Clinical trial NCT03292731
Conditions: Preterm Birth
Sponsor: Steve N. Caritis, MD
Start date: 2018-02-12; Completion date: 2021-09-02
Enrollment: 159 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The study plans to determine the relationship between plasma concentrations of 17-OHPC hydroxyprogesterone caproate (17-OHPC) and the rate of preterm birth. The study is an open label study of pregnant women with one or more prior spontaneous preterm births who are receiving either 250mg of 500 mg of 17-OHPC as a weekly single injection. The safety of the 500 mg dose will also be assessed.

Detailed description:
The study will determine the association between plasma concentrations of 17-OHPC (hydroxyprogesterone caproate) and the rate of preterm birth and will evaluate the impact of several potential covariates on plasma concentrations of 17-OHPC and its efficacy. 17-OHPC (hydroxyprogesterone caproate) administration has proven effective in reducing preterm births in high risk groups but the current dose of 250mg administered intramuscular (IM ) is thought to be an inadequate for a substantial portion of women receiving the therapy. The potential benefit of identifying a therapeutic concentration range and of optimizing the dosage of 17-OHPC are substantial.

One cohort of pregnant subjects with a history of a prior spontaneous preterm birth with be randomized to either the 250mg or 500mg weekly intramuscular injections. All subjects will have trough blood samples collected immediately prior to their second injection of the 17-OHPC, at 26-30 weeks (but only after a minimum of 7 injections have been administered) , 6-9 weeks later and at the time of delivery. Another tube of maternal blood will be collected during one of the scheduled blood samples for genotyping. A cord blood specimen will also be collected and with consent, a cord blood specimen will be collected for genetic studies of the infant. Investigators will also collect a small sample of the placenta after delivery.

In order to enhance sample size for this trial, investigators will also enroll a second cohort of subjects (ancillary cohort) who are not in the randomized clinical trial (RCT) described above. Women already receiving 250 mg 17-OHPC weekly from their healthcare provider as part of their standard of care will be approached prior to 26 weeks gestation. This will be an observational cohort and subjects enrolled in the ancillary cohort will not be randomized, as they are already receiving the 250 mg dose. Research staff will not administer the study drug to subjects enrolled in the ancillary cohort. These subjects will be asked to provide two blood samples: one at 26-30 weeks and one 6-9 weeks later, which will be utilized to address the primary objective of the study.

In response to the addition of the ancillary cohort, randomization for subjects in the RCT will be 2:1 for the 500 mg vs the 250 mg dose. The ancillary study will be implemented initially at the UPITT site only but may be expanded to other sites, as required.

Arms: arm 1: For safety assessment of the 500 mg dose, subjects will be randomized to the 500 mg dose of 17-OHPC. arm 2: For safety assessment of the 500 mg dose, subjects will be randomized to the 250mg dose of 17-OHPC. arm 3: Receiving 250 mg as part of routine care
Interventions: intervention 1: 17-Hydroxyprogesterone caproate 250mg or 500 mg. intervention 2: Safety study of 500 mg dose.
Intervention descriptions: intervention 1: For the pharmacodynamic study, subjects receiving either 250mg or 500 mg dose will be studied to relate the plasma concentration at 26-30 to the rate of sPTB. For the safety study, subjects will be randomized to either the 250mg or 500 mg dose. intervention 2: Subjects randomized to 250 mg or 500 mg dose

Protocol documents:
Prot_SAP_019.pdf: 
1 
 
 7-15-2020 
 
Relationship between Plasma Concentration of 17-hydroxyprogesterone caproate (17-
OHPC) and Preterm Birth 
Protocol version 3.4 
NCT 03292731 
 
Obstetric-Fetal Pharmacology Research Centers (OPRC) Network 
The Eunice Kennedy Shriver National Institute of Child Health and Human Development 
(NICHD) 
 
 
 
 
 
 
 
Principal Investigator:  
Steve N. Caritis, MD 
Magee-Womens Hospital 
300 Halket Street Room 2229 
Pittsburgh, PA 15213 
 
 
 
 
 
 


2 
 
Revision History: 
 
Version/Protocol 
Date 
Revision Description 
Version 2.3 
20 Aug 2017 
Original version approved by UPITT IRB 
Version 2.4 
11 Oct 2017 
- Added the option for subjects assigned to the 500 mg dose to either receive one 2 ml 
injection (500 mg) or two 1 ml injections (250 mg each) administered at the same time 
- Clarified that screening can occur up to 20 6/7 weeks and that the latest gestational age to 
start treatment is 21 6/7 weeks 
- Added information regarding randomization procedures 
- Added information for removing subjects from the study or stopping treatment, as well as 
under what scenarios subjects removed from the study will be replaced 
- Added statement that data from subjects removed from the study will not be used for 
analyses related to plasma 17-OHPC concentration 
- Added SAE reporting requirements 
- Added clarification to the patient management section regarding study non-compliance 
- Other minor changes were made to improve clarity and ensure consistency among all study 
documents. 
Version 2.5 
1 Dec 2017 
Addition of information relating to the use of the REDCap Database 
Revised storage of biological samples to -70° to -80° C. 
Version 2.6 
11 Jan 2018 
- Allow weekly 17-OHPC injections to be administered by research staff, physician's office 
staff, or a person selected by the subject who can administer the medication in the subject's 
home.    
- Research staff will monitor compliance through weekly discussion with the subject and 
empty vial counts.  
- Recruited subjects will be seen at MWH every 4-6 weeks to provide additional medication 
and to perform other aspects of the study. 
- An additional blood sample will be collected at 6-9 weeks after the 26-30 week specimen. 
Version 2.7 
26 Feb 2018 
- Corrected the size of the placenta sample that will be collected after delivery from 3x3 inch 
to 3x3 cm.  
- Clarification that the person administering home injections will attend a study visit 
conducted prior to the start of home injections for training 
Version 2.8 
2 Apr 2018 
- Section C.2: Enrollment may differ substantially between centers 
- Additional subsections were added to C.7: criteria for stopping treatment, non-compliant 
subjects, and subjects that withdraw from the study.  
- The former section C.9 "Criteria for Removing Subjects from the Study or Stopping 
Treatment" was deleted, as the content from this section is part of Section C.7 
Version 2.9 
7 May 2018 
- Section E.3: The assay for measuring 17-OHPC is being validated to measure lower 
concentrations and will be performed using plasma and whole blood samples.  
Note: Analysis of 17-OHPC will be performed using plasma and whole blood samples at 
the Magee-Womens Hospital site. The other sites will collect plasma samples only. 
- Section C.2: Added secondary research question: Does the 17-OHPC concentration in 
maternal plasma or whole blood correlate better with certain physiological parameters? 
- Section D.1: Added secondary outcome: The correlation of progesterone and 17-
hydroxyprogesterone in maternal plasma and whole blood to clinical outcomes 
 
 


3 
 
Version 3.0 
29 Aug 2018 
- Section C.6.i: study drug “may be administered by...physician’s office staff or other qualified 
medical personnel or a person selected by the subject who can be trained to administer the 
medication in the subject’s home.”  
- Section C.6.iii: examination of the injection sites during the in-person study visits will be 
performed as needed 
- Section C.2: revised the last bullet under secondary research questions to replace “certain 
physiological parameters” with “certain pharmacological and clinical outcomes”.  
- Section D.1: revised the last bullet under secondary outcomes to replace “clinical 
outcomes” with “pharmacological and clinical outcomes”.  
- Exclusion Criteria: added current or history of thrombosis, liver tumors, liver disease/failure, 
renal disease/failure, breast cancer, other hormone sensitive cancers, or a history of these 
conditions, undiagnosed vaginal bleeding unrelated to pregnancy, moderately severe 
depression (PHQ-9 score of ≥ 15 or suicidal ideation) 
- Section C.6.iii: Added screening tests (PHQ-2 and PHQ-9) for depression 
- Section C.7.i: added uncontrolled hypertension and suicidal ideation to the criteria for 
stopping treatment 
Version 3.1 
06 Nov 2018 
- Removed the list of Steering Committee members from the cover page 
- Section C.6.i: subject will be given 6-9 weeks’ worth of medication to take home with her 
until the next visit with research staff  
- Section C.6.iii: frequency of in-person study visits changed to 6-9 weeks 
- Section C.6.v: added clarification that delivery samples will only be collected if possible 
- Section C.7.i: removed subjects with hypertension requiring 2 or more agents as criteria for 
stopping treatment 
- Section E.1: we will save an aliquot from each lot of the study drug rather than from each 
patient 
Version 3.2 
31 July 2019 
- Section C.3.1: Added an EPDS score of >13 to the exclusion criteria 
- Section C.6.iii: Added clarification to depression screening and added EPDS as a test for 
depression 
- Section C.6.v: The first maternal blood sample will be drawn prior to the 2nd injection, 
ideally on day 7 from the first injection. Added clarification that the third maternal blood 
sample must be drawn no later than 37 weeks 
Version 3.3 
3 Jan 2020 
- Introduction, Rationale, Sections C.2, C.3, C.5, and C.6 were revised to include the addition 
of the Ancillary Cohort.  
- Introduction: Added information related to the FDA Advisory Committee decision 
- Study Design: Added the University of Utah and the University of Texas – Houston as 
additional study sites 
Version 3.3 
16 April 2020 
- Correction to EPDS question 10: a score of ≥1 indicates suicide risk 
Version 3.4 
15 July 2020 
- Section C.6.1 was revised to allow subjects in the ancillary cohort to receive the Makena 
study drug 
 
