reduced evoked pain 
responsiveness among individuals lower in EO function to levels statistically comparable 
to those observed in participants with higher spontaneous EO function in the absence of 
any morphine.  We propose that these findings fit what we term an “opioid supplement 
model,” in which opioid analgesics supplement EO function, making up the difference 
necessary to maximize opioid receptor-mediated pain inhibition.  If the opioid 
supplement model is valid, then the desired degree of pain relief might be obtained by a 
combination of lower EO activity and higher amounts of opioid analgesics, or conversely 
– and in most cases preferably - higher EO activity and lower amounts of opioid 
analgesics.  In this latter context, we propose that aerobic exercise may be used to 
enhance EO function, with subsequent beneficial effects on the dose of opioid analgesic 
needed to achieve desired pain relief.   
Rationale for use of an aerobic exercise manipulation to enhance EO function 
derives from several sources.  First, aerobic exercise is used clinically in CP 
management, and results of a number of controlled studies document that it 
significantly reduces evoked pain responses and clinical pain intensity in CP 
patients46,72,94,103,107.  Second, a review by Co-Inv Dr. Koltyn documents the analgesic 
effects of exercise with regard to laboratory evoked pain responses76, with several opioid 
blockade studies conducted in individuals free of CP supporting a role for enhanced EO 
activity in these effects55,68,74,98,99.  These latter studies demonstrated that reductions in 
evoked pain responsiveness induced by acute exercise when healthy subjects are 
administered placebo do not occur when EO function is blocked by pharmacological 
opioid antagonists.  Third, recent brain imaging research also supports these findings, 

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indicating that an acute exercise manipulation reduced evoked pain responses and 
enhanced pain-related brain activations in an important EO pain modulatory brain region 
(periaqueductal gray), with parallel increases in circulating levels of beta-endorphin, a 
key EO analgesic104.  Fourth, potential importance of EO mechanisms in reducing CP via 
regular aerobic exercise training is suggested by opioid blockade work in animals.  
Regular exercise (from 5 days to 5 weeks) was found to significantly reduce, via EO 
mechanisms, behavioral indicators of CP in animal models of chronic muscle pain and 
neuropathic pain6,110.  Interestingly, significantly increased brain levels of beta-endorphin 
were noted during the exercise period, with exercise-related analgesia persisting for 5 
days following exercise discontinuation110.  The latter finding is particularly intriguing, as 
it suggests that regular aerobic exercise training may enhance EO function not only 
immediately after acute exercise, but for an extended period of time after acute exercise 
has ceased.  Recently published work in humans further indicates that self-reports of 
greater physical activity, including exercise, is associated with enhanced conditioned 
pain modulation96, a marker of descending pain inhibitory function which some work 
suggests has an EO-mediated component75,122. 
Plasma endocannabinoid levels will be assayed in vitro from blood samples 
obtained in the study.  Endocannabinoids can produce analgesia, may potentially 
interact with and influence opioid responses (thus serving as a potential confound to 
tests of primary study hypotheses), and may represent a previously unexplored 
mechanisms of change for analgesic effects of exercise.73  .   
 
4.0 
Inclusion/Exclusion Criteria 
 
Inclusion criteria for study participation will be: 
 
Intact cognitive status and ability to provide informed consent 
 
Ability to read and write in English sufficiently to understand and complete study 
questionnaires  
 
Age 18-55 inclusive 
 
Presence of persistent daily low back pain of at least three months duration and 
of at least a 3/10 in average intensity 
 
Exclusion criteria will be:  
 
Engagement in > 2 days/wk and > 60 min/wk of moderate or vigorous intensity 
activity based on responses to 6 validated survey questions at screening (CDC 
BRFSS), and confirmed by exercise-specific questions in the electronic diary 
obtained prior to the exercise intervention period 
 
History of renal or hepatic dysfunction 
 
Current or past alcohol or substance dependence 
 
A history of PTSD, psychotic, or bipolar disorders 
 
Chronic pain due to malignancy (e.g., cancer), autoimmune disorders (e.g., 
rheumatoid arthritis, lupus), or fibromyalgia 
 
Recent daily opiate use 
 
Use of any opioid analgesic medications within 72 hours of study participation 
(confirmed through rapid urine screening conducted prior to study participation)  
 
Females who are pregnant 
 
History of cardiovascular disease (including myocardial infarction) 
 
