Clinical trial NCT02359006
Conditions: Opioid Dependence Pain
Keywords: Analgesia pain analgesia methadone opiates
Sponsor: Yale University
Start date: 2015-03-12; Completion date: 2017-04-19
Enrollment: 27 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Opioids are the most commonly utilized pharmacological treatment for moderate to severe pain. However, their clinical value is hindered by the development of opioid-induced hyperalgesia (OIH). OIH manifests as heightened pain sensitivity, and is an increasingly challenging drawback to the efficacy of opioid treatment. Although the mechanism of action modulating OIH is not completely understood, previous animal studies suggest that this phenomenon is a result of proinflammatory responses. Thus, administering an adjunct anti-inflammatory agent may attenuate OIH. Minocycline is one such agent; it is a tetracycline derivative antibiotic that inhibits microglia activation, nitric oxide (NO) production, and the release of pro-inflammatory cytokines and chemokines. In fact, recent evidence suggests that minocycline may attenuate the neuroinflammatory effects of opioids while enhancing their antinociceptive effects. Therefore, the investigators will determine if minocycline will mitigate OIH in methadone-maintained patients.

Detailed description:
Sixty completers will be recruited through the VA methadone clinic, as well as through the APT Foundation Methadone Maintenance Program. After the initial phone screening, potential subjects will undergo a comprehensive evaluation which will include medical, psychiatric, and drug use histories as well as physical, psychiatric, and laboratory examinations. Laboratory examination will include CBC, liver and thyroid function tests, serum electrolytes, BUN, creatinine, PT, PTT, urine analysis (including urine pregnancy for women) and urine toxicology screening.

Participants will be terminated from the study following opioid relapse, or use of any other psychotropic medications. If participants are noncompliant (no-show, positive urine screening, noncompliance with medication protocol/missing more than one dose of minocycline/placebo), participation will be terminated.

This double-blind, randomized clinical trial will randomize male and female veterans and non-veterans currently undergoing methadone maintenance treatment for opioid dependence to either minocycline (200mg/day) or placebo for 15 days. Upon inclusion, participants will be subjected to a pain assessment to evaluate baseline pain thresholds and tolerance: the Cold Pressor Test. An experimental treatment of either minocycline or placebo will then be initiated and maintained for 15 days. Additionally, at the beginning of Week 2 of treatment, participants will be given a Personal Digital Assistant (PDA) an HP iPAQ Pocket PC 2003 Pro that will administer Ecological Momentary Assessments (EMA). Using EMA, we can assess change in pain sensitivity, withdrawal symptoms and cognitive performance in the participants' natural environment, which increases the ecological validity of the study. Participants will be asked to return to the laboratory several times a week for the 15 consecutive days that they are taking minocycline in order to receive the study medication and to assess changes in pain thresholds and tolerance (i.e. to assess the presence, or lack thereof of hyperalgesia). Upon completion of experimental treatment, participants will be asked to return a final time to undergo pain measurement once more, to assess any changes in pain sensitivity after completion of minocycline.

Arms: arm 1: 200mg minocycline arm 2: Sugar pill
Interventions: intervention 1: Minocycline intervention 2: Placebo
Intervention descriptions: intervention 1: Minocycline will be compared with placebo intervention 2: Placebo will be compared with minocycline

Protocol documents:
Prot_000.pdf: 
Title of Study: The Effect of Minocycline on Opioid-Induced Hyperalgesia in Opioid-
Maintained Patients 
 
NCT#: 02359006 
 
Document: Study protocol 
 
Date of Document: Last updated and approved by IRB on 7/11/2017 
 
 


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1.  Principal Investigator: Mehmet Sofuoglu, M.D. Ph.D. 
 
2.  Co-Investigators (including VA and WOC staff): While all research staff is trained to 
provide informed consent, those identified with an asterisk (*) will provide informed consent for 
this study. The PI and the listed research team will have access to PHI. 
Co-Investigators:.; Ismene Petrakis, M.D. 
Authorized Prescribers: Mehmet Sofuoglu, M.D. Ph.D., Kevin Sevarino, M.D., or 
Gerald Valentine, M.D. 
Nursing Staff: *Ellen Mitchell, RN 
Research Staff: *Lance Barnes, *Stacy Minnix, B.S.W., *Christopher Cryan, 
*Michael Stephens 
 
3.  Title of Project: The Effect of Minocycline on Opioid-Induced Hyperalgesia in Opioid-
Maintained Patients 
 
4.  Purpose: Opioids are the most commonly utilized pharmacological treatment for moderate to 
severe pain. However, their clinical value is hindered by the development of opioid-induced 
hyperalgesia (OIH). OIH manifests as heightened pain sensitivity, and is an increasingly 
challenging drawback to the efficacy of opioid treatment. Although the mechanism of action 
modulating OIH is not completely understood, previous animal studies suggest that this 
phenomenon is a result of proinflammatory responses. Thus, administering an adjunct anti-
inflammatory agent may attenuate OIH. Minocycline is one such agent; it is a tetracycline 
derivative antibiotic that inhibits microglia activation, nitric oxide (NO) production, and the 
release of pro-inflammatory cytokines and chemokines. In fact, recent evidence suggests that 
minocycline may attenuate the neuroinflammatory effects of opioids while enhancing their 
antinociceptive effects. Therefore, we will determine if minocycline will mitigate OIH in opioid-
maintained patients. 
 
Hypothesis:  We hypothesize that adjunct minocycline (200mg) will attenuate hyperalgesia often 
observed in patients undergoing opioid maintenance treatment. 
 
5-6. Background and Significance 
 
Introduction 
 
Opioids are the most prominent pharmacological treatment for moderate to severe pain that 
results from health issues such as cancer, chronic injuries, and surgical procedures. However, 
this drug class has an infamously high abuse liability, which casts a dark cloud over their clinical 
utility. Prescription of opioids over the past two decades has increased dramatically, surpassing 
257 million in 2009 (Food and Drug Administration (FDA), 2010). Subsequent to this increase in 
prescription comes a drastic increase in the number opioid dependent persons. Consequently, 
there has been a striking increase in the number of deaths due to prescription opioids, from 
approximately 4,000 in 1999, to over 16,000 in 2010 (Centers of Disease Control and 
Prevention, 2012). Furthermore, opioid dependence has an estimated economic cost of $72.5 
billion in health care services (Coalition Against Insurance Fraud, 2007), and those who abuse 
this drug class generate an average annual health care cost that is 8.7 times higher than non-


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abusers (White et al., 2005). With that said, opioid abuse, addiction, and overdose have become 
part of a major societal epidemic. 
 