 
 


4 
 
 
 
Table of Contents 
B.  Background ....................................................................................................................................................................................... 7 
B.1. Significance of Preterm Birth ............................................................................................................................................ 7 
B.2. Prevention of PTB with 17-OHPC ................................................................................................................................. 7 
B.3. Issues with 17-OHPC for PTB Prevention ................................................................................................................ 7 
B.4. Pharmacology of 17-OHPC .............................................................................................................................................. 9 
B.5. Preliminary Data Relevant to the Primary Outcome .................................................................................. 11 
B.6. Rationale for Proposed Study ....................................................................................................................................... 13 
C. Study Design .................................................................................................................................................................................. 19 
C.1. Infrastructure ......................................................................................................................................................................... 19 
C.2.   Primary and Secondary Objectives ........................................................................................................................ 19 
C.3. Screening Procedures ...................................................................................................................................................... 20 
C.3.i. Eligibility Criteria ........................................................................................................................................................... 20 
a. RCT Eligibility Criteria ............................................................................................................................................................ 20 
b. Ancillary Cohort Eligibility Criteria ................................................................................................................................... 21 
C.3.ii. Gestational Age Determination ..................................................................................................................... 22 
C.4. Informed Consent Procedures and Documentation ......................................................................................... 22 
C.5. Randomization Procedures ........................................................................................................................................... 23 
C.6. Study Procedures ............................................................................................................................................................... 23 
C.6.i. Study Drug Dispensation ......................................................................................................................................... 23 
C.6.ii. Randomization and Baseline ................................................................................................................................ 24 
C.6.iii. Study Visits ................................................................................................................................................................... 25 
C.6.iv. Delivery ........................................................................................................................................................................... 27 
C.6.v. Specimen Collection ................................................................................................................................................. 27 
a. RCT Specimen Collection ................................................................................................................................................... 27 
b. Ancillary Cohort Specimen Collection ........................................................................................................................... 28 
C.7. Patient Management ....................................................................................................................................................... 28 
C.7.i Criteria for Stopping Treatment ............................................................................................................................. 28 
C.7.ii Non-Compliant Subjects .......................................................................................................................................... 28 
C.7.iii Subjects that Withdraw from the Study ........................................................................................................... 29 
C.8. Study Halting Criteria ......................................................................................................................................................... 29 
C.9. Medical monitor and Logistic Coordinating Core ................................................................................................. 31 


5 
 
D. Statistical Analysis and Sample Size Justification ...................................................................................................... 31 
D.1. Primary and Secondary Outcomes ......................................................................................................................... 31 
D.2. Sample Size and Power .................................................................................................................................................. 33 
E. Data Management and Quality Assurance ..................................................................................................................... 34 
E.1. Investigational Product Supply ..................................................................................................................................... 34 
E.2. Clinical Data Management ............................................................................................................................................. 35 
E.3. Laboratory Test Results ................................................................................................................................................... 35 
F. References ....................................................................................................................................................................................... 37 
 
 
 


6 
 
A.  Introduction 
Preterm birth (PTB) remains one of the most important problems in obstetrics today, 
affecting more than 530,000 babies each year in the United States. PTB is the leading 
cause of newborn death and a major cause of lifelong disability. A recent report by the 
Institute of Medicine estimated that the annual societal economic burden associated with 
PTB in the United States to be at least $26.2 billion per year, or $51,600 per infant born 
preterm. A single preterm birth is associated with a recurrence rate of 14-22%, two prior preterm 
births with a recurrence of 28-42% and women with more than two prior preterm births face an 
astounding 67% risk of having another preterm birth. Over the last five decades attempts to 
reduce the PTB rate have failed despite a wide variety of approaches. Recently, however, 
administration of intramuscular 17-hydroxyprogesterone caproate (17-OHPC) has proven 
effective in reducing the recurrence of preterm births in this high-risk group. Currently, the 
medication is offered to all women with a prior spontaneous PTB. A dose of 250 mg is 
administered intramuscularly weekly from weeks 16-21 until week 36 of gestation. This 
dosing regimen does not have a sound pharmacologic basis. Recent data on the 
pharmacology of 17-OHPC suggest that plasma concentration is closely related to the efficacy 
of this treatment. As many as half the women receiving this medication achieve an inadequate 
plasma concentration with the standard 250 mg weekly dose and may not benefit from this 
treatment. 
 
In this proposed study, we will determine the relationship between plasma concentrations of 
17-OHPC and the rate of preterm birth (PTB). The study is a randomized open label study of 
pregnant women with one or more prior spontaneous preterm births. Subjects will be 
randomized to a weekly single injection of either 250 or 500 mg 17-OHPC. The randomization 
will be 2:1 for the 500mg vs the 250 mg dose. The recruited cohort of women will be sufficiently 
large to describe the association between plasma concentration of 17-OHPC and the rate of 
preterm birth and to evaluate the impact of several potential covariates on plasma 
concentration of 17-OHPC and its efficacy.  
Primary Objective: To assess the relationship between the plasma concentration of  
17-OHPC and rates of preterm (<37 weeks) birth.  


7 
 
B.  Background   
B.1. Significance of Preterm Birth 
Preterm birth (PTB) remains one of the most important problems in obstetrics today, 
affecting more than 530,000 babies each year in the United States.
1 PTB is the leading 
cause of newborn death and a major cause of lifelong disability. A recent report by the 
Institute of Medicine estimated that the annual societal economic burden associated with 
PTB in the United States to be at least $26.2 billion per year, or $51,600 per infant born 
preterm.
2 A single preterm birth is associated with a recurrence rate of 14-22%, two prior 
preterm births with a recurrence of 28-42% and women with more than two prior preterm births 
face an astounding 67% risk of having another preterm birth.
3    
B.2. Prevention of PTB with 17-OHPC 
Over the last five decades attempts to reduce the PTB rate have failed despite a wide variety 
of 
approaches. 
Recently, 
however, 
administration 
of 
intramuscular  
17-hydroxyprogesterone caproate (17-OHPC) has proven effective in reducing preterm births 
in this high-risk group.
4 The target population of 17-OHPC is the woman with a history of 
one or more prior spontaneous preterm births. A study from the Maternal-Fetal Medicine Units 
(MFMU) Network
4 demonstrated a 33% reduction in preterm birth among women with a prior 
spontaneous PTB who were treated with (17-OHPC) as opposed to placebo. The American 
College of Obstetricians and Gynecologists (ACOG) supports use of 17-OHPC to reduce the 
rates of PTB in women with a prior spontaneous PTB.5 Most insurers endorse the use of 17- 
OHPC as they see a benefit in controlling rising health care costs associated with preterm 
delivery. It has been estimated that universal use of 17-OHPC in at risk women would reduce 
the societal cost of prematurity by $2 billion annually
6,7and may actually reduce prematurity 
rates.
8,9   
B.3. Issues with 17-OHPC for PTB Prevention 
Currently, 17-OHPC is offered to all women with a prior spontaneous PTB, which accounts 
for roughly 133,000 women annually.
10 A dose of 250 mg is administered intramuscularly 
weekly from weeks 16-21 until week 36 of gestation. This dosing regimen does not have a 
sound pharmacologic basis and is based on a meta-analysis
11 and a single clinical trial
12 