History of seizure disorder 

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 
Prior allergic reaction/intolerance to morphine or its analogs 
 
Presence of cardiac disease or any other medical condition that would make 
engaging in the aerobic exercise manipulation unsafe   
 
5.0 
Enrollment/Randomization 
 
Participants will be recruited from a list of potentially qualifying participants from past 
studies in our lab who had previously indicated a desire to be contacted about future 
studies, as well as via local newspaper advertising, posted flyers (including at the 
Vanderbilt Interventional Pain Center and the Osher Center for Integrative Medicine at 
Vanderbilt),and e-mail advertisements distributed throughout campus via the Vanderbilt 
mass e-mail system.  Interested individuals will be able to contact the designated study 
research assistant by phone or e-mail. Random assignment to both study conditions will 
be stratified by community vs. clinic population recruitment source to control for possible 
influence of this variable on results. Approximately equal numbers of males and females 
of all races will be recruited for the study.  Individuals expressing an interest will first be 
provided verbally with information on the study procedures, risks, and benefits, and if 
interested, will be given the opportunity to read the IRB-approved informed consent 
form.  All questions from potential subjects will be answered by the study representative 
as accurately as possible. All individuals agreeing to participate will provide written 
informed consent prior to beginning any study procedures. Subject recruitment and 
consent procedures will be carried out by individuals designated and trained by the PIs 
and the IRB to carry out these procedures (i.e., the research assistant).  
 
The study will employ a mixed between/within-subjects design using double-blind, 
counterbalanced, placebo-controlled administration of both an opioid antagonist 
(naloxone) and an opioid agonist (morphine). The study will use a 6 week supervised 
aerobic exercise manipulation, with subjects randomly assigned to the exercise protocol 
or a no exercise control condition. All participants will undergo three identical laboratory 
pain-induction sessions (each ≈5 days apart) prior to randomization to experimental 
condition, and again at the end of the 6 week exercise manipulation period (regardless 
of exercise group assignment) during which they will receive the 3 study drugs and 
participate in controlled laboratory evaluation of evoked thermal pain responsiveness.   
 
6.0 
Study Procedures 
 
Screening/Consent visit (Day 1 of Study Participation): Potential participants will be 
screened to determine eligibility for participation in the study. This screening, supervised 
by the study physician, will include evaluation to characterize the nature of the chronic 
back pain and to confirm eligibility to engage in the exercise manipulation. Potential 
participants will also be screened for the exercise exclusion criteria by answering 
questions related to moderate-vigorous physical activity from the CDC Behavioral Risk 
Factor Surveillance Survey to classify potential subjects as active or inactive.  Following 
informed consent, participants will complete study questionnaires. Questionnaires will 
include assessment of occupational status (Employed, Not Working, Disabled), and for 
working individuals, number of hours worked per week and nature of the job for use as 
an additional measure of activity. Participants will also complete the International 
Physical Activity Questionnaire (IPAQ) to gather additional relevant physical activity 

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information prior to beginning participation in the study, to supplement diary-based data 
regarding exercise activity.  Questionnaires assessing factors related to back pain, 
anger, depression, anxiety, pain coping, and pain medication responses will be 
completed.  Participants will then complete the 5-day pre-manipulation electronic diary 
assessment procedures.   
 
Pre-Manipulation 5 day Electronic Diary Monitoring Period (Days 2-6): Daily chronic back 
pain intensity will be assessed for 5 days pre-manipulation via an electronic pain diary 
using handheld Palm® Zire 22 PDAs, running the Palm OS platform. To provide a 
representative sampling of daily back pain intensity, participants will be prompted by an 
audible alert at 9am and again at 6pm daily. At each prompting, participants will be 
asked to electronically record ratings of their current back pain, recent sleep, activity 
levels, and mood using 9 point numeric scales.  Participants will begin the laboratory 
portion of the study following completion of this first diary monitoring period. 
 