 
Hyperalgesia as a clinical problem 
 
In addition to abuse, opioids have a range of unfavorable side effects that include respiratory 
depression, as well as cardiovascular and gastrointestinal effects. Furthermore, opioids 
notoriously elicit a paradoxical state of increased pain sensitivity, known as hyperalgesia. 
Hyperalgesia is defined as an increased sensitivity to painful stimuli, as well as allodynia, which 
is pain evoked by a stimulus that is not under normal circumstances considered painful (Heger, 
Mair, Otter, Helwig, & Suttorp, 1999).  Such a phenomenon is typically evident following 
administration of titrated doses of opioids, which is almost always necessary after the inevitable 
development of tolerance (Pasero & McCaffery, 2012). While hyperalgesia can be reliably 
elicited in preclinical models (Woolf, 1981; Mao et al., 1994; Ossipov, Lai, Vanderah, & 
Porreca, 2003; Waxman et al., 2009; Juni et al. 2010; Waxman et al., 2010), in humans this 
paradoxical state is less predictable and a quite serious complication of clinical opioid treatment 
(Compton, 2008; De Conno et al., 1991; Ossipov et al., 2004; Sjøgren et al., 1994; Sjøgren et al., 
1998). 
 
Although a majority of research on OIH is comprised of animal studies, the obvious application 
of such research is the use of opioids in clinical settings involving treatment of pain, where this 
phenomenon inserts a complication. The success of pain treatment in humans is hindered by the 
appearance of hyperalgesia, where opioid titration and rotation become a potentially dangerous 
guessing game. Further complicating the issue is the fact that clinicians cannot effectively treat 
OIH, as there is no consensus regarding the mechanism by which hyperalgesia manifests. Since 
opioids are the cornerstone for treatment of pain, understanding the mechanisms underlying OIH 
is imperative. As we advance our understanding of OIH, clinicians will be better able to 
effectively address patient needs, improve patient care, and prevent further medical 
complications arising from opioid use. 
 
Human studies of OIH 
 
Clinical investigations of OIH tend to focus on defining the contextual characteristics in which 
OIH appears. Investigations have been sporadically conducted in humans using observational 
case studies, as well as cross-sectional methodology.  A majority of these studies focus on a few 
particular cohorts; for example, patients suffering from chronic illnesses such as cancer (Sjøgren 
et al., 1994; Mercadante & Acuri, 2005), those with acute pain following various surgical 
procedures (Lee, Lee, & Kim, 2013; Chia et al., 1999), populations of opioid addicts (both 
former and current) (Doverty et al., 2001a; Compton et al., 2010), opioid-naïve pain patients 
beginning opioid therapy for chronic pain (Chu et al., 2006), and healthy volunteers undergoing 
human experimental pain testing (Compton et al., 2003). Results of these studies indicate that 
opioid rotation is sometimes sufficient to eliminate hyperalgesia in the aforementioned 
populations. Others suggest that complete discontinuation is the most efficacious way to relieve 
this opioid-induced increased pain sensitivity. However, while it might be an ideal solution, 
opioid discontinuation is not always realistic or appropriate. As such, a solution that attenuates 
OIH such that the patient may continue benefitting from chronic opioids is needed. For example, 


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recent evidence suggests the addition of adjunct pregabalin to effectively diminish OIH in 
patients receiving chronic opioids (Compton et al., 2010; Lee et al., 2013). 
 
Clinical models of hyperalgesia 
  
Doverty et al. (2001a) attributed inconsistencies in the literature to the array of pain modalities 
and tests used to assess opioid-induced hyperalgesia. Indeed, a complicating factor exists in the 
wide variance of assessments and populations used to measure this phenomenon, ranging from 
cold pain, to chemical pain, to pressure pain models in patients with acute surgical pain to those 
maintained on methadone. Illustrated in both animal and human studies, manipulations that 
produce profound OIH on one pain assay in one individual may show no or varying hyperalgesic 
liabilities on other assays in another individual, suggesting that OIH is drug and modality 
specific, and likely influenced by varying genetic backgrounds (Angst & Clark, 2006; Mogil et 
al., 1999a; Mogil et al., 1999b; Doverty et al., 2001a; Chu, Clark, & Angst, 2006; Krishnan et al., 
2012).  
 
The most common assessments used to measure pain are the cold pressor test, electrical 
stimulation, and heat-capsaicin. In the most reliable test for assessing opioid effects (Krishnan et 
al., 2012), the cold pressor test requires that study participants submerge their hands and 
forearms into ice water, and are instructed to report initial detection of pain as well as the point at 
which they can no longer tolerate said pain. This test is quite robust and reliable when measuring 
pain related to opioid administration (Modir & Wallace, 2010; Olesen et al., 2012). Another 
common measure for analyzing opioid effects is electrical stimulation. In this assessment, 
cutaneous electrodes are attached to the participants’ ear lobe, whereby increasing pulses of 
electricity are delivered. Participants verbally indicate the first perception of the stimulus as 
painful, and resultant increases in pain up until they can no longer tolerate it (Doverty et al., 
2001a; Olesen et al., 2012). However, this measure yields unfavorable results in regard to opioid-
induced hyperalgesia in both methadone and buprenorphine maintenance patients (Doverty et al., 
2001a; Luginbühl et al., 2003; Compton et al., 2012). Finally, the capsaicin model produces 
primary and secondary hyperalgesia after a subcutaneous injection of capsaicin is applied. This 
model is widely used in opioid-induced pain research, but is not ideal for repeated use. 
 