8 
 
suggesting benefit but no dose-ranging studies or concentration controlled trials on the safety 
or efficacy of 17-OHPC have been performed to date. Considerable skepticism about the 
efficacy 
and 
safety 
of  
17-OHPC exists despite the drug’s approval by the FDA. Skepticism regarding the findings in 
the Meis trial stem from the unexpectedly high rate of preterm birth in the placebo group (52%) 
compared with the 17-OHPC treated group (37%).
4, 11
 In other studies of women having a prior 
preterm birth, the rate of recurrent preterm birth in untreated women has been closer to the 
37% range. As part of the conditional FDA approval for Makena, the sponsor was asked to 
complete another randomized placebo-controlled trial.  That study was recently completed 
and the results were reviewed by the FDA-Advisory Committee. There was no benefit of 17-
OHPC over placebo in that study. The FDA Advisory Committee on October 29, 2019 voted 9 
to 7 to withdraw approval of Makena (17-hydroxyprogesterone caproate, 17-OHPC). The FDA 
will decide on whether or not to accept the Advisory Committee’s recommendation; the close 
vote suggests that the FDA choice is far from clear. Furthermore, there are several issues 
related to the trial that confound the issue of 17-OHPC efficacy. The Prolong study had difficulty 
recruiting in the United States since 17-OHPC had been considered ‘standard of care’ for all 
women with a singleton pregnancy and a prior PTB. Consequently, most subjects were 
recruited overseas primarily in Russia and the Ukraine. The sample size was large (1708 
subjects) but the primary outcome (delivery<35 weeks) was uncommon occurring in only 12% 
of the subjects and rates were similar in both treatment groups. The populations studied were 
very different in the MFMU study which showed a benefit and the Prolong study which did not. 
In the MFMU study 59% of enrollees were African American vs 7% in the Prolong trial. Also 
risk factors for PTB such as smoking, >1 prior preterm birth and other PTB risk factors were 
greater in the MFMU trial , 20 vs 8%, 32 vs 12%, 91 vs 48%, respectively. Importantly, no 
significant harms from 17-OHPC were identified. 
      The challenge faced by the FDA as well as providers of care is how to integrate the Prolong 
data into clinical care for women with a prior preterm birth. Currently, both the Society for 
Maternal-Fetal Medicine (SMFM) and the American College of Obstetricians and Gynecologist 
(ACOG) support continuing use of 17-OHPC for women with a prior PTB. The perspective is 
that the Prolong trial does not negate the findings of the MFMU (Meis) trial. Those skeptical of 
the effectiveness of 17-OHPC have suggested using vaginal progesterone in women with a 
prior spontaneous PTB despite the fact that  the  largest  placebo  controlled  randomized  


9 
 
control trial demonstrated  that  vaginal  progesterone  was  ineffective  in  women  with  a  
prior  PTB.13 This suggestion, however, is appealing to some and limits enthusiasm for a large 
clinical trial of 17-OHPC. 
The findings from our current trial may provide important information on the effectiveness of 
17-OHPC. In particular, if there is a relationship between 17-OHPC concentration and the rate 
of PTB, a higher dose may be needed. The failure of the Prolong trial and the relatively low risk 
reduction (33%) in the Meis trial may be due to an inadequate dose. Our recent data, which 
are summarized below, suggest that the 250 mg dose may be inadequate in a substantial 
portion of women receiving this therapy.
14 From a safety point of view, doses as high as 
1000 mg weekly have been used in pregnant women without any apparent adverse effects; 
this provides substantial latitude in dosing.
15-17  
B.4. Pharmacology of 17-OHPC 
Until recently, the only pharmacological data published on 17-OHPC were obtained in non-
pregnant women who were receiving the medication for treatment of uterine cancer.
18 The 
doses used varied widely and as much as 1000 mg was administered daily for 5 days. 
The dosing regimen used in non-pregnant women with uterine cancer is not applicable to 
pregnant women and cannot be used as a justification for the use of 250 mg weekly for 
prevention of preterm delivery. In the MFMU study reported by Meis et al, blood was not 
collected.
4 Therefore, there was no way to evaluate the relationship between plasma 
concentrations of 17-OHPC and the outcome of interest, the rate of PTB. We have utilized 
one small and two large clinical cohorts to evaluate the pharmacokinetics and 
pharmacodynamics of 17-OHPC.
14,19-21 The terminal half-life of 17-OHPC administered 
intramuscularly was estimated to be around 16 days in women with a singleton gestation. There 
was a good correlation (r = 0.83) between the trough plasma concentrations of 17-OHPC and 
the area under the plasma concentration vs time curve (AUC), a measure of patient exposure 
to the drug at steady state. In all cohorts, a fixed dose of 250 mg resulted in a large 
variation in the trough plasma concentrations of 17-OHPC suggesting that there was a large 
variation in drug exposure in these patients. At least part of the observed variability in the 
trough plasma concentrations of 17-OHPC in these patients may be attributed to maternal 
BMI with heavier women having lower plasma concentrations. Variation in the expression and 
activity of the CYP3A4/5 enzyme that is primarily responsible for the metabolism of 17-OHPC 


10 
 
does not appear to impact plasma concentrations (unpublished data). We have analyzed data 
from two large multicenter trials from the Maternal-Fetal Medicine Units Network (STTARS and 
Omega-3 trials) and one multicenter study from the Obstetrical-Fetal Pharmacology Research 
Units Network (Caproate PK study).
14,19-21 Two of these were performed in women with 
singleton gestation and one study was performed in women with twin gestation. The twin 
study performed in the MFMU Network (STTARS Trial) was the first study of 17-OHPC in 
pregnant women. Both pharmacokinetic and a pharmacodynamic studies were performed in 
women with twin gestation.
19,20 More relevant to the present proposal are the two studies 
performed in women with singleton gestation.
14,21 In the OPRU Caproate PK study21, 
we defined the pharmacokinetic characteristics of 17-OHPC in a small (n=61) cohort of 
women with singleton gestation and a prior preterm birth. These women received weekly 
injections of 17-OHPC and underwent 2 
pharmacokinetic studies at 20 0/7 to 24 6/7 
weeks' gestation (PK1) and again at 31 0/7 to 
34 
6/7 weeks' gestation (PK 2). Figure 1 depicts 
the 
mean and SD of 17-OHPC concentrations in 
these women. Concentrations of 17-OHPC were 
higher during PK 2 than during PK 1. In 18 
women, blood samples were obtained over a 
28-
day period beyond the last injection (extended 
study, Figure 2).
21 Maternal and/or cord blood 
were obtained at delivery. The half-life (median 
±SD) of 17-OHPC was 16.2 ± 6 days. Body 
mass 
index 
affected 
maternal 
17-OHPC 
concentrations. The trough plasma concentration 
of 
17-OHPC was a good surrogate marker of drug 
exposure AUC (r=0.83). The cord to maternal 17-
OHPC concentration ratios averaged 0.2; 17-
OHPC was detectible in cord plasma 44 days 
after the last maternal injection. The trough plasma concentration of 17-OHPC was a good 
surrogate marker of drug exposure AUC (r=0.83). This study also showed  


11 
 
a large variation in the trough plasma concentrations of 17-OHPC in subjects receiving  
a fixed 250 mg weekly dose. Some of that variation was attributable to maternal BMI (Figure 
2) which seems to account for < 30% of the variability in plasma concentrations when POPPK 
analysis is performed.  
 
We have also studied the pharmacology of 17-OHPC in the laboratory. Using human liver 
microsomes, 
hepatocytes 
and 
expressed 
enzymes 
we 
have 
demonstrated 
that  
17-OHPC is primarily metabolized by the CYP3A enzymes.
22 Using adult and fetal hepatocytes 
we have shown that CYP3A4, CYP3A5 and CYP3A7 are capable of metabolizing 17-OHPC.
23 
Using human liver microsomes we have recently shown that the metabolism of 17-OHPC is 
inhibited by progesterone
24 and several other drugs commonly used in pregnancy. Fourteen 
co-medications listed below were  identified  as  inhibitors  (>30%  inhibition)  of  the  
metabolism  of  17-OHPC:  ketoconazole, fluconazole, itraconazole, voriconazole, nelfinavir, 
ritonavir, esomeprazole, montelukast, bergamottin, sertraline, trazodone, haloperidol, 
tacrolimus, and fluticasone.
25 We have also demonstrated that transporters are involved in the 
disposition of 17-OHPC which is transported into and out of hepatocytes by an active efflux 
process which is significantly inhibited by cold temperature, cyclosporine, verapamil and 
rifampin.
23  The active efflux mechanism was observed in both adult and fetal hepatocytes.  
 