Lab Visit 1 (≈Day 7): Participants will undergo a urine screening for opiate use (females 
will also be screened for pregnancy); complete psychometric instruments; training for 
heat pain stimulus (Visit 1 only); 5 minutes of seated rest; participants rate their current 
low back pain intensity; nurse inserts i.v. cannula; nurse obtains a 4ml blood sample via 
cannula to assess endocannabinoid levels (Visits 1 and 4 only); nurse administers 
randomized drug dose #1.  Administration of randomized drug dose #1 (specific drug 
protocol is summarized below) will be followed by 10 minutes of seated rest to achieve 
peak drug levels; participant rating of chronic pain intensity; thermal pain task #1 
(described in detail below); assess thermal pain intensity; assess drug side effects and 
psychoactive effects; nurse administers randomized drug dose #2. Administration of 
drug dose #2 will be followed by 10 minutes of seated rest to achieve peak drug levels; 
participant rating of chronic pain intensity; thermal pain task #2; assess thermal pain 
intensity; assess drug side effects and psychoactive effects; nurse administers 
randomized drug dose #3 .  Administration of drug dose #3 will be followed by 10 
minutes of seated rest to achieve peak drug levels; participant rating of chronic pain 
intensity; thermal pain task #3; assess thermal pain intensity; assess drug side effects 
and psychoactive effects; nurse administers randomized drug dose #4.  Administration 
of drug dose #4 will be followed by 10 minutes of seated rest to achieve peak drug 
levels; participant rating of chronic pain intensity; thermal pain task #4; assess thermal 
pain intensity; assess drug side effects and psychoactive effects; seated rest until side 
effects remit. 
 
(each session ≈ 5 days apart) 
 
Lab Visit 2: Participants will undergo a urine screening for opiate use (females will also 
be screened for pregnancy); 5 minutes of seated rest; participants rate their current low 
back pain intensity; nurse inserts i.v. cannula; nurse administers randomized drug dose 
#1.  Administration of randomized drug dose #1 will be followed by 10 minutes of 
seated rest to achieve peak drug levels; participant rating of chronic pain intensity; 
thermal pain task #1; assess thermal pain intensity; assess drug side effects and 
psychoactive effects; nurse administers randomized drug dose #2. Administration of 
drug dose #2 will be followed by 10 minutes of seated rest to achieve peak drug levels; 
participant rating of chronic pain intensity; thermal pain task #2; assess thermal pain 

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intensity; assess drug side effects and psychoactive effects; nurse administers 
randomized drug dose #3 .  Administration of drug dose #3 will be followed by 10 
minutes of seated rest to achieve peak drug levels; participant rating of chronic pain 
intensity; thermal pain task #3; assess thermal pain intensity; assess drug side effects 
and psychoactive effects; nurse administers randomized drug dose #4.  Administration 
of drug dose #4 will be followed by 10 minutes of seated rest to achieve peak drug 
levels; participant rating of chronic pain intensity; thermal pain task #4; assess thermal 
pain intensity; assess drug side effects and psychoactive effects; seated rest until side 
effects remit. 
 
(each session ≈ 5 days apart) 
 
Lab Visit 3: Participants will undergo a urine screening for opiate use (females will also 
be screened for pregnancy); 5 minutes of seated rest; participants rate their current low 
back pain intensity; nurse inserts i.v. cannula; nurse administers randomized drug dose 
#1.  Administration of randomized drug dose #1 will be followed by 10 minutes of 
seated rest to achieve peak drug levels; participant rating of chronic pain intensity; 
thermal pain task #1; assess thermal pain intensity; assess drug side effects and 
psychoactive effects; nurse administers randomized drug dose #2. Administration of 
drug dose #2 will be followed by 10 minutes of seated rest to achieve peak drug levels; 
participant rating of chronic pain intensity; thermal pain task #2; assess thermal pain 
intensity; assess drug side effects and psychoactive effects; nurse administers 
randomized drug dose #3 .  Administration of drug dose #3 will be followed by 10 
minutes of seated rest to achieve peak drug levels; participant rating of chronic pain 
intensity; thermal pain task #3; assess thermal pain intensity; assess drug side effects 
and psychoactive effects; nurse administers randomized drug dose #4.  Administration 
of drug dose #4 will be followed by 10 minutes of seated rest to achieve peak drug 
levels; participant rating of chronic pain intensity; thermal pain task #4; assess thermal 
pain intensity; assess drug side effects and psychoactive effects; seated rest until side 
effects remit. 
 