Mechanisms of Hyperalgesia 
 
Several prominent hypotheses of opioid-induced hyperalgesia have emerged over the past two 
decades. While there is no consensus on a definitive mechanism underlying OIH, a theory that is 
steadily gaining popularity postulates that hyperalgesia is a result of glial cell-regulated immune 
responses via action at the toll-like receptor-4 subtype (TLR4) (Hutchinson et al., 2009; Lewis et 
al., 2010; Watkins, Wiertelak, Goehler, Mooney-Heiberger, Martinez, et al., 1994; Watkins, 
Hutchinson, Rice, & Maier, 2009). Opioids have been shown to activate TLR4s in addition to 
their well-known action at opioid receptors. Of particular interest is the fact that two of the three 
intracellular pathways activated by TLR4s are responsible for proinflammatory glial responses. 
Furthermore, there is some evidence to suggest that the aversive side effects of opioids are 
modulated by this receptor population (Watkins et al., 2009). As such, glial-regulated TLR4 
pathways present a logical potential culprit for the mechanism underlying OIH (Hutchinson et 
al., 2009). 


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Indeed, the molecular mechanisms underlying OIH are not well understood (Bian et al., 2012). 
Aforementioned cumulating evidence suggests that glial cells, particularly microglia, play an 
essential role in development of OIH. About 70% of the cells in the CNS are glial cells, and 
microglia represent 5 to 10% of these glial cells. Microglia are intrinsic immune cells of the 
brain and express many neurotransmitter receptors (Pocock & Kettenmann, 2007). Stress, 
alcohol, and drugs of abuse such as cocaine, methamphetamine, and opioids activate microglia 
(Coller & Hutchinson, 2012), which results in the release of cytokines (TNF-α,IL-1β, and IL-6 
and others), chemokines, lipid mediators of inflammation, matrix metalloproteases (MMPs) and 
nitric oxide (Pocock and Kettenmann, 2007). It has been suggested that activation of microglial 
cells in spinal and cortical areas contributes to development of OIH. Pharmacological treatments 
that inhibit microglial activation have been shown to block development of morphine tolerance 
in rodents (Mika et al., 2009), and increase analgesic potency (Hutchinson et al., 2008). Such 
findings suggest that pharmacological treatments targeting microglia activation may have utility 
in preventing OIH. One such medication is minocycline. 
 
Minocycline: an antibiotic with multiple CNS effects 
 
Minocycline is a second-generation tetracycline analog antibiotic commonly used for the 
treatment of acne. Minocycline is a highly lipophilic molecule that easily penetrates the blood-
brain barrier (Kim & Suh, 2009). In addition to its antimicrobial effects, minocycline has 
significant neuroprotective effects and is currently under investigation for the treatment of 
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, stroke, schizophrenia, 
depression and more recently for methamphetamine dependence (Chaudhry et al., 2012; Kim and 
Suh, 2009; Pae et al., 2008).  
 
Both preclinical and clinical studies have demonstrated the anti-inflammatory and 
neuroprotective properties of minocycline (Kim & Suh, 2009). Inhibition of microglia activation 
is likely the main mechanism for these effects. The mechanism of minocycline’s anti-
inflammatory and neuroprotective effects may be at least partly due to its capacity to inhibit P38 
mitogen-activated protein kinases (MAPK). The p38 MAPKs modulate multiple cell functions 
including proliferation, gene expression, mitosis, cell survival and apoptosis. Of particular 
interest is the fact that they are critically involved in the activation of microglia (Coller & 
Hutchinson, 2012). In neurons, they play an important role in neuronal death. Inhibition of the 
p38 MAPK may contribute both the anti-inflammatory and neuroprotective effects of 
minocycline.  
 
Significance: As evidenced, OIH is a complicated phenomenon that remains nebulous. As there 
is no clear consensus on a theoretical model or mechanism, treating OIH in clinical settings 
remains quite challenging. It is possible that the increased pain sensitivity resulting from chronic 
opioid treatment is directly related to upward dose titration, tolerance, and addiction. With opioid 
use, abuse, addiction, and related deaths steadily rising, variables inciting this epidemic must be 
addressed. While there are active efforts to find more efficacious treatment alternatives for 
chronic pain (evidenced by the recent FDA approval of Phase 2 clinical trials for two glial-
targeting drugs for neuropathic pain) (Watkins et al., 2009), research is needed that will aid the 
utility of currently approved and available pharmacotherapies. If a way to treat OIH can be 


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elucidated, this will allow clinicians to continue prescribing opioids without concern over 
paradoxically increasing the very symptom they set out to treat: pain. 
 
7.  Subjects 
 
Veterans and non-veterans who meet the following criteria will be eligible for participation: 
 
Inclusion criteria: 
• Males and females, between the ages of 18 and 60 
• Diagnosed with opioid dependence and currently enrolled in methadone or buprenorphine 
maintenance treatment  
• Compliant in opioid maintenance treatment and on a stable dose for two weeks or greater 
• No current dependence or abuse of any other drugs (other than tobacco or marijuana) 
• No current medical problems deemed contraindicated for participation by physician 
investigator 
• For women, not pregnant as determined by pregnancy screening; not breast feeding; 
using acceptable birth control methods; not experiencing moderate to severe premenstrual 
symptoms (may interfere with pain assessment); regular menstrual cycles 
 
Exclusion criteria: 
• Current major untreated, unstable psychiatric illnesses including mood, psychotic, or 
anxiety disorders 
• History of major medical illnesses, including liver diseases, heart disease, or other 
medical conditions that the physician investigator deems contraindicated for inclusion in 
the study 
• Current use of over-the-counter or prescription psychoactive drugs  known to affect pain 
threshold or pain tolerance (including NSAIDS, serotonin-norepinephrine reuptake 
inhibitors (SNRIs), (e.g.,venlafaxine, duloxetine), tricyclic antidepressants (e.g., 
nortriptyline, amitriptyline), anticonvulsant medications (e.g., topiramate, tegretol), 
benzodiazepines (e.g., alprazolam, diazepam), and other opioid drugs)  
• Liver function tests (ALT or AST) greater than 3x normal 
• Allergy to minocycline or other tetracyclines 
 
8.  Privacy 
All information that is obtained from subjects will be used for the specifically stated purposes 
that are described in this Project Description and have been approved by the HSS. The personal 
identifiers that are necessary for this research and that will be obtained are the following: Name, 
Medical Record Number, Age, Gender, Medical and Psychiatric History, and Laboratory 
Examination. The procedures for data collection and recruitment of subjects, described 
elsewhere in the project description, are the least intrusive consistent with obtaining the 
information necessary to complete this project. Medical records will be reviewed to extract 
research information by Mehmet Sofuoglu M.D., Ph.D., and Ellen Mitchell R.N. All members of 
the research team will review, use, and record the minimum amount of information necessary to 
accomplish the goals outlined in the protocol. All members of the research team have been 


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trained on VHA privacy regulations and policies and training are current. The settings in which 
informed consent discussions, other subject interviews or research procedures occur will provide 
that same privacy protections that would exist if these discussions, interviews, or procedures 
were carried out for required clinical care (private rooms, drawn curtains, etc.). The information 
will be used for this project, or disclosed to others, only as permitted by the Privacy Act, the 
HIPAA Privacy Rule, and VA policy. 
 