B.5. Preliminary Data Relevant to the Primary Outcome 
The overall efficacy of 17-OHPC (33% reduction in preterm birth rates), might be improved 
if a pharmacologically-based dosing regimen was utilized rather than a single dose “one 
fits 
all” 


12 
 
strategy. To better understand the relationship between drug exposure and outcome, we 
have recently performed a secondary analysis of a cohort of women with a singleton 
gestation and a prior PTB using data obtained in the Omega-3 study from the MFMU 
Network.
14  
In this study, a single blood sample was obtained at 24-28 weeks’ gestation in 331 
subjects who received weekly injections of 250 mg 17-OHPC. The trough plasma 
concentration of 17-OHPC in this cohort of women varied widely, ranging from  
4-56 ng/ml (Figure 3) with a median value of 9.9 ng/ml. The rate of preterm birth was 
significantly associated with the plasma 17-OHPC concentration with the highest rates seen 
in women with the lowest quartile of plasma concentrations (Figure 4). The significant 
relationship between quartiles of the plasma concentrations and the outcome of preterm 
birth persisted after adjustment for potential confounders such as body mass index (BMI), 
gestational age at study entrance, gestational age at blood sampling, race and ethnicity (Figure 
5). The apparent plateauing effect of efficacy with concentrations above 10 ng/ml (Figure 4) 
could be due to a true plateau effect on preterm birth rates or alternatively it may be due to the 
small sample size at the higher concentrations. To address this issue, we examined the 
relationship between the rate of preterm birth and plasma concentrations of 17-OHPC 
expressed as a continuous variable rather than as quartiles.  
After log transformation of the concentration data we demonstrated a significant relationship 
(p<0.04) between the outcome of preterm birth and plasma concentrations of 17-OHPC; the 
hazard ratio for preterm birth decreased by 0.49 for every unit increase in the log of the 17-
OHPC concentration (Figure 6). This relationship is linear and demonstrates that the rate of 
PTB does not appear to plateau as suggested in Figure 4. In the secondary analysis of the 
Omega-3 study, women with plasma concentrations of 17-OHPC >10 ng/ml had a rate of 
preterm birth of 25% while women with concentrations < 10ng/ml had a preterm birth rate 
Figure 6.  Relationship between Plasma 17-OHPC and PTB  


13 
 
of 37% (Figure 4). Importantly, only 50% of the women in this study achieved plasma 
concentrations > 10 ng/ml and only 5% of subjects achieved concentrations greater than 20 
ng/ml (Figure 3). The median concentration of 17-OHPC at the highest quartile was only  
15 ng/ml (Figure 4). These data suggest that that the rate of preterm birth could be reduced 
further with higher plasma concentrations of 17-OHPC. The higher concentrations could impact 
two aspects of 17-OHPC efficacy. First, the percentage of women below what appears to be 
the lowest level of efficacy, i.e., 10 ng/ml would be increased with a higher dose. Secondly, the 
apparent plateauing effect may disappear with more concentrations in excess of 20 ng/ml. If 
indeed there is no plateauing of efficacy at higher concentrations, then the overall success rate 
could be increased beyond the reported 33% overall efficacy of 17-OHPC.  This study provided 
proof of the efficacy of 17-OHPC because the pharmacodynamic end point, PTB was 
dependent on plasma 17-OHPC concentrations. This evidence addresses those skeptics of 
the utility of 17-OHPC in preventing recurrent preterm birth.  
 
Using data from the Omega-3 study, we are able to model 17-OHPC pharmacokinetics. 
Figure 7 depicts plasma concentrations expected from both a 250 and 500 mg weekly dose of 
17-OHPC. With the higher doses of OHPC in the proposed study, we anticipate that plasma 
concentrations will range from ~ 4 -110 ng/ml with a median concentration of 10 ng/ml with the 
250 mg dose and 20 ng/ml with the 500 mg dose based on these simulated data.  
 
B.6. 
Rationale for Proposed Study 
Clearly, research that evaluates the dose/ concentration relationship between 17-OHPC and 
PTB rates is urgently needed. Such a study should correlate a measure of drug exposure such 
as steady state trough plasma concentrations with the outcome of preterm birth. The potential 
benefit of identifying a therapeutic concentration range and of optimizing the dosage of 17-


14 
 
OHPC are substantial but several challenges exist for such investigations. The first challenge 
relates to the fact that animal models cannot be used to identify a therapeutic concentration 
range of 17-OHPC for several reasons. In all animals, other than monkeys and guinea pigs, 
progesterone decreases dramatically prior to birth.
26 Only in humans, monkeys and guinea 
pigs is plasma progesterone withdrawal not an antecedent to parturition. However, neither the 
monkey nor guinea pig is a good model for human preterm labor since in the human, preterm 
birth is multifactorial and in many cases related to infection/inflammation, circumstances not 
typical in these animals. This challenge will be met by performing the study in humans. 
A second challenge to establishing a therapeutic concentration and optimizing the dosage of 
17-OHPC is the lack of availability of 17-OHPC at a higher concentration than the standard 
250 mg/ml concentration that is currently used. The FDA-approved product, Makena, is 
available but only at a concentration of 250 mg/ml and this product costs anywhere from $900- 
$1600 per dose.  We considered several options for administering a 500 mg weekly dose of 
17-OHPC using the 250 mg Makena product including: twice weekly injection of 1 ml of the 250 
mg product, administering 2 injections of 250 mg Makena at one time or administering a single 
injection of 2ml of Makena for a total of 500 mg. All of these options would be very costly as 
insurance would not necessarily cover the 250 mg dose at all centers and none would cover the 
500 mg dose. We explored the option of utilizing a compounding pharmacy to prepare both a 
250 mg/ml and a more concentrated 500 mg/1.4 ml product. We also considered utilizing the 
compounding pharmacy to supply only the more concentrated solution for those subjects 
assigned to the 500 mg weekly dose while those assigned to the 250 mg dose would use 
whatever product is used at their respective institutions. Use of a compounding pharmacy 
requires one that is appropriately qualified based on FDA requirements. We did identify such a 
pharmacy in Tennessee that prepared 17-OHPC for one of the MFMU studies. We have been 
unable however, to obtain the 17-OHPC powder from a qualified distributor in the United States 
making the compounding option moot. We initiated discussions with AMAG Pharmaceuticals 
the distributor of Makena and they have agreed to provide us the Makena product free of charge 
for the entire study. Women assigned to the 250 mg/week group will receive a 1 ml injection 
(250 mg) weekly while women assigned to the 500 mg/week group will receive either a single 2 
ml weekly injection or two 1 ml weekly injections administered at the same time. The option of 
using the Makena product is by far the best for this RCT as all participants will get the same 
FDA-approved product, the study leadership will not need to negotiate with a 17-OHPC supplier 


15 
 
or a compounding pharmacy. The primary disadvantage is that women randomized to the 
500mg injection rather than the standard 250 mg injection may experience more discomfort. We 
are somewhat encouraged by our reproductive endocrinology colleagues who typically 
administer 2 ml progesterone in oil on a daily basis to women undergoing Assisted 
Reproductive Technologies. Thus, the second challenge, the availability of a 250 and 500 mg 
product, has been met. 
 
A third challenge to further explorations regarding optimization of the dose of 17-OHPC relates 
to the challenges in conducting a large clinical trial in pregnant women. A large sample size is 
needed to demonstrate significant improvement in preterm birth rates with a different dose of 
17-OHPC since the current dose of 250 mg of 17-OHPC appears to reduce preterm birth rates 
by 33%. A large sample size is also required because several maternal characteristics may 
affect plasma concentrations of 17-OHPC
14,19-21
 and several single nucleotide polymorphisms 
of the progesterone receptor gene appear to impact the drug’s efficacy both positively and 
negatively.
27
 This challenge is met by the participation of 5 centers with large populations of 
women receiving 17-OHPC. All three centers have participated in multiple trials of 17-OHPC by 
the MFMU. 
 