Exercise Manipulation (Weeks 4 – 10 of study participation): Following the third 
laboratory visit, participants will be randomly assigned to study condition, stratified by 
recruitment source (clinic vs. community). Participants randomly assigned to the 
exercise condition will then complete the 18 session aerobic exercise manipulation 
supervised by an ACSM-certified personal trainer at the Dayani Center (3 exercise 
sessions per week for 6 weeks).  Participants assigned to the control condition will not 
undergo any manipulation during this 6 week period, and will be asked to continue their 
current activity levels and not engage in any additional exercise activity during the study 
period (confirmed via electronic diary).  All subjects will complete brief pain and 
psychosocial questionnaires once weekly by phone.   
 
Lab Visits 4 – 6 (≈Weeks 11-12):  Identical procedures to those described above for Lab 
Visits 1-3.  Drug order is again randomized and counterbalanced.  
 
Post-Manipulation 5 day Electronic Diary Monitoring Period (≈Week 11, between Lab 
Visits 4-5):  Identical to pre-manipulation electronic diary procedures described above.   
 

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Description of Laboratory Visit Procedures: 
All laboratory procedures will be conducted with participants seated upright in a 
comfortable chair.  Each participant’s series of three laboratory sessions (placebo, 
naloxone, morphine) carried out both pre- and post-manipulation will be scheduled 
within an ≈10 day period, at the same time of day to control for variance due to 
circadian rhythms. 
 
Study Drug Protocol: Study drugs will be administered (in double-blind fashion) in 
randomized, counter-balanced order.  The assigned study drug will be administered 
within a given session over 4 infusions (2 min each) according to the following protocol, 
depending on the assigned drug for that visit: 1) 4 doses of saline placebo (20ml each), 
2) an 8mg dose of naloxone (in 20ml saline vehicle), followed by saline, 4mg naloxone, 
and saline, or 3) morphine sulfate (0.03 mg/kg in 20ml saline vehicle initially, followed 
by 3 incremental doses of 0.02mg/kg each).  To avoid excessive morphine doses (and 
increased side effect risks) for individuals who are obese, a modified weight adjustment 
will be employed for individuals more than 30% over ideal body weight based on 
subjects’ observed weight in clinic compared to appropriate ideal body weight for males 
(50kg+2.3kg for each inch over 5 feet) or females (45.5kg +2.3kg for each inch over 5 
feet) [from http://www.globalrph.com/].  For individuals meeting this obesity criterion, 
morphine dosage will be based on Adjusted Body Weight rather than actual body 
weight, as calculated by: Ideal Body Weight + 0.4 (actual weight – Ideal Body Weight).     
 
Thermal Pain Task Procedures: The heat pain task will use a computerized Medoc TSAII 
NeuroSensory Analyzer to assess heat pain threshold and tolerance using an ascending 
method of limits protocol.  Ten minutes after each drug infusion, three trials will be 
conducted for heat pain threshold and three trials for heat pain tolerance, with each trial 
conducted sequentially at one of three non-overlapping sites on the non-dominant 
ventral forearm.  An interval of 30 seconds between successive stimuli will be employed.  
For pain threshold trials, the probe will start at an adaptation temperature of 32○C, with 
temperature increasing at a ramp rate of 0.5○C/sec until the participant indicates that 
the stimulus has begun to feel “painful.”  For each tolerance trial, the probe will start at 
an adaptation temperature of 40○C, with the temperature increasing at a ramp rate of 
0.5○C/sec until the participant indicates maximum tolerance has been reached. 
Immediately upon completion of the final heat pain tolerance trial at each drug dosage, 
participants will be asked to rate the pain just experienced using the MPQ. 
 
Patient instructions for this task: “During this next part of the experiment, I’ll be testing 
your sensitivity to the heat pain stimulus I showed you earlier. First we will do the pain 
threshold test, which asks you to indicate when the heat FIRST begins to feel painful. 
The probe will start out slightly cool, and when we start the test, you will hear a beep 
and the probe will begin feeling warm and the temperature will steadily increase. During 
the test, keep your finger on the LEFT button of this mouse, and press this button the 
moment the heat first becomes painful. Keep in mind that this is not a test of how much 
pain you can tolerate, but rather, we want to know the instant at which the heat 
stimulus first feels painful. Please stay alert and concentrate throughout the test. 
Pressing the button will turn the heat off, so don’t press it until the heat first feels 
painful to you. We will do this test several times. Do you have any questions about this 
test? The next test we will do is the heat pain tolerance test. Again, each trial will be at 

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a different spot on your forearm. During this test, the probe will start out slightly warm. 
The temperature will then begin slowly increasing, and I’d like you to go as long as you 
can, and then indicate when the heat pain has reached the maximum you can tolerate 
by clicking the LEFT button on this mouse. Pressing the LEFT button will turn the heat 
off, so don’t press it until the heat becomes intolerable. We’ll repeat this procedure 
several times. Do you have any questions?”               
 