9.  Selection 
 
Sixty completers will be recruited through the VA methadone and buprenorphine clinics, as well 
as through the APT Foundation Methadone and Buprenorphine Maintenance Programs. After the 
initial phone screening, potential subjects will undergo a comprehensive evaluation which will 
include medical, psychiatric, and drug use histories as well as physical, psychiatric, and 
laboratory examinations. Laboratory examination will include CBC, liver and thyroid function 
tests, serum electrolytes, BUN, creatinine, PT, PTT, urine analysis (including urine pregnancy 
for women) and urine toxicology screening.  
Participants will be terminated from the study following opioid relapse, or use of any other 
psychotropic drugs/medications. If participants are noncompliant (no-show, positive urine 
screening, noncompliance with medication protocol/missing more than one dose of 
minocycline/placebo), participation will be terminated. 
 
10.  Research Plan 
 
A. Overview:  This double-blind, randomized clinical trial will randomize male and female 
veterans and non-veterans currently undergoing opioid maintenance treatment for opioid 
dependence to either minocycline (200mg/day) or placebo for 15 days. Upon inclusion, 
participants will be subjected to a pain assessment to evaluate baseline pain thresholds and 
tolerance: the Cold Pressor Test. An experimental treatment of either minocycline or placebo 
will then be initiated and maintained for 15 days. Additionally, at the beginning of Week 2 of 
treatment, participants will be given a Personal Digital Assistant (PDA) an HP iPAQ Pocket PC 
2003 Pro that will administer Ecological Momentary Assessments (EMA). Using EMA, we can 
assess change in pain sensitivity, withdrawal symptoms and cognitive performance in the 
participants’ natural environment, which increases the ecological validity of the study. 
Participants will be asked to return to the laboratory several times a week for the 15 consecutive 
days that they are taking minocycline in order to receive the study medication and to assess 
changes in pain thresholds and tolerance (i.e. to assess the presence, or lack thereof of 
hyperalgesia). Upon completion of experimental treatment, participants will be asked to return a 
final time to undergo pain measurement once more, to assess any changes in pain sensitivity after 
completion of minocycline. 
 
B. Medication 
 
Minocycline: In this study, minocycline will be administered at 200 mg/day, as a single dose, for 
15 days, similar to the dosing in our previous studies (Sofuoglu et al., 2011; Sofuoglu et al., 
2009). This is the usual daily dose of minocycline used in clinical trials for the treatment of 


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infections. Following oral administration, peak plasma levels of minocycline are reached within 
1- 4 hours. The elimination half-life of minocycline ranges from 11 to 24 hours.  
The most common side effects associated with minocycline include dizziness, vertigo, nausea 
and vomiting. Photosensitivity manifested by an exaggerated sunburn reaction has been has been 
reported rarely with minocycline. Patients who experience CNS side effects including light 
headedness, dizziness, or vertigo should be cautioned about driving vehicles or using hazardous 
machinery while on minocycline therapy. These symptoms may disappear during therapy and 
usually disappear rapidly when the drug is discontinued. Concurrent use of tetracycline may 
render oral contraceptives less effective. Subjects will need other forms of contraception. 
Minocycline is contraindicated in pregnant women and can be harmful to the fetus. To minimize 
these risks, subjects will have frequent outpatient visits, where they will receive take-home oral 
minocycline capsules sufficient to last until their next scheduled outpatient visit, and any adverse 
effects from the study medication will be monitored under the nurses’ supervision. Female 
subjects will be tested for pregnancy before each test session. As minocycline should be taken on 
an empty stomach, subjects will be advised not to eat for one hour prior to coming to the clinic.  
 
C. Study Procedures 
 
Cold Pressor Test (CPT): Adapted from Eckhardt et al. (1998), the CPT has good predictive 
validity for the analgesic effects of opioids (Conley et al., 1997). For this test, two water coolers 
filled with either warm (100.04ºF/37.8ºC) or cold water (32.9-34.7ºF/0.5-1.5ºC) are used. To 
begin the CPT, participants first immerse their forearm into the warm-water bath for 2 min. 
Participants are then instructed to immerse their forearm into the cold water bath and  report the 
first time they experience pain. They are also instructed to keep their arm submerged as long as 
they can but were permitted to withdraw their arm from the water if the pain gets uncomfortable. 
Latency to first pain sensations (threshold) and to arm withdrawal from the water (tolerance) is 
recorded. During testing and following arm withdrawal, heart rate, blood pressure is monitored. 
After arm withdrawal, the McGill Pain Questionnaire will be completed. Before, during, and 
after pain testing, the VAS will be completed. 
 
EMA Procedures: Participants will be given a PDA at the beginning of Week 2. The Smartphone 
will be programmed to beep four times each day at random times (Random Assessment; RA) for 
one week.   
 
EMA Assessments:  Participants will be asked if they feel any pain “at this moment” on seven-
point Likert scales (1=strongly disagree to 7=strongly agree). Craving for heroin (e.g., “At this 
moment, I am craving heroin”) and difficulty concentrating will be assessed with single items. 
Several additional items will assess opioid withdrawal. At each time point, the PDA will 
administer the Sustained Attention to Response Test (SART). 
 
1) Physiological 
 
Heart rate and blood pressure: Heart rate and blood pressure will be measured throughout the 
session. 
 
Breathalyzer: A breathalyzer reading will be obtained before the start of each session. 
 