In order to enhance sample size for this trial, we will enroll a separate cohort of subjects 
(ancillary cohort) who are not in the parent trial described above.  Women already receiving 
250 mg 17-OHPC weekly based on their clinical history will be approached prior to 26 weeks 
gestation. Eligible subjects either refused the Randomized Controlled Trial (RCT) or they were 
never approached for the trial. These subjects will be asked to provide two blood samples: one 
at 26-30 weeks and one 6-9 weeks later to assure steady state. This ancillary study will be 
implemented initially at the UPITT site only but may be expanded to other sites, as required.  
Thus, the current design appears to address the challenges described above. Given the 
observation reported here from the Omega-3 study, it appears that many women receiving 17-
OHPC are inadequately treated.  It is therefore imperative that this study be performed to 
establish the relationship between plasma 17-OHPC concentration and the rate of PTB and to 
determine if higher plasma concentrations will reduce the risk of PTB to a greater degree than 
reported in the Meis trial. An understanding of the relationship between plasma concentrations 
of 17-OHPC and PTB rates will lead to an optimized dosing regimen that will benefit a higher 


16 
 
percentage of patients who would otherwise deliver babies prematurely. Preterm birth rate is 
the only acceptable outcome of interest in assessing the optimal dosing regimen of 17-OHPC 
as no surrogate biomarker for PTB has been identified at this point. Alternative methods for 
addressing this problem do not exist.  
 
The findings of the proposed study will have a dramatic impact on clinical practice and will 
energize the research community to further explore the potential mechanism of action and 
target organ(s) of this therapy. Demonstration that the rate of PTB is related to plasma 17-
OHPC concentration will lead to a different dosing regimen for women with a prior preterm 
birth. This would impact not only practice in the United States but throughout the world as 
treatment with 17-OHPC is internationally embraced. Positive findings from this project will 
have a substantial public health impact. Approximately 133,000 pregnant women annually with 
a history of a prior preterm birth would be eligible for this treatment.10 If the preterm birth rate 
is reduced even 10% from its current rate with the optimized dosing regimen the impact would 
be substantial. In dollar terms each preterm birth cost nearly $52,000. Preventing 10% of these 
13,000 preterm births will save $663 million annually. Incremental reductions in preterm birth 
would lead to comparable changes in financial savings. 
 
Some may suggest that the striking relationship between plasma 17-OHPC concentration and 
the rate of preterm birth we have reported from the MFMU Omega-3 trial provides sufficient 
proof to adopt a strategy of merely doubling the standard dose of 17-OHPC. We strongly 
disagree with such a strategy because all women in our study received Omega-3 
supplementation and the impact of this supplement on the metabolism and plasma 
concentration and efficacy of 17-OHPC is not completely clear. Furthermore, as a secondary 
analysis the Omega-3 study was not designed to address the relationship between plasma 
concentrations of 17-OHPC and the outcome of preterm birth. The Omega 3 trial utilized only 
weekly injections of 250 mg of 17-OHPC and therefore data from this trial cannot address the 
issues of what impact higher plasma concentrations would have on the PTB rate. Recently, 
another study evaluated the relationship between 17-OHPC concentrations and preterm birth and 
a concentration relationship with the rate of preterm birth could not be demonstrated 28. These 
findings emphasize the need for a prospective study that relates plasma 17-OHPC concentrations 
and PTB rates. 


17 
 
. 
 
The proposed research will shift both fundamental research and clinical practice paradigms. 
Paradigm is defined by Webster as “a model for something that may be copied or a group of 
ideas about how something should be done, or thought about”. The proposed study will clearly 
challenge two existing paradigms; one related to the regulation of parturition and the second 
related to the study of medications in pregnancy. The demonstration of a relationship 
between dose or concentration of 17-OHPC and the outcome of preterm birth will not only 
impact the clinical care of women at risk for preterm birth but will also impact the scientific 
community and the long held concepts of what regulates human parturition. Progesterone 
withdrawal preceding parturition is a characteristic of most mammalian species except humans, 
monkeys and guinea pigs.
26 In humans, progesterone concentrations increase as pregnancy 
progresses and do not fall prior to labor onset. Plasma progesterone concentrations are high 
and in excess of concentrations required to half saturate (EC50) the progesterone receptor. 
The suggestion that progesterone supplementation might delay human parturition, therefore, 
seems biologically improbable. However, several clinical trials have demonstrated a benefit 
with progesterone supplementation in certain conditions. A benefit is seen with 17-OHPC 
in women with prior preterm birth but not in women with twin or triplet gestation or a 
shortened cervix.
4,29-31 Vaginal progesterone appears to confer benefit to women with a 
shortened cervix but not to women with prior preterm birth.
13,32.33 Each of these clinical trials 
has been subject to criticism and skepticism abounds. For 17-OHPC, only a single large trial 
has shown benefit and in that trial the rate of preterm birth in the control group was much higher 
than expected leading some to suggest that the medication did not provide a benefit as the 
preterm birth rate in the 17-OHPC group was identical to the rate reported in other studies in 
women with a prior preterm birth and no treatment.
4,34 Vaginal progesterone appears to reduce 
preterm birth in women with a shortened cervix but this treatment failed to gain FDA approval 
because, in the opinion of the FDA, the study failed to demonstrate benefit in women residing 
in the United States. Thus, although clinical trials suggest that progesterone supplementation 
may impact the parturitional process, proof of concept is clouded. If the proposed study 
demonstrates a concentration relationship between 17-OHPC and preterm birth rates, 
then the role of progesterone as a regulator of human parturition will be established 


18 
 
beyond doubt.  This will be innovative because it will dramatically “change ideas about how 
something should be done, made, or thought about”. 
 
Likewise, demonstration that the pharmacologic characteristics of a medication can impact 
drug concentration and efficacy should set a new standard in Obstetric Pharmacology as 
to what evidence is required before medications are prescribed for pregnant women. The 
proposed study brings concepts to Obstetric Pharmacology that are not innovative by scientific 
standards in other disciplines but in dealing with pregnant women and their fetuses, Obstetrical 
Pharmacology is far less advanced. The FDA does not allow medications to be administered 
to non-pregnant individuals without knowledge of the proper dose or the risks and benefits 
of treatment. Efficacy studies are not undertaken without first establishing an optimal dosing 
regimen. These rules do not apply to medications used by pregnant women. Drug 
administration regimens for pregnant women are based on dosage regimens derived from men 
and non-pregnant women. Few of the medications taken by pregnant women have been 
studied adequately. At a time when the most precise information is needed to protect the 
pregnant woman and her baby, data are sparse and the patient and provider are uninformed 
about proper dosing regimens and the risks or benefits of any treatment. Too many 
pharmaceutical interventions in obstetrics have been tested and failed because of inadequate 
pharmacological assessment of the medication. Ritodrine was approved as a labor inhibition 
treatment but inadequate pharmacological data led to maternal deaths due to an excessive 
intravenous dosing regimen while an inadequate oral maintenance dose led to treatment 
failures.
35,36 The approach   taken in the proposed study  that  seeks  to  establish a relationship 
between drug concentrations and the outcome of preterm birth while exploring the impact of 
maternal covariates as modifiers of maternal:fetal drug  concentrations  and efficacy will  be 
innovative and, in fact, transformational in the field  of Obstetric Pharmacology. Knowledge of 
the relationship between maternal:fetal drug concentration ratios and metabolic activity 
should be fundamental to any drug administered to pregnant women. 
 
 


19 
 
 
C. Study Design 
C.1. Infrastructure 
The OPRC program is comprised of representatives from the NICHD (Bethesda, MD) and three 
Clinical Sites: Northwestern University (Chicago, IL), University of Texas Medical Branch 
(Galveston, TX) and Magee-Womens Hospital (Pittsburgh, PA). Two other sites, The 
University of Texas at Houston and the University of Utah, were added as additional sites; 
however, each site is conducting the study under separate INDs. The research is overseen by 
an internal Steering Committee, an external Data Monitoring Committee (DMC), the local 
Institutional Review Boards (IRBs) of each clinical site and the FDA. Details regarding the 
composition and responsibilities of the Steering Committee and OPRC locations are provided 
in the Network Policy Manual. Details regarding the composition and responsibilities of the 
DMC are provided in its charter.  
C.2.   Primary and Secondary Objectives 
We will recruit 300 pregnant women with one or more prior preterm births. Recruitment 
will occur at the three OPRC clinical sites and the two additional sites. Ideally, each center 
will enroll approximately 100 subjects; however, enrollment may differ substantially between 
centers. Enrolled subjects will be randomized in a 2:1 ratio to receive a single weekly injection of 
500 mg or 250 mg 17-OHPC. The 500 mg dose can be administered either as a single 2 ml weekly 
injection (500 mg) or two 1 ml (250 mg) weekly injections administered at the same time. Those 
assigned to the 250 mg weekly dose will receive 1 ml of 250 mg of 17-OHPC. 
The Primary Objective of this study is to determine whether the rate of preterm birth is related 
to the plasma concentration of 17-OHPC. 
Secondary Research Questions include the following: 
1. What is the impact of race or BMI on plasma 17-OHPC concentrations? 
2. Are maternal side effects increased at higher doses or higher plasma concentrations of 
17-OHPC? 
3. Is there an increase in adverse fetal effects from a higher dose or higher concentrations 
of 17-OHPC? 
4. What is the relationship between dose of 17-OHPC and plasma concentrations?  