Exercise Manipulation Protocol: Participants randomly assigned to the exercise training 
group will undergo aerobic exercise training 3 times/wk for 6 wks. To enhance and 
monitor adherence, all of the exercise sessions will be conducted at the Dayani Center 
and supervised by ACSM certified personal trainers thoroughly trained in study 
procedures. Each exercise session will consist of a 5 minute warm-up, 30 minutes of 
aerobic exercise, followed by a 5 minute cool-down period. Aerobic exercise will consist 
of treadmill walking/running, stepping, elliptical, or cycling exercise as preferred by the 
participant. This will allow participants to choose the type of aerobic exercise most 
tolerable to them to minimize symptom exacerbation (i.e., acute increases in pain) while 
maximizing adherence to the training program. Effort levels across the different types of 
aerobic exercise will be standardized using heart rate (HR) and perceived exertion (RPE) 
monitoring as recommended by the American College of Sports Medicine (ACSM). Target 
heart rate zones will be established using the Karvonen formula and heart rate reserve 
(HRR). Duration of exercise will be standardized at 30 minutes with a target exercise 
intensity between 70-85% HRR (RPE = 15, hard). Because of the focus on de-
conditioned individuals with CLBP, the duration and intensity of exercise will be 
progressively increased up to target during the first two weeks to avoid symptom 
exacerbation and minimize study drop-out. Participants will begin with 10-15 minutes of 
exercise between 40-55% HRR (RPE = 11-12, light) during the first week, 20-30 
minutes of exercise between 55-70% HRR (RPE = 12-13, somewhat hard) during the 
second week, and then 30 minutes of exercise between 70-85% HRR (RPE= 14-16, 
hard) for the duration of the study. To ensure that participants are exercising within 
their prescribed workload during each session, HR and RPE will be assessed and 
recorded every 5 minutes during exercise using Polar HR monitors and Borg’s 6-20 RPE 
scale. In addition, pain will be assessed every 5 minutes during exercise using a pain 
rating scale developed for this purpose to monitor back pain, with adjustments to the 
workload in the event of back pain during exercise. 
 
Endocannabinoid Assay Protocol:  Outside of the participants’ laboratory sessions, we 
will also separately conduct in vitro assessment of plasma endocannabinoid levels from 
sampled blood.  All samples will remain de-identified using only assigned study subject 
numbers.  Assays will be carried out on plasma extracted from blood samples 
designated for this purpose obtained before and after the 6 week exercise intervention 
(sessions 1 and 4 only).  Assays will be conducted under the supervision of Co-
Investigator Dr. Kelli Koltyn (Univ. of Wisconsin), given her prior experience with these 
assays73.  The endocannabinoids N-arachidonylethanolamine (AEA) and 2-
arachidonoylglycerol (2-AG), key endogenous agonists of the CB1 receptor, as well as 4 
structural lipid analogs (palmitoylethanolamide [PEA], oleoylethanolamide [OEA], N-
docosahexaenoylethanolamine [DEA], and 2-oleoylglycerol [2-OG]), will be quantified 
using isotope-dilution, liquid chromatography–electrospray ionization–mass spectrometry 
based on published methods.     

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7.0 
Risks 
 
There are several potential risks to subjects. First, subjects will experience brief, 
moderate intensity acute pain upon application of the thermal pain stimuli used for 
evaluation of acute pain responsiveness. However, subjects have total control over the 
duration of their exposure to this pain because they terminate the task by indicating 
when they have reached their tolerance limit. Previous research indicates that this task 
is safe, but to further maximize safety, individuals experiencing cardiovascular problems 
will be excluded from this study. Because subjects have total control over the duration 
of the task, its psychological impact is expected to be minimal. 
 
Participants will experience very brief, mild pain upon insertion of a cannula (for study 
drug administration) during each experimental session. Insertion will be performed by a 
trained nurse or physician to minimize discomfort associated with insertion of the 
cannula. There is a risk of infection and local inflammation at the site of cannula 
insertion. Precautions will be taken to insure that such risks are minimized.   
 