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Blood Samples: 
 
Serum Cytokines: Serum levels of pro-inflamatory cytokines will be assayed using 
electrochemiluminescence multi-array technology (Meso Scale Discovery, Gaithersburg, MD) as 
described in the methods of DellaGioia et al. (2013). These include Interleukin-1 beta (IL-1β), 
Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Interleukin 12 p70 (IL12p70), 
Interferon gamma (IFNg), Tumor Necrosis Factor alpha (TNFα), Granulocyte-Macrophage 
Colony Stimulating Factor (GM-CSF). Serum samples will be obtained at baseline, and once 
more at the end of minocycline/placebo treatment. Statistical analysis will examine if serum 
cytokine levels change with medication treatment. 
 
2) Cognitive Performance 
For cognitive performance, we chose three tests that are likely to be sensitive to minocycline’s 
effects in our subject population: the Rapid Visual Information Processing (RVIP), Sustained 
Attention to Response Test (SART), and the Digit Symbol Substitution Test (DSST). 
The RVIP is widely used as a measure of sustained attention with a working memory 
component. In this task, subjects are asked to respond to any of three digit sequences in a 
continuous stream of digits lasting for 7 minutes. A white box appears in the center of the 
computer screen, inside which digits from 2 to 9 appear in a pseudo-random order at the rate of 
100 digits per minute.  Subjects are instructed to detect consecutive odd or even sequences of 
digits (e.g., 2-4-6, 3-5-7, 4-6-8, 5-7-9, etc.) and to register responses using a press-pad.   
 
The SART (Robertson et al., 1997) is a Go No-Go task. It assesses the ability to withhold 
responses to an infrequently occurring target (No-Go trials). A total of 225 single digits (25 x 9 
digits) are presented on a computer monitor for 250 ms each, immediately followed by a mask 
for 900 ms. Subjects must press a spacebar in response to every digit except the “3”. 
 
The DSST is a test of psychomotor performance, which measures motor persistence, sustained 
attention, response speed and visuomotor coordination. The task is to fill in blank spaces with the 
symbols that are paired with the number above the blank space as fast as possible for 90 sec. 
DSST was chosen because several studies have demonstrated its sensitivity to opioid effects 
(Cooper et al., 2012; Zacny and Lichtor, 2008). 
3) Self-report 
Intake Measures: 
 
Structured Clinical Interview for DSM-IV (SCID) for DSM-IV Axis I disorders: This semi-
structured interview based on DSM-IV (APA, 1994) and will be administered to diagnose Axis I 
disorders. 
Session Measures: 
 
Brief Pain Inventory – Short Form: BPI-SF is a self-report questionnaire that assesses severity of 
pain, impact of pain on daily function, location of pain, pain medications, and amount of pain 
relief in the past 24 hours or the past week (Cleeland and Ryan, 1994). 
 


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Opioid Withdrawal Symptom Checklist (OWSC): Withdrawal signs and symptoms will be 
assessed using a 22-item withdrawal instrument that has been reliably used to assess opiate 
withdrawal. This scale rates the following items from 0 to 4: lacrimation, nasal congestion, 
yawning, sneezing, coughing, throat clearing, restlessness, nausea/vomiting, gooseflesh, 
sweating, stomach cramps, muscle cramps, and feeling hot/cold. 
 
Time-line Follow-Back Assessment Method (TLFB): Drug use for each day during the 30-day 
period prior to the study, throughout the period of study participation, and at the follow-up 
session will be obtained using this self-report measure. Participants are given a blank calendar 
covering the specified time interval, and are asked to retrospectively reconstruct their drug use 
over that time interval. The process is facilitated by establishing anchor points (e.g., holidays, 
anniversaries, major national events, etc.). It can be scored to provide the number of days on 
which various levels of drug use occurred. The time-line method has good test-retest reliability 
and good validity for verifiable events. It has been used in numerous studies to assess drug use 
habits. 
 
Short-Form McGill Pain Questionnaire (SF-MPQ): The SF-MPQ will be used to capture the 
sensory and affective dimensions of the pain experience immediately following the CPT  
(Melzack, 1987). In this 15-item questionnaire, participants describe their experience of pain by 
choosing among a series of possible answers [none (score=1), mild (score=2), moderate 
(score=3), or severe (score=4)]. The items to describe the pain include ‘Throbbing’,‘Shooting’, 
‘Stabbing’, ‘Sharp’, ‘Cramping’, ‘Gnawing’, ‘Hotburning’, ‘Aching’, ‘Heavy’, ‘Tender’, 
‘Splitting’, ‘Tired,’ ‘Exhausting’, ‘Sickening’, ‘Fearful’, and ‘Punishing-Cruel’. Scores are 
added across all 15 items to generate a sum score, which ranging from 15 and 60. 
 
Visual Analog Scale (VAS): The VAS will be used by participants as a secondary rating scale 
for subjective pain severity before, during, and after pain testing. This scale contains a horizontal 
line, anchored by verbal descriptors of “no pain” and “pain as bad as it could be”. Participants 
will place a vertical line at the point that best indicates their present pain  (Burckhardt & Jones, 
2003). 
 
Drug Effects Questionnaire (DEQ): Subjects will rate the following items from "not at all" to 
"extremely" on a 100 mm scale: “alert,” “calm,” “confused,” “depressed,” “confused,” “high,” 
“anxious,” “sedated,” “tired,” “social,” “self-confident,” “talkative,” “hungry,” “feeling the drug 
strength,” “feel good drug effects,” “feel bad drug effects,” and “want more drug.” These items 
are commonly used to assess the subjective effects of drugs (Fischman and Foltin, 1991).  
 
Profile of Mood States (POMS): This 72–item questionnaire consists of adjectives commonly 
used to describe momentary mood states, has been found to be sensitive to the mood-altering 
effects of drugs including amphetamine (de Wit and Griffiths, 1991; Levin et al., 1998), and is 
widely used as a research tool in behavioral pharmacology (Fischman and Foltin, 1991). Its 
reliability and validity have been extensively evaluated (McNair et al., 1971). The POMS rates 
eight mood states on a 5–point rating scale (0=not at all to 5=extremely): (1) Anger, (2) Anxiety, 
(3) Confusion, (4) Depression, (5) Elation, (6) Fatigue, (7) Friendliness and (8) Vigor. The 
POMS will be used during each session to measure the effects of minocycline on mood. 
 