20 
 
5. Do single nucleotide polymorphisms in the maternal progesterone receptor,  
P-glycoprotein affect efficacy or the placental transfer of 17-OHPC? 
6. Is efficacy of 17-OHPC affected by the gestational age at therapy initiation, the number of 
prior PTBs or the gestational age at delivery of prior PTBs? 
7. What is the relationship between cord blood concentrations of 17-OHPC and maternal 
plasma concentrations? 
8. Does the 17-OHPC concentration in maternal plasma or whole blood correlate better with 
certain pharmacological and clinical outcomes?  
Subjects in the ancillary cohort will already be receiving 250 mg 17-OHPC weekly as part of 
their standard of care, based on their clinical history. The primary objective in the ancillary 
cohort is identical to the RCT.  
The secondary research questions are similar to the RCT except for numbers 2, 3, and 7 
above. Data will be collected from subjects in the ancillary cohort, but will not be utilized for 
secondary research questions 2 and 3 above. These research questions cannot be adequately 
addressed because subjects in the ancillary cohort are not randomized.  Research questions 
5 (cord blood only) and 7 cannot be addressed because biological fluids will not collected at 
delivery in the ancillary cohort. 
C.3. Screening Procedures 
All patients who present for prenatal care before 20 6/7 weeks are eligible for screening. The 
latest gestational age to start treatment in the RCT is 21 6/7 weeks. Subjects who either refuse 
the RCT or are not approached may be eligible for participation in the ancillary cohort. The 
inclusion/exclusion criteria will be reviewed with the patient’s chart. Potentially eligible subjects 
will be told about the study and asked to sign a medical records release so that the medical 
records of her previous deliveries may be obtained. Documentation of the previous deliveries 
will be reviewed to ensure the patient meets the inclusion criteria. If a patient appears to meet 
the criteria for enrollment and expresses interest in the study, she will be told about the 
study and asked to sign the informed consent form. A copy of the consent form will be provided 
to the patient. 
C.3.i. Eligibility Criteria 
a. RCT Eligibility Criteria 
Inclusion Criteria - Subjects in the RCT must meet all of the following: 


21 
 
1.  Pregnant female with documented prior birth between 16 0/7- 35 6/7 week gestation from 
spontaneous preterm labor or preterm premature rupture of membranes 
2.  Gestational age (GA) < 22 weeks, based on study determined GA (as treatment must start 
between 16 0/7 and 21 6/7 weeks) 
3.   Singleton gestation 
4.   Age between 18 – 45 years  
5.   Able to give informed consent and undergo study procedures 
 
Exclusion Criteria - Subjects in the RCT must not meet any of the following: 
1.   Plan for cerclage 
2.   Plan for progesterone treatment other than study medication 
3.  Known major fetal anomaly or chromosomal anomalies that might affect gestational age at 
delivery 
4.  Malformation of uterus (uterine didelphus, septate uterus or bicornuate uterus) or known 
cervical length <2.5 cm 
5.  Participation in another trial that may affect gestational age at delivery  
6.   Planned delivery at other institution where pregnancy outcome data cannot be obtained 
7.  Medical or obstetrical complication that might affect gestational age at delivery, such as 
active ulcerative colitis, liver tumors, liver disease/failure, renal disease/failure, 
undiagnosed vaginal bleeding unrelated to pregnancy, or hypertension requiring 2 or more 
agents 
8.  Current or history of thrombosis or thromboembolic disorders 
9.  Known or suspected breast cancer, other hormone-sensitive cancer, or a history of these 
conditions 
10. Moderately severe depression (PHQ-9 score ≥15, EPDS score of >13, or suicidal ideation) 
b. Ancillary Cohort Eligibility Criteria 
Inclusion Criteria - Subjects in the ancillary cohort must meet all of the following: 
1.  Pregnant female with documented prior birth between 16 0/7- 35 6/7 week gestation from 
spontaneous preterm labor or preterm premature rupture of membranes 
2.  Receiving 250 mg 17-OHPC weekly- must be compliant with that treatment based on 
interview and reviewing the medical record 
3.  Gestational age (GA) <26 weeks, based on study determined GA  


22 
 
4.   Singleton gestation 
5.   Age between 18 – 45 years  
6.   Able to give informed consent and undergo study procedures 
 
Exclusion Criteria - Subjects in the ancillary cohort must not meet any of the following: 
1. Inclusion in the RCT of 250 vs 500 mg OPRC study 
2. Cerclage in place 
3. Plan for progesterone treatment other than study medication 
4. Known major fetal anomaly or chromosomal anomalies that might affect gestational 
age at delivery 
5. Malformation of uterus (uterine didelphus, septate uterus or bicornuate uterus) or 
known cervical length <2.5 cm 
6. Participation in another trial that may affect gestational age at delivery  
7.  Planned delivery at other institution where pregnancy outcome data cannot be obtained 
8. Medical or obstetrical complication that might affect gestational age at delivery, such 
as active ulcerative colitis, liver tumors, liver disease/failure, renal disease/failure, 
undiagnosed vaginal bleeding unrelated to pregnancy, or hypertension requiring 2 or 
more agents 
9. Current or history of thrombosis or thromboembolic disorder. 
10. Known or suspected breast cancer, other hormone-sensitive cancer, or a history of these 
conditions. 
11. Moderately severe depression (PHQ-9 score ≥15, EPDS score of >13, or suicidal 
ideation)- based on criteria in the RCT 
C.3.ii. Gestational Age Determination 
We will use the criteria recommended by the American Congress of Obstetricians and 
Gynecologists (ACOG) 37. 
C.4. Informed Consent Procedures and Documentation 
Written informed consent will be obtained before entry into the trial. Full disclosure of the 
nature and potential risks of participating in the trial will be made. Each center will develop its 
own consent form according to the requirements of its IRB. Women who are not fluent in 
English will be enrolled by a person fluent in their language and both verbal and written 


23 
 
informed consent obtained in that language; if such are not available, they will not be 
included. 
C.5. Randomization Procedures 
 
Eligible and consenting women will be enrolled and randomized by clinical site and prior 
preterm birth history (based on earliest SPTB, dichotomized to 30 weeks or less or >30 
weeks) to the standard 17-OHPC dose of 250 mg or to a dose of 500 mg intramuscularly 
weekly starting at 16 0/7 – 21 6/7 weeks and continuing till  
36 6/7 weeks gestation. We will stratify at randomization by site and pregnancy 
history due to the dramatic ethnic, racial and socioeconomic differences in the 
population at the 3 centers and the demonstrated impact of gestational age at a 
prior preterm birth on subsequent pregnancy duration. This will be an open label 
study. Blinding is not required since the primary outcome of interest is the rate of preterm 
birth according to plasma concentration achieved. Knowledge of the dose by the provider 
or subject does not impact the primary outcome, i.e., the rate of preterm birth or the 
plasma concentration. We will not stratify subjects at randomization by any maternal 
variables since an objective of the study is to define which maternal characteristics might 
impact 17-OHPC concentrations or efficacy. Our prior work demonstrates a clear impact of 
maternal BMI but this effect is modest i.e., ~17%.38 
Subjects enrolled in the ancillary cohort will not be randomized as they are already on the 250 
mg dose.  
C.6. Study Procedures 
C.6.i. Study Drug Dispensation 
• Randomized Clinical Trial:  For subjects in the RCT, the weekly injections will be 
administered into the buttocks/hip by either research staff, physician’s office staff or 
other qualified medical personnel, or a person selected by the subject who is trained 
to administer the medication in the subject’s home. That person will attend a study 
visit with the subject, conducted prior to the start of home injections, where research 
staff will demonstrate the technique of injection and withdrawal of medication from the 
vial in a fashion that maintains sterility. The subject will be given 6-9 weeks’ worth of 
medication to take home with her until the next visit with research staff. Every injection 
will be recorded as the research staff will be in contact with the subject weekly. The 


24 
 
medication will be the standard 250 mg of 17-OHPC in 1 ml castor oil, or 500mg 17-
OHPC in 2 ml castor oil. In order to minimize the discomfort associated with the 500mg 
dose, we will offer women the option of either a single 2 ml injection (500 mg) or two 1 
ml injections (250 mg) for a total dose of 500 mg. All of the 17-OHPC will be dispensed 
in a 5 ml multi-dose vial. Information regarding drug dispensation will be recorded at 
each visit/encounter with the participant from randomization up until the last encounter 
at 36 6/7 weeks, or delivery, whichever occurs first.  
 