The medication used for opioid blockade, naloxone, is not an experimental or new drug, 
and is FDA-approved. Previous studies indicate that it is safe for individuals who are not 
opiate dependent, do not have liver disease, and do not have cardiovascular problems. 
Potential subjects experiencing these types of problems will be excluded from this study 
to insure a maximal level of safety with drug administration. Subjects taking daily 
opiates, even if not dependent, will also be excluded from the study to avoid 
precipitating minor withdrawal symptoms. In some individuals, naloxone may increase 
pain sensitivity to the acute pain tasks somewhat, but again, participants may terminate 
these tasks if they’ve reached their tolerance limit. Based on previous studies, naloxone 
is expected to have limited if any direct effects on clinical pain intensity among pain 
patients, and therefore, there appears to be little risk of exacerbating patients’ chronic 
pain conditions. Even if such changes do occur, the brief half-life of naloxone (on 
average, approximately 60-80 minutes) insures that any pain exacerbation will be of 
short duration. With the exception of possible effects on pain sensitivity, naloxone is not 
known to be associated with other clinically significant effects in healthy individuals who 
are not opiate dependent or using daily opiates.   
 
The other study drug, morphine, is an FDA-approved opioid analgesic with a very long 
history of use in standard clinical practice. It can produce dose-dependent side effects 
including sedation, somnolence, nausea, brief vomiting, euphoria, and respiratory 
depression. The cumulative dose to be used in the proposed study (0.09 mg/kg) will be 
weight adjusted, and represents a relatively low dose within the normal range of 
dosages used in standard clinical practice, representing a dosage only slightly larger 
than in our recent work (0.08 mg/kg; Bruehl et al., 2013). This should help limit any 
negative side effects. The half-life of morphine is approximately 2 hours, and study 
participants will be kept under observation in each study session until any side effects 
remit to the point where it is safe for them to leave unassisted. If morphine side effects 
are severe, these effects will be immediately reversed by administration of naloxone as 
determined appropriate by the study physician.   
 

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As part of this study, half of the participants will be randomly assigned to undergo a 6-
week supervised aerobic exercise manipulation as described above.  To maximize safety 
with this type of exercise, individuals with a history of cardiovascular disease are 
excluded from participating in this study.  The intensity of this aerobic exercise training 
will start low but progressively increase over the 6-week intervention period.  Safety and 
possible discomfort associated with these increases in exercise intensity will be 
minimized via close monitoring by a certified personal trainer of participant’s heart rate, 
perceived exertion levels, and back pain intensity during exercise.  Exercise intensity will 
be adjusted as appropriate to maximize the participant’s level of conditioning while 
minimizing exacerbation of back pain.  In addition, participants will be able to choose 
your preferred form of exercise from several available options.   
 
In the event new information becomes available that may affect the risks or benefits 
associated with this study, participants will be notified so that they can make an 
informed decision whether or not to continue their participation in this study. There may 
be unknown or unanticipated adverse effects in addition to those listed.  
 
 
8.0  
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
 
The PI and co-investigators will carefully monitor adherence to the study protocol on a 
regular basis via direct supervision, monitoring of obtained study materials, and regular 
study team meetings. All adverse events and unanticipated problems will be immediately 
communicated across the two study sites.  Deviations from protocol will be immediately 
corrected, and if appropriate, will be reported to the IRB and the program officer at 
NIH/NIDA (sponsoring agency). 
 
9.0 
Study Withdrawal/Discontinuation 
 
Subjects will be informed that they can withdraw for any reason from the study at any 
time without penalty. Subjects may be withdrawn from the study if the study physician  
determines that their medical condition makes their participation in the study unsafe. 
Upon withdrawal from the study, subjects will be compensated for their participation 
(prorated as appropriate).   
 
10.0 Statistical Considerations 
 
Given the project focus on testing indirect (mediated) effects of the aerobic exercise 
manipulation via EO enhancement (mediator) on CP intensity (Aim 1), morphine 
analgesic requirements (Aim 2), and associated abuse relevant subjective effects (Aim 
3), the project is powered specifically for the proposed bias-corrected bootstrap 
mediation analyses.  Empirical power estimates for this mediation analysis approach are 
available64.  The mediation analyses will test the mediation model presented in Figure 2 
below.  The power estimates below jointly take into consideration the effect sizes for the 
a and b paths in Figure 2.  The effects of exercise on EO analgesia (a path) can be 
derived from 3 studies in healthy individuals reporting differences in pain sensitivity 
following acute exercise between placebo and EO blockade (naloxone) conditions55,68,74.  