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Systematic Assessment of Side Effects (SAFTEE): In order to monitor adverse events from the 
study medications, the SAFTEE will be administered before and after each session. This is a 
symptom checklist (Levine and Schooler, 1986) that has been used successfully in our previous 
studies to assess possible side effects of study medications. It includes information regarding 
severity of any presenting side effects, as well as the course of action taken by study staff in 
response. 
 
4) Genetics:  
DNA Extraction: The PAXgene blood DNA Kit (PreAnalytiX Gmbh) will be used for peripheral 
blood DNA extraction. The isolation procedure is easy to perform, and rapid, and results in high 
yield and high quality genomic DNA. Briefly, 8.5 ml of human whole blood is collected into 
PAXgene Blood DNA tubes, which contain a proprietary blend of reagents, optimized for 
isolation of high-quality genomic DNA. Genomic DNA is then isolated from peripheral blood 
using the PAXgene Blood DNA Kit. The extraction is performed in a single processing tube to 
minimize the risk of sample mix-up and cross-contamination. High yields of pure DNA are 
obtained that perform well in a wide range of downstream applications (e.g., PCR, genotyping, 
restriction enzyme digestion, Southern-blotting, and pharmacogenomic studies). Typically, 150-
500 ug of genomic DNA (A260nm/A280nm: 1.7~1.9) can be obtained. In this study, we will 
examine genetic variation that may influence variations in opioid-induced hyperalgesia, as well 
as variations in minocycline’s effects. These will include genes that are related to glutamate, 
GABA, dopamine, opioid receptors, as well as other targets. 
 
Procedure: 
After an initial scripted phone screening to determine obvious exclusions, potential participants 
will be screened in person, which will include discussion of basic inclusion and exclusion criteria 
and a brief description of the study. Details of the Cold Pressor Test pain assessment will be 
discussed. If potential participants are still interested, a full screening will be completed. A 
research assistant and a clinician will perform the initial screening. During this time, informed 
consent will be obtained along with a signed release to allow study personnel to monitor opioid 
maintenance, and laboratory examinations will commence. This comprehensive evaluation will 
include medical, psychiatric, and drug use histories, along with physical, psychiatric, and 
laboratory examinations. Laboratory examination will include a complete blood count, liver and 
thyroid function tests, serum cytokines, serum electrolytes, BUN, creatinine, PT, PTT, RPR, 
random glucose, urine analysis (including urine pregnancy for women), and urine toxicology 
screening and genetics. Women will be asked to report the first day of their last menstrual cycle, 
and note will be taken of any ongoing oral contraceptive prescriptions. In addition to this 
standard clinical evaluation of psychiatric and medical histories, mental status examination, 
physical examination, and laboratory assessment, potential participants will undergo a clinical 
diagnostic interview, the Structured Clinical Interview for DSM-IV Disorders (SCID). 
 
All sessions will occur at the West Haven VA Connecticut Healthcare Campus. During an 
adaptation session, all participants will undergo a baseline urine toxicology screen. On test days, 
all participants will be asked to refrain from caffeine, nicotine, and from eating within one hour 
of arrival. Participants will be asked to come to the testing site for a total of ten times: one initial 
screening day (~ 3 hours), one day for adaptation (~ 1.5 hours), three testing days (~ 2 hours), 


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four outpatient days to receive medication and check in with research and medical staff (~1 
hour), and one final day for follow-up (~ 0.5 hour).  Participants will be asked to arrive at 
approximately the same time each morning, coordinating with attendance at their opioid 
maintenance clinic. A study nurse will confirm with their respective program that participants 
did not receive either methadone or buprenorphine that morning, and will call the program when 
testing is complete to permit dispersal of that day’s methadone or buprenorphine dose. On the 
initial screening day and at the end of medication treatment, blood will be drawn to determine 
serum cytokine levels. Participants will undergo a variety of cognitive and self-report measures, 
as well as assessments to confirm restraint from illicit drug use and lack of adverse effects of 
medication. Prior to their daily methadone or buprenorphine dose and thus at trough plasma 
levels of opioid, participants will receive either minocycline (200mg) or placebo. Subsequently, 
all participants will perform the Cold Pressor Test. During all pain assessment sessions, heart 
rate, and blood pressure will be monitored. Participants will complete the McGill Pain 
Questionnaire and a VAS after the completion of pain assessment. On outpatient medication visit 
days, participants will have vital signs checked, undergo a urine screen, and receive their study 
medication for that day, as well as sufficient take-home minocycline/placebo capsules to last 
until their next scheduled visit. During their follow-up visit, approximately one week after 
completing minocycline/placebo, participants will undergo the same procedures as the adaptation 
session to monitor any changes in pain response following cessation of study medication. 
Table 1. Experimental Design 
 
 
Week 1 
Week 2 
Week 3 
Wee
k 4 
IS 
AS 
TS 
Out-patient 
Session 
Out-patient 
Session 
TS 
Out-patient 
Session 
Out-patient 
Session 
TS 
Discharge 
FU 
Abbreviations: IS: Initial Screening; AS: Adaptation Session; Out: Outpatient Session;  
TS: Test Session; FU: Follow-up 
 
 
 
Table 2A. Schedule of Events: Adaptation Session 
Time 
Measures and Events 
-60 min 
Vital Signs; breathalyzer; urine screen 
-45 min 
OWSC; TLFB; BPI-SF; DEQ; POMS; 
SAFTEE 
-30 min 
RVIP, SART, DSST 
-15 min 
HR/BP; VAS 
0-15 min 
CPT; HR/BP; McGill; VAS; Discharge  
Lunch or Snack 
 
 


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Table 2B. Schedule of Events: Test Session (once per week) 
Time 
Measures and Events 
-45 min 
Vital Signs; breathalyzer; urine screen; blood sample* 
-30 min 
OWSC; TLFB; BPI-SF; DEQ; POMS; SAFTEE 
-15 min 
RVIP, SART, DSST 
0 min 
Minocycline/placebo administration, Snack  
15 min 
HR/BP; DEQ 
30 min 
HR/BP; DEQ 
45 min 
HR/BP; DEQ; VAS 
60-75 min 
RVIP, SART, DSST, CPT; HR/BP; McGill; VAS; 
SAFTEE; Discharge 
Lunch 
 