• Ancillary Cohort: Subjects in the ancillary cohort will have 17-OHPC prescribed by 
their healthcare provider as part of their standard of care.  Subjects will be offered the 
GMP produced Makena drug for consistency with the RCT and also to potentially 
enhance recruitment if the prescribed 17-OHPC medication is not covered by the 
subject's insurance. Although it is preferred that subjects use the Makena medication, it 
is not a requirement for the ancillary cohort. We will document the source of the 
medication. Study drug will be distributed to subjects during routine OB visits or during 
study visits. 
 Research staff will not be responsible for administering injections to subjects enrolled 
in the ancillary cohort; however, they may administer the injections on an as needed 
basis. Study drug will be distributed to subjects during routine OB visits or during study 
visits. 
 
C.6.ii. Randomization and Baseline 
At randomization, we will record height and weight from the medical record and collect 
information about demographics (e.g., age and race), social history (e.g., marital status, 
years of education, alcohol use, tobacco use and illicit drug use), medical and obstetric 
history, prenatal labs and ultrasound findings from the index pregnancy, and concomitant 
medication and supplement use. Self-reported information will be confirmed with the medical 
record. This visit is anticipated to last approximately 30 minutes. 
 
 


25 
 
C.6.iii. Study Visits 
• Randomized Clinical Trial:   
Each center will see subjects enrolled in the RCT every 6-9 weeks to provide additional 
medication and to perform other aspects of the study including examination of the 
injection sites, as needed. All subjects in the RCT will have venous blood draws 
performed at the times described in the protocol. Research staff will contact subjects 
weekly to record injection information, ask about possible side effects of the medication 
experienced since the last injection, and about the use of other medications. They will also 
ask about any hospitalizations and any pregnancy complications that have been diagnosed 
since the last contact. If complications of the pregnancy arise, such as need for a cervical 
cerclage, or need for vaginal progesterone, they will be recorded. If the patient is 
hospitalized for any reason including preterm labor, treatment should continue. The local 
principal investigator will evaluate the event’s expectedness, relatedness and severity. 
Events will be reported to the local IRBs per institutional procedures and the participating 
investigators and FDA, as required. Details of monitoring, documenting and reporting 
serious adverse events (SAEs) will be documented in the study’s safety monitoring plan. 
Weekly communication will occur until the patient delivers.  
 
Subjects in the RCT will be assessed for depression using either the Patient Health 
Questionnaires (PHQ-2 and PHQ-9, if required) or the Edinburgh Postnatal Depression Scale 
(EPSDS) 40 at the following study visits when maternal blood samples are collected: Screening 
Visit, 26-30 week visit, and 6-9 weeks after the 26-30 week visit.  
The Patient Health Questionnaire-2 (PHQ-2) will be administered as a screen for depression.  
If the PHQ-2 score is 3 or greater, major depressive disorder is possible and subjects will be 
further evaluated with the Patient Health Questionnaire-9 (PHQ-9).  The PHQ-9 is a 
multipurpose instrument for screening, diagnosing, monitoring and measuring the severity of 
depression. Alternatively, the EPDS consists of 10 questions and is a valuable and efficient 
way of identifying patients at risk for perinatal depression. 
o 
A PHQ-9 score of ≥ 15 or an EPDS score >13 represents moderately severe 
depression. Subjects with a PHQ-9 score of ≥ 15 or an EPDS score >13 at the Screening 
Visit will be excluded from enrolling in the study and will be referred to a mental health 
provider.  Currently enrolled subjects who develop depression and have a PHQ-9 score 


26 
 
≥ 15 or an EPDS score >13 will not automatically be withdrawn from the study, but a 
risk-benefit discussion for 17-OHPC treatment is appropriate. Subjects will be offered a 
referral to a mental health provider, if not already in treatment. If the patient is in 
treatment, we will continue to monitor her depression and notify her treatment 
professional, as appropriate.   
  
Note: Question 9 on the PHQ-9 and question 10 on the EPDS are single screening 
questions on suicide risk. If a subject answers yes to question 9 on the PHQ-9 or has a 
score ≥1 for question 10 on the EPDS, they are ineligible to continue with the study and 
will be immediately referred to a mental health provider for further assessment. 
o 
A PHQ-9 score of ≥ 10 and < 15 or an EPDS score >10 and ≤13 indicates moderate 
depression. Subjects with moderate depression will not be excluded or withdrawn from 
the study, but a risk-benefit discussion for 17-OHPC treatment is appropriate. Patients 
will be offered a referral to a mental health provider, if not already in treatment. If the 
patient is in treatment, we will continue to monitor her depression and notify her 
treatment professional, as appropriate.   
 
• Ancillary Cohort:  
All subjects in the ancillary cohort will have venous blood draws performed at the times 
described in the protocol. Study drug will be dispensed to subjects during routine OB visits 
or during study visits. Subjects will be given enough medication to last 6-9 weeks.  The 
administration of the injections will be managed by the prescribing physician. 
Research staff will contact subjects weekly to record injection information, ask about 
possible side effects of the medication experienced since the last injection, and about the 
use of other medications. They will also ask about any hospitalizations and any 
pregnancy complications that have been diagnosed since the last contact. If complications 
of the pregnancy arise, such as need for a cervical cerclage, or need for vaginal 
progesterone, they will be recorded.  
 


27 
 
 
C.6.iv. Delivery 
At delivery, pregnancy and neonatal outcome information will be obtained from the medical 
record. Biological samples will not be collected at delivery for subjects in the ancillary cohort. 
C.6.v. Specimen Collection 
a. RCT Specimen Collection 
Maternal blood will be collected on four occasions by a health care provider or research staff 
trained in phlebotomy:  
1. Prior to the 2nd injection (1 tube of 7 ml), ideally on day 7 from the first injection 
2. Between 26-30 weeks - but only after a minimum of 7 injections have been administered 
(1 tube of 7 ml will be drawn just prior to the next injection; this will be a trough level)   
3. Between 6-9 weeks after the 26-30 week specimen but not later than 37 weeks  
(1 tube of 7 ml will be drawn just prior to the next injection; this will be a trough level) 
4. At delivery (1 tube of 7 ml), when possible 
A second tube of blood (7 mL) will be drawn with any of the first 3 blood draws for genotyping 
of various genes related to preterm birth if mother consents to this optional test.  
 
Umbilical cord blood (7 mL) will be collected at delivery.  A second cord blood specimen (7 ml) 
will be drawn for genetics studies in the baby if mother consents. These blood specimens will 
be collected, when possible, and used to determine the concentrations of various biochemicals, 
17-OHPC and its metabolites, and for genetic studies. 
 
A 3 x 3 cm piece of placenta will be collected, when possible, for p-glycoprotein and other 
transporters.  
 
Labor and Delivery biological specimens will be collected when possible. All biological 
specimens will be frozen at -70 to -80° C and stored locally until assayed at the University of 
Pittsburgh. 
 
 


28 
 
 
b. Ancillary Cohort Specimen Collection 
Maternal blood will be collected on two occasions by a health care provider or research staff 
trained in phlebotomy:  
1. Between 26-30 weeks - but only after a minimum of 7 injections have been administered 
(1 tube of 7 ml will be drawn just prior to the next injection; this will be a trough level)   
2. Between 6-9 weeks after the 26-30 week specimen but not later than 37 weeks  
(1 tube of 7 ml will be drawn just prior to the next injection; this will be a trough level) 
A second tube of blood (7 mL) will be drawn with any of the first 2 blood draws for genotyping 
of various genes related to preterm birth if mother consents to this optional test. 
 