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Effect sizes ranged from d=.6 to d=1.2 in directions indicating that opioid blockade 
reduced post-exercise analgesia, suggesting that EO function partly mediated the effect 
of acute exercise on pain intensity. The mean effect size across these studies for  the 
impact of EO mechanisms on exercise-related  analgesia was d= .93 (equivalent to 
r=0.42).  This effect size is key to initial analyses of Specific Aim 1.  The proposed 
sample size will result in a power exceeding 0.90 for testing effects of the exercise 
manipulation on EO function (two-sided p<.05 significance criterion).  Effect size 
estimates for the b path in the mediational model reflecting influences of EO function on 
morphine responses can be derived from our prior work, which indicated an average 
effect size of r=0.27 for associations between natural variations in EO function (opioid 
blockade effects) and analgesic responses to a single morphine dose across all pain 
measures and both laboratory evoked pain tasks18.  Effect size for the b path reflecting 
influences of EO function on chronic back pain intensity can be estimated from Bruehl et 
al.25, with an effect size of r=0.26 for associations between EO analgesic function in the 
laboratory and diary ratings of chronic back pain intensity (greater EO function was 
associated with lower daily back pain intensity). Given these effect sizes for the a and b 
paths in the mediation model, the total sample size for the proposed project was 
selected as n=116, resulting in a power of 0.80 (two-tailed p<.05 criterion for 
significance) for testing mediation effects hypothesized in Aims 1-3 using the proposed 
bootstrap approach (based on Fritz and MacKinnon64).    
 
Data Reduction and Analyses 
Blockade effect variables reflecting EO function will be derived by subtracting evoked 
pain responses during placebo from comparable responses during opioid blockade 
(separately for pre-manipulation baseline [BL] and post-manipulation). Given no 
theoretical rationale for expecting dose response effects with repeated dosing for 
placebo or naloxone conditions, we will average over the 4 pain stimuli within each of 
these drug conditions (separately for pre- and post-manipulation sessions) as a means 
of reducing random measurement error in deriving blockade effects.  Blockade effects 
will be derived such that larger positive values reflect greater EO analgesia. To reduce 
number of variables, a mean value of the standardized EO function variables (blockade 
effects) will be derived and will be used initially in analyses (as in Bruehl et al.18).   
Chronic pain intensity pre- and post-manipulation will be defined as mean chronic pain 
ratings during the respective electronic diary sampling periods.  As manipulation checks, 
we will determine whether degree of spontaneous EO function pre-manipulation is 
correlated significantly with evoked pain sensitivity and chronic pain intensity at BL.  
Total number of exercise minutes recorded during the post-intervention diary monitoring 
period (obtained in both groups) will be explored as a potential covariate for use in 
primary group analyses.   
 
Primary Analyses. 
Aim 1: To determine whether an aerobic exercise manipulation increases EO activity in 
CLBP patients, and whether these EO changes mediate manipulation-induced reductions 
in evoked pain responsiveness and daily CP intensity.  We will conduct Group (exercise, 
control) x Time (BL, post-manipulation) mixed design ANOVAs to determine whether 
evoked pain sensitivity and CP intensity decreased significantly in the aerobic exercise 
group only.  We will also conduct Group (exercise, control) x Time (BL, post-
manipulation) mixed design ANOVAs on EO function (blockade effect variables pre- and 