Table 2C. Schedule of Events: Follow-Up (Week 4) 
Time 
Measures and Events 
-60 min 
Vital Signs; breathalyzer; urine screen 
-45 min 
OWSC; TLFB; BPI-SF; DEQ; POMS; SAFTEE 
-30 min 
RVIP, SART, DSST 
-15 min 
HR/BP; VAS 
0-15 min 
CPT; HR/BP; McGill; VAS; Discharge 
Lunch or Snack 
Abbreviations: COWS: Clinical Opiate Withdrawal Scale; TLFB: Time-line Follow-Back; BPI-
SF: Brief Pain Questionnaire – Short Form; DEQ: Drug Effects Questionnaire; POMS: Profile of 
Mood States; SAFTEE: Systematic Assessment of Side Effects; RVIP: Rapid Visual Information 
Processing; SART: Sustained Attention to Response Test; DSST: Digit Symbol Substitution 
Test; CPT: Cold Pressor Test; HR: Heart Rate; BP: Blood Pressure; VAS: Visual Analog Scale 
*On the last test day only 
 
11. Statistical Considerations 
a. Statistical analysis. 
The primary dependent variables will be pain sensitivity, cognitive performance, subjective drug 
effects, and physiological measures. Pain sensitivity will be assessed with the Cold Pressor Test 
(CPT). The cognitive performance will be assessed with the Digit Symbol Substitution Test 
(DSST) and a Go-No/Go task. Subjective drug effects will be assessed with the Drug Effects 


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Questionnaire (DEQ). The physiological responses will include heart rate, and blood pressure. 
The independent variable, Treatment, will be categorical with 2 levels: minocycline or placebo. 
The primary analysis will be done with repeated measures of variance. In these analyses, effects 
for treatment (minocycline or placebo) and session and the interaction between treatment and 
time will be included. Adjustments will be made (e.g., Bonferroni correction) for the multiple 
testing of the data.  
b. Sample size and power analysis:  
There are no previous studies that examined minocycline’s effects on OIH, as told by the CPT, in 
opioid-maintained patients. For power calculations, we referenced a previous study that 
examined the effect of gabapentin on the CPT in MM patients (Compton et al., 2010). As such, 
to allow for an approximate 15% drop out rate, we plan to recruit 60 participants, with 30 in each 
condition. This will yield over 80% power with a moderate effect size (d=0.5) for the between-
subjects comparison of minocycline or placebo effects on opioid-induced hyperalgesia. 
 
 
12. Risks and benefits 
Potential risks 
1. Minocycline: The most common side effects associated with minocycline dizziness, 
vertigo, nausea and vomiting. Photosensitivity manifested by an exaggerated sunburn 
reaction has been has been reported rarely with minocycline. Patients who experience 
CNS side effects including light headedness, dizziness, or vertigo should be cautioned 
about driving vehicles or using hazardous machinery while on minocycline therapy. 
These symptoms may disappear during therapy and usually disappear rapidly when the 
drug is discontinued. Concurrent use of tetracycline may render oral contraceptives less 
effective. Subjects will need other forms of contraception. Minocycline is contraindicated 
in pregnant women and can be harmful to the fetus. To minimize these risks, subjects will 
be administered minocycline in the clinic daily and any adverse effects from the study 
will be monitored. Female subjects will be tested for pregnancy before each test session. 
 
2. Blood Drawing: Subjects will have approximately 50 cc of blood drawn as a result of 
their participation in the study. Blood drawing can cause some pain and result in a 
hematoma. 
 
Protection of subjects 
a. Our inclusion and exclusion criteria will be applied by experienced professionals who 
will be carefully trained and monitored in order to accept only appropriate subjects into 
the study. Thus, effective screening will exclude subjects who would be placed at a 
greater risk. This is determined by the medical and psychiatric history, drug use history, 
the physical examination, and the laboratory studies done prior to beginning this research 
protocol. 
 
b. Confidentiality will be protected by having records identified by code number only with 
the master list including names kept in a sealed envelope in a locked file in the Principal 
Investigator's office. Confidentiality in this study is of the utmost importance to us. All 
information obtained will be stored in coded form. The paper files will be kept in locked 


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file cabinets in Bldg. 36 and the electronic records will be kept in VA intranet (M-drive). 
 
Risk Benefit ratio 
Participation in the study may lead to continued successful treatment for opioid addiction, as 
well as a decrease in OIH, which will be beneficial to subjects participating in the study. Given 
the anticipated benefits to subjects and society, the moderate to low risk to subjects is reasonable. 
 
13. Safety 
The study will be monitored by a Data and Safety Monitoring Board (DSMB) because this 
population might be considered vulnerable due to their substance abuse. Risks associated with 
participating in this protocol are moderate. There is adequate surveillance and protections to 
discover adverse events promptly and keep their effects minimal. The Board will be composed of 
persons who are experienced in the conduct of clinical trials for the treatment of addictive 
disorders and who have appropriate expertise in substance abuse and psychopharmacology.  
There will be at least one physician on the board, who is not directly involved in this trial but 
who has the requisite expertise. In order for the DSMB to fulfill its mission of assuring the safety 
of human subjects and the scientific integrity of the study being conducted, the Board will review 
adverse event data no less than three times per year. During the meeting the DSMB will review a 
table of adverse events at these four-month intervals. Following each DSMB meeting, written 
minutes will be prepared and distributed, summarizing any recommendations including interim 
analyses, as described above. These written reports will insure accurate preparation of any 
protocol amendments that might be necessary. After each DSMB meeting, this written report 
will describe all recommendations including additional safety steps. All serious adverse events 
such as deaths, hospitalizations and unexpected toxicity will be reported to the IRB as well as 
our local hospital risk management committee under expedited reporting, i.e., immediately by 
telephone as the information is available to us, followed by written report within 48 hours. The 
procedures for the written reporting include written documentation using the clinical notes 
related to the adverse event and specific forms detailing the event with a sign-off by all 
appropriate supervisory personnel. Communication of recommendations and decisions are made 
back to the investigator in a timely manner. These reports will also circulate to the DSMB as the 
safety monitor. 
 
