Effects of Intramuscular Oxytocin on Pupil Diameter and Heart Rate Variability
Phase: PHASE4
Conditions: Chronic Pain Due to Injury
Sponsor: Wake Forest University Health Sciences

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NCT04105998 
Title: Effects of Intramuscular Oxytocin on Pupil Diameter and Heart Rate 
Variability 
Date: 10/18/2021 
 

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BACKGROUND 
 
A large body of evidence in animals and in humans suggest that oxytocin can 
produce analgesia to experimental pain stimuli and relieve pain in acute and chronic 
clinical pain states.  For the past 8 years we have, under an NIH MERIT award, tested 
the role of the spinal cord in oxytocin’s actions by studies in rodents and clinical trials, 3 
of which are ongoing, of spinally injected oxytocin. 
 
The Pain Mechanisms Laboratory at Wake Forest is now in the process of 
assembling an NIH program project grant (P01) on the topic of oxytocin sites, 
mechanisms, and clinical applications for analgesia, and Dr. Eisenach will lead the 
clinical project in that proposal.  In order to acquire adequate preliminary data to support 
this application, we are examining the effects of oxytocin in several small clinical 
studies. The first one of these (IRB 00056413) has been completed, and described the 
pharmacokinetics of oxytocin, 10 IU, after intravenous administration in men and 
women.  The second one (IRB00056660) is ongoing and examines intramuscular 
oxytocin, 10 IU, compared to placebo in a double blinded study on sensory changes 
after controlled UV-B sunburn.  We have obtained a waiver of IND from the FDA for 
oxytocin at these doses by these routes of administration in men and women. 
 
The goal of this third protocol is to test central effects of Intramuscular oxytocin 
on noninvasive measures which can be easily and repeatedly assessed.  The first two 
studies are providing information related to dosing of oxytocin and measurement of its 
effects on sensory nerves in the periphery.  Oxytocin also enters the central nervous 
system after systemic administration, and the goal of the current study is to test the 
effects of oxytocin on heart rate variability and pupil diameter, both of which affect 
subtle effects on autonomic tone and which have been proposed in earlier studies by 
others to be altered by oxytocin. Taken together, these 3 studies will provide most of the 
information required to support larger clinical studies to be proposed in the P01 
application. 
RATIONALE 
 
Nearly all clinical studies of oxytocin involve intranasal administration, which 
results in minimal or no increase in circulating oxytocin concentration, but effects that 
are presumed to reflect actions in the brain.  The focus of most of these studies involves 

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presumed social effects of oxytocin, typically measured by brain activity or subjective 
report when viewing images of differing social valence.  These studies universally show 
an effect of oxytocin, although their rigor has been questioned because of extremely 
small sample size and lack of a priori statistical analysis plan [16].  Most studies 
demonstrate small effects of oxytocin close to a threshold P value of 0.05 for statistical 
significance, and these measures can typically only be assessed once, requiring a 
between subject design inappropriate to pharmacokinetic-dynamic modeling.  For these 
reasons we do not plan to use these kinds of outcome measures to assess central 
effects of oxytocin. 
 
This protocol focuses on two measures of autonomic control, both in use in 
current studies by our group, to quantify central actions of oxytocin.  First, we propose 
to examine the influence of oxytocin on heart rate variability with focus on its effect on 
high frequency variability, most commonly ascribed to parasympathetic nervous system 
activity acting to brake sympathetic tone.  Intranasal oxytocin has been demonstrated to 
have a large effect size on this measure which can be repeated at frequent intervals [7]. 
As a secondary assessment of parasympathetic activity, we will measure high 
frequency fluctuation in pupil diameter, termed hippus, which also reflects 
parasympathetic nervous system activity [14].  This outcome measure can be assessed 
in 3 seconds using equipment currently being used under IRB approved protocols. 
 
  
 
 

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Protocol 
Effects of intramuscular oxytocin on pupil diameter and heart rate 
variability 
 
PRIMARY GOAL:  Provide an estimate of effect size of intramuscular oxytocin on tests of 
parasympathetic nervous system activity using variations in pupil diameter and in heart 
rate. 
 
GENERAL METHODS:  Equal numbers of healthy adult men and women will be recruited 
(22 total) and will have 2 visits in the Pain Research Unit at Piedmont Plaza 2.  A non-
invasive, chest band heart rate monitor will be applied and baseline pupil diameter 
measures obtained with a pupilometer.  Subjects will receive an intramuscular injection 
of either oxytocin (Pitocin®), 10 IU, or saline.  The study will be randomized and double 
blind and all subjects will receive both treatments. 
 
STUDY DESIGN 
Inclusion Criteria: We request permission to study up to 30 subjects so that we will 
have 22 evaluable subjects who have completed both visits. 
1. Male or female > 18 and < 66 years of age, Body Mass Index (BMI) <40. 
2. Generally in good health as determined by the Principal Investigator based on 
prior medical history, American Society of Anesthesiologists physical status I or II 
3. Normal blood pressure (systolic 90-140 mmHg; diastolic 50-90 mmHg) resting 
heart rate 45-100 beats per minute) without medication 
4. Female subjects of child-bearing potential and those < 1 year post-menopausal, 
must be practicing highly effective methods of birth control such as hormonal methods 
(e.g., combined oral, implantable, injectable, or transdermal contraceptives), double 
barrier methods (e.g., condoms, sponge, diaphragm, or vaginal ring plus spermicidal 
jellies or cream), or total abstinence from heterosexual intercourse for a minimum of 1 
full cycle before study drug administration. 
5. All subjects must be vaccinated against SARS-CoV-2, with proof via vaccination 
card or the NC HHS form. 
 