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post-manipulation) to determine whether EO function increased in the aerobic exercise 
condition only.  We will then test whether group differences in pre- to post-manipulation 
EO changes mediate group differences in pre- to post-manipulation reductions in evoked 
pain responsiveness and CP intensity using the bias-corrected bootstrap mediation 
testing approach of Preacher and Hayes100.  This approach is statistically powerful and 
not dependent on any assumptions about the sampling distribution of the variables, and 
was used successfully in our recently published work18.  Per Preacher & Hayes100, the 
only precondition for testing mediation will be presence of an effect to be mediated (i.e., 
the exercise condition is associated with significantly greater reductions in evoked pain 
responses and CP intensity than the control condition).  For any outcomes for which this 
precondition is met, bootstrap tests of mediation will proceed using the Indirect 
procedure for SPSS100,101 (http://www.afhayes.com/spss-sas-and-mplus-macros-and-
code.html#sobel) as in our recent work18.  If the 95% CI for the indirect effect in a 
given  model does not include zero, this will indicate that the hypothesized indirect 
(mediated) effect is significant at p<.05. In this case, a significant indirect path will 
indicate that the link between aerobic exercise condition and reduced evoked pain 
responsiveness or CP intensity is significantly mediated by exercise-related EO 
enhancements.  Significant direct and indirect paths jointly for the same outcome will 
indicate partial mediation.  A significant direct path in the absence of a significant 
indirect path will be interpreted as indicating that the effects of the exercise 
manipulation on that outcome are mediated via non-opioid mechanisms. 
 
Aim 2: To determine whether the exercise manipulation is related to achieving analgesic 
targets at lower opioid analgesic dosages, and whether these changes in analgesic 
requirements are mediated by pre-post EO changes.   We will calculate dose response 
curves (per Prentice102) based on morphine condition evoked pain responses at the four 
incremental morphine dose levels (separately for pre- and post-manipulation). Based on 
results of our prior work18, a 25% reduction in pain sensitivity relative to pre-exercise 
placebo pain responses will be the target.  The total dosage of morphine needed at pre-
manipulation BL to achieve a 25% reduction in pain responses (based on the dose 
response curve) will be compared to the dose of morphine needed to achieve a 25% 
pain reduction at post-exercise.  We will conduct Group (exercise, control) x Time (BL, 
post-manipulation) mixed design ANOVAs to determine whether the total morphine dose 
required to achieve 25% pain relief decreased significantly in the exercise group only.  
We will then use mediation analysis procedures as described above to test whether 
group differences in pre- to post-manipulation EO changes mediate group differences in 
pre- to post-manipulation reductions in morphine dosage required to achieve 25% relief.  
The only precondition for conducting these mediation tests will be that there is an effect 
to be mediated, i.e., the exercise manipulation significantly alters the morphine dosage 
required to achieve 25% pain relief.   
 
Aim 3: To determine whether the exercise manipulation is related to achieving a 25% 
morphine-induced reduction in evoked pain responsiveness with fewer negative side 
effects and abuse-relevant effects, and whether these changes are mediated by pre- to 
post-manipulation EO changes. Aim 3 analyses will parallel Aim 2 except that side 
effects and abuse-relevant effects at the 25% pain reduction target will be the DV.    
 
Secondary Analyses. 

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Secondary exploratory analyses will examine potential moderator effects of sex and BL 
EO function on effects observed in primary analyses. These analyses will test whether 
the direction and magnitude of Group effects and/or Group x Time interaction effects for 
DVs depend on the sex of the participant and/or on degree of BL EO analgesia. To 
illustrate, GLM procedures will be used to test for a significant Group x Time x BL EO 
function interaction effect for morphine dosage outcomes in Aim 2. If significant, simple 
effects will be used to test whether Group was related to changes in required morphine 
dosages from BL to post-manipulation differently for those low vs high in BL EO 
function.  Within the exercise group only, we will also examine associations between 
exercise intensity (i.e., average number of minutes ≥ target 70% HRR during sessions) 
and pre-post manipulation EO changes and changes in morphine responsiveness.  
Finally, we will test whether: a) BL depression and anxiety symptoms predict analgesic 
responses; b) the exercise manipulation reduces these symptoms; c) such changes 
contribute to manipulation-related changes in analgesic responses. 
 
11.0 Privacy/Confidentiality Issues 
 
All published data will be reported in a manner in which individual data for specific 
subjects are not identifiable. Data will be coded on paper and in the computer by 
subject number rather than name to help further insure confidentiality. All hardcopy 
study records from each site will be maintained in a filing cabinet in a locked office at 
each PI’s office suite, and will be accessible only to the principal investigators and their 
designees. All other subject data records will be maintained on password-protected 
computers stored in the locked offices of each PI. Hardcopies of the data will be 
maintained for 6 years, after which they will be destroyed.   
 
12.0 Follow-up and Record Retention 
 
The expected duration of this study is 4 years. Hardcopies of the data will be maintained 
for 6 years after the study is completed, after which they will be destroyed.  
  

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