Reporting of Adverse Events 
An adverse event is any physical or clinical change or disease experienced by the subject at any 
time during the course of the study, whether or not considered related to the use of the study 
drug. This includes the onset of new illness and the exacerbation of pre-existing conditions. 
Subjects will be questioned and/or examined by the investigator or his/her designee for evidence 
of adverse events. The questioning of subjects with regard to the possible occurrence of adverse 
events will be generalized such as, “How have you been feeling since your last visit?”  The 
presence or absence of specific adverse events will not be solicited from subjects. 
All adverse events will be recorded in the subject’s medical and/or research records. The onset 
and end dates, severity, and relationship to the study drug will be recorded for each adverse 
event. The severity of the adverse event will be assessed according to the following grading 
system. Any action or outcome (e.g., hospitalization, discontinuation of therapy, etc.) will also be 
recorded for each adverse event. 


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Grading. For the severity of adverse events, the following definitions will be used: 
• Mild:  awareness of sign, symptom, or event, but easily tolerated, 
• Moderate: discomfort enough to cause interference with usual activity and may warrant 
intervention, 
• Severe: incapacitating with inability to do usual activities or significantly affects clinical status, 
and warrants intervention, 
• Life-threatening: immediate risk of death 
 
Attribution. The investigator will also assess the relationship of any adverse event to the use of 
the study drug, based on available information, using the following guidelines: 
• Definite:  Adverse event(s) will clearly be related to investigational agent(s) or other 
intervention 
• Probable: Adverse event(s) will likely be related to investigational agent(s) 
• Possible: Adverse event(s) may be related to investigational agent(s) 
• Unlikely: Adverse event(s) will doubtfully be related to investigational agent(s) 
• Unrelated: Adverse event(s) will clearly not be related to the investigational agents(s) 
 
Definition of Serious Adverse Events. All serious adverse events, whether or not deemed drug-
related or expected, will be reported by the principal investigator or designee to the IRB within 
24 hours (one working day) by telephone. A serious adverse event is any event that: is fatal, is 
life-threatening (life-threatening is defined as the patient was at immediate risk of death from the 
AE as it occurred), is significantly or permanently disabling, requires inpatient hospitalization or 
prolongs hospitalization, is a congenital anomaly or birth defect.  
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject, and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition.  
A written report will follow as soon as possible which includes a full description of the event and 
any sequelae. This includes serious adverse events that occur any time after the inclusion of the 
subject in the study (defined as the time when the subject signs the informed consent through 
discharge from the study (i.e., either subject completed or discontinued their study participation). 
The subject is considered discharged either after the completion of the last visit or contact (e.g., 
phone contact with the investigator or designee). Discontinuation is the date a subject and/or 
investigator determines that the subject can no longer comply with the requirements for any 
further study visits or evaluations (e.g., the subject is prematurely discontinued from the study). 
All serious but unanticipated adverse events will be reported to the HSS and HIC within 48 
hours; and serious anticipated adverse events will be reported immediately to the HSS and HIC 
whenever their magnitude or frequency exceeds expectations.  
Serious adverse events will get reviewed immediately by the PI. Otherwise, this moderate risk 
study will be monitored quarterly by the PI and the members of the DSMB, and reported to the 
IRB if trends in AEs emerge. 
 
14. Informed consent: Recruitment will be by word-of-mouth, referrals from area programs and 
by advertisement. A research staff member will interview opioid dependent individuals that are 


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interested in participating in the study over the phone.  If subjects pass the initial screening for 
the study, they will then come into the clinic for a full screening evaluation. Upon arrival, 
research assistant will read the detailed consent form and will ask questions to make sure that the 
subjects understand the procedure and their rights, and informed consent will be obtained.     
 
15. Confidentiality: Confidentiality in this study is of the utmost importance to us. All 
information obtained will be stored in coded form. The names of the subjects will be used in 
hospital records.   
 
16. Location of Study: This study will be conducted at the West Haven VA Connecticut 
Healthcare System. 
 
17. Payment: All participants will be compensated for their participation. Participants will be 
paid $30.00 for the initial screening and $50 for the adaptation session. Participants will also 
receive $50 for each of the three test sessions, and $20 for each of the four outpatient sessions as 
well as $50 for the follow-up visit. In addition, participants will be paid $20.00 a week to help 
off- set transportation cost, and $1.50 for each completed assessment on the PDA. Thus, 
participants can potentially receive a total of $482.00. 
 
18. Source of Funds: This protocol is being funded by MIRECC. 
 
19. Duration: The entire study will take approximately two years to complete.    
 
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VA Healthcare System  
 
MINOCYCLINE in OIH 
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SAP_001.pdf: 
Title of Study: The Effect of Minocycline on Opioid-Induced Hyperalgesia in Opioid-
Maintained Patients 
 
NCT#: 02359006 
 
Document: Statistical Analysis Plan 
 
Date of Document: Last updated on 7/1/2017 
 
 


Statistical Considerations for Minocycline in OIH  
 
a. Statistical analysis. 
The primary dependent variables will be pain sensitivity, cognitive performance, subjective drug 
effects, and physiological measures. Pain sensitivity will be assessed with the Cold Pressor Test 
(CPT). The cognitive performance will be assessed with the Digit Symbol Substitution Test 
(DSST) and a Go-No/Go task. Subjective drug effects will be assessed with the Drug Effects 
Questionnaire (DEQ). The physiological responses will include heart rate, and blood pressure. 
The independent variable, Treatment, will be categorical with 2 levels: minocycline or placebo. 
The primary analysis will be done with repeated measures of variance. In these analyses, effects 
for treatment (minocycline or placebo) and session and the interaction between treatment and 
time will be included. Adjustments will be made (e.g., Bonferroni correction) for the multiple 
testing of the data.  
b. Sample size and power analysis:  
There are no previous studies that examined minocycline’s effects on OIH, as told by the CPT, in 
opioid-maintained patients. For power calculations, we referenced a previous study that 
examined the effect of gabapentin on the CPT in MM patients (Compton et al., 2010). As such, 
to allow for an approximate 15% drop out rate, we plan to recruit 60 participants, with 30 in each 
condition. This will yield over 80% power with a moderate effect size (d=0.5) for the between-
subjects comparison of minocycline or placebo effects on opioid-induced hyperalgesia.