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Exclusion Criteria: 
 
1. Hypersensitivity, allergy, or significant reaction to any ingredient of Pitocin® 
2. Any disease, diagnosis, or condition (medical or surgical) that, in the opinion of 
the Principal Investigator, would place the subject at increased risk (active gynecologic 
disease in which increased tone would be detrimental e.g., uterine fibroids with ongoing 
bleeding), compromise the subject’s compliance with study procedures, or compromise 
the quality of the data 
3. Women who are pregnant (positive result for urine pregnancy test at screening 
visit), women who are currently nursing or lactating, women that have been pregnant 
within 2 years. 
4. Subjects with neuropathy, chronic pain, diabetes mellitus, or taking 
benzodiazepines or pain medications on a daily basis. 
5. Subjects with history of eye surgery, with cataracts, or taking medications for eye 
conditions. 
 
Study Visit 1:  The participant will report to Piedmont Plaza 2. The participant 
will review and sign the Informed Consent. After informed consent is adequately 
obtained, a detailed medical history will be obtained from the participant, female 
participants will provide a urine sample to determine pregnancy status.  The research 
nurse will have the subject place the chest belt for heart rate monitoring, familiarize the 
subject with the pupilometer, and calibrate the system for gaze by having the subject 
look at each of 9 gray Xes on a monitor located in front of the chin rest and pupilometer 
camera. 
Baseline blood pressure, heart rate and respiratory rate will be measured. Pupil 
diameter measurements will be obtained for 20 seconds every 5 minutes for 20 
minutes. The subject will only need to position their head with chin on the chin rest 
during times of pupil measurements.  During the pupil measurements we will record the 
measurements using an infrared camera.   
 
The subject will then receive an intramuscular injection of oxytocin (Pitocin®), 10 
IU or the same volume (1 ml) of saline.  Pupil measurements will be made for 20 
seconds every 5 minutes until 120 minutes after study drug administration. 

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Study Visit 2 
 
All procedures from Study Visit 1 will be repeated with the cross-over study drug 
solution. The visits will be at least 24 hours apart. 
 
Safety and Monitoring:  Given that oxytocin is available over the counter in much larger 
systemic doses than proposed in this application and since multiple scientific reports 
have examined much larger systemic doses than in this application using only 
subjective reporting, we propose to only request subjects to report any unusual 
sensations and query them at the end of each drug administration study day if they 
experienced any unusual sensations. Blood pressure, heart rate and respiratory rate will 
be measured at both study visits at baseline and then at 30, 60, and 90 minutes after 
the injection. 
RISKS 
 
Risks of intramuscular injection include pain and bruising.  Risks of oxytocin are 
primarily restricted to uterine contraction in the presence of pregnancy, since oxytocin 
receptors are not present in the non-pregnant uterus (Package insert attached). 
Additionally we noted in our previous study of intravenous oxytocin there is the 
possibility of a feeling of being flushed, headache and increased heart rate with no 
significant change in blood pressure during or immediately after the infusion. These 
events were reported by previous participants in the intravenous study, but were short 
lived, lasting approximately 12-15 minutes. We have received no reports of these 
effects in our current study of intramuscular oxytocin. The use of infrared video camera 
will does not expose participants to a greater amount of light than expected in normal 
use of an infrared camera. 
Data Safety Monitoring Plan 
 
Although the use of oxytocin in this protocol is outside FDA approval for oxytocin 
administration during the postpartum period, the dose to be studied is similar to or less 
than that of multiple recent studies in healthy volunteers with intranasal oxytocin 
purchased over the counter in the US [1-6; 8-13; 15-19] and without description of 

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adverse events.  For these reasons, we do not propose a data safety monitoring 
committee or special safety evaluation beyond those required by IRB regulations. 
Minority, Gender, and Children Participation 
Both sexes and races and ethnicities will be actively recruited in this small study.  
Children under age 18 are not included in these protocols because this protocol 
because the safety of this product has not been established in children.  
 
Volunteer Payment 
Participants will be paid a total of $200 according to the following payment 
schedule which we have used throughout the last 2 cycles of this grant's protocols. We 
believe that this payment schedule is fair and appropriate, paying for each procedure 
attempted and an additional payment for completion of the entire study.  
Completion of study visit 1: $50 
Completion of study visit 2: $150 
Long-term Follow-up 
Volunteers will be contacted one and 7 days after the last study visit and 
questioned about adverse events from the study. 
 
Data Share Plan Stanford Pilot Data   
Scope of Work:  The Dr. Steven Shafer at Stanford University will establish and direct 
a pharmacokinetic/dynamic (PK/PD) modeling to facilitate future modeling in a NIH 
grant to be funded late 2021/early 2022. The PK/PD modeling will generate a 
mathematical model of oxytocin uptake, distribution, and elimination following 
intravenous delivery in humans. The PK/PD Core will use these mathematical models to 
describe the relationship between concentration at the site of effect and therapeutic 
response. The models will facilitate dosing regimens for future studies.  The dose effect 
of intramuscular oxytocin will be modeled by the relationship noted on pupil diameter 
and heart rate variability and pupillary hippus measurements. 
 
 
 

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STATISTICAL RATIONALE 
Co-Primary outcome measures are magnitude of power at the dominant frequency in 
the Fourier transform of pupil diameter in the .1-2 Hz range, termed hippus, and heart 
rate variability in the high frequency range (HF-HRV).  These are complementary 
measures which are both affected by parasympathetic nervous system activity and by 
non-overlapping other factors.  We request a convenience sample of 22 subjects for the 
purpose of preliminary data to provide variance and effect size estimates that will inform 
federal grant submission for use of this model to a larger sample size to adequately test 
the plasma concentration response of computer controlled infusions of oxytocin to 
targeted plasma concentrations with intravenous infusions.  Thus, we propose 
descriptive statistics as the primary analysis of this pilot study.  
 
 
 
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