C.7. Patient Management 
No attempt will be made to alter or mandate clinical management of the subjects.  
C.7.i Criteria for Stopping Treatment 
In the event that either of the two scenarios below occur, treatment with 17-OHPC will stop and 
the subject will be retained in the study but their data related to 17-OHPC concentration will not 
be utilized since they will have stopped the medication.  Additional subjects will be recruited to 
maintain the sample size.   
a. Systemic drug allergy, thrombosis during pregnancy, cholestatic jaundice, suicidal 
ideation, or other concern regarding the patient's safety 
b. Pregnancy termination (not planned at the time of eligibility determination) 
The ancillary cohort is an observational cohort.  This section is not applicable to the ancillary 
cohort, as the 17-OHPC medication is prescribed and managed by the subject’s healthcare 
provider. 
C.7.ii Non-Compliant Subjects 
Compliance with drug administration will be documented by discussion with the subject during 
the weekly contact with the subject and by counting the used vials. Non-compliant subjects will 
be retained in the study and treatment with the randomized dose of 17-OHPC will continue; 
however, their data related to 17-OHPC concentration will not be utilized for the primary 
outcome. Additional subjects will be recruited to maintain the sample size. Non-compliance will 
be defined as:  


29 
 
a. Subjects who miss any injections in the 4 weeks preceding the 26-30 week blood draw. 
b. Subjects who, after enrollment, admit to missing 2 or more injections after the first 
injection. 
C.7.iii Subjects that Withdraw from the Study 
If a subject in the RCT withdraws their consent, we will continue to provide the standard dose 
of 250 mg until treatment can be transitioned to the patient’s healthcare provider, if desired, to 
ensure there is no lapse in patient care. Additional subjects will be recruited to maintain the 
sample size.  
Patients who are greater than 21 6/7 weeks GA when they elect to withdraw from the study 
may not be eligible for insurance coverage of the standard 250 mg weekly dose administered 
by their healthcare provider. In this case, the study will continue to provide the standard 250 If 
mg weekly dose until 36 6/7 weeks gestation or until delivery.   
If a subject in the ancillary cohort withdraws from the study, her treatment will continue to be 
managed by her healthcare provider. Any data collected will be retained and used as 
appropriate.  
C.8. Study Halting Criteria 
We have established study halting criteria since the 500 mg dose is not FDA approved. We will 
use subjects in the 250 mg RCT group as a comparator for adverse effects and to establish 
criteria for stopping the study. We will not use subjects in the ancillary study who are receiving 
the 250 mg dose as a comparator since they will not have been randomized.  
 
The DMC will review enrollment and AEs on a regular basis (every 4 months). Enrollment will 
not stop unless the Network is directed to stop by the DMC or the FDA. Criteria that warrant 
discussion of halting the study by the DMC will include more than expected occurrence of the 
SAEs previously known to be associated with 17-OHPC use, or SAEs unexplained by the 
patient’s medical condition. Preterm birth is an expected pregnancy complication in this group 
of subjects and is associated with significant and serious fetal and neonatal morbidities and 
mortality mostly related to severe prematurity. Therefore, when the DMC evaluates SAEs, a 
distinction will be made as to whether the SAE is thought to be related to the patient’s medical 


30 
 
or pregnancy condition (e.g. severe preeclampsia, extreme prematurity, etc) or whether it is 
thought to be related to study medication. Study halting criteria include: 
 
1. Fetal and neonatal deaths.  
A safety signal for an excessive number of early pregnancy losses and stillbirths was identified 
in the Meis study by the FDA in their review of the Makena NDA. The rate of loss was 3.6 in 
the 17-OHPC group and 1.3 % in the control group. If this signal is related to plasma 
concentrations of 17-OHPC, then a greater number of these adverse outcomes might be seen. 
Fetal and neonatal deaths will be evaluated on an ongoing basis. Study coordinators will report 
serious adverse events to the site PI, the LCC, and the study Lead PI within 24 hours of 
notification. Information not available at the time of the initial report should be submitted as a 
follow-up report within 48 hours of notification. It would appear reasonable to set some criteria 
to alert the Medical Monitor and the DMC that a review is necessary. We have set this alert at 
a rate of pregnancy loss (20 0/7 weeks till delivery) or stillbirth of 1.5 times higher in the 500 
mg weekly group than in the 250 mg group. The DMC should review all deaths to determine 
the relationship to study drug and to determine what if any action is needed. Enrollment in the 
study will continue during this review. After the review the DMC, in conjunction with NICHD and 
the Medical Monitor, will determine whether the study should be stopped or continued.  
 
2. Thromboembolic Events (deep vein thrombosis or pulmonary embolus) 
There is a potential risk of thrombosis from progestin treatment but this risk is low. Genetic 
predisposition to clotting is seen in over 5% of the population and pregnancy increases the risk 
of thrombosis so clotting during pregnancy is uncommon but NOT an unexpected event. Doses 
of 17-OHPC of 250 mg weekly have not been associated with an increased risk of clotting but 
the potential exists that higher doses of 17-OHPC may increase the risk of thrombosis. We 
would expect no more than 3 cases of thrombosis (3%) in the 150 subjects recruited to the 250 
mg weekly group. If the number of cases of deep vein thrombosis or pulmonary embolus in the 
500 mg weekly group exceeds the number of cases in the 250 mg group by more 3% (3 cases) 
then these cases will be identified by the Medical Monitor and reviewed by the DMC for 17-
OHPC causality. All SAEs are reported promptly to the site PIs, Medical Monitor and study lead 
and are reviewed by the DMC. The DMC should review all the thrombotic events to determine 
the relationship to study drug and to determine what if any action is needed. 


31 
 
 
C.9. Medical monitor and Logistic Coordinating Core 
A Medical Monitor has been selected for this trial. The Medical Monitor will receive reports from 
each clinical center which will be compiled by the NICHD funded Logistic Coordinating Core 
(LCC). The LCC facilitates and coordinates the OPRC clinical study activities and ensures that 
all activities are implemented. 
The role of the Medical Monitor will include:  
• Ongoing monitoring of SAE reports submitted by the clinical centers to identify 
   
safety concerns and suggest protocol modifications, if necessary  
• Advise IND sponsor, Principal Investigator(s), and DMC regarding SAEs and safety 
  concerns  
• Provide reports when necessary to the Principal Investigator and DMC  
D. Statistical Analysis and Sample Size Justification 
D.1. Primary and Secondary Outcomes 
The primary outcome is the rate of preterm birth (delivery prior to 37 0/7 weeks) 
according to 17-OHPC concentration. 
Secondary outcomes include: 
• The rate of spontaneous PTB prior to 32 0/7 and 35 0/7 weeks according to the 
concentration of 17-OHPC. 
• The impact of dose, race, BMI and co-medications on plasma 17-OHPC 
concentrations 
• Plasma concentrations of 17-OHPC, mono-hydroxylated metabolite of 17-OHPC 
• Concentrations of progesterone and 17-hydroxprogesterone in maternal and cord 
blood according to the weekly dose of 17-OHPC 
• The frequency of maternal side effects according to dose and plasma concentration 
• The frequency of neonatal complications according to dose and plasma concentration 
• Impact of dose on rates of spontaneous PTB 
• The correlation of progesterone and 17-hydroxyprogesterone in maternal plasma and 
whole blood to pharmacological and clinical outcomes 


32 
 
The primary outcome will be the rate of preterm birth (<37 weeks) according to the plasma 
concentration of 17-OHPC (as a continuous variable) from the 26-30 week sample.  The 
distribution of plasma concentration is expected to be highly positively skewed; thus, a natural 
logarithmic transformation will be applied to plasma concentrations to produce normally 
distributed values.  A Cox proportional hazard model will be employed to analyze the effect of 
continuous log-transformed plasma concentration on event-free survival (in this case, “event-
free survival” meaning “not birthed until term”). Specifically, the hazard of delivering preterm 
will be modeled as a function of log-transformed plasma concentration and additional 
covariates that may influence treatment effectiveness (e.g., BMI, race, gestational age at 
study entry).  Adjusted survival curves will be calculated using the relationship between the 
hazard function and the survival function.  A Wald test will be performed to determine whether 
the hazard ratio corresponding to a one-unit increase in log-transformed plasma concentration 
is significantly different from zero.  Wald and likelihood ratio tests will also be employed to test 
for the significance of additional covariates, and stratified analyses may be considered if 
appropriate (i.e., race-specific analyses).  This semi-parametric modeling approach will allow us 
to analyze the effect of, and not just the presence of, covariates on event-free survival in a robust 
manner.   
 
Secondary outcomes that are also survival outcomes (such as spontaneous PTB, spontaneous 
PTB before 32 weeks and before 35 weeks, and indicated PTB) will be analyzed in a similar 
fashion to the primary outcome analysis described above. For other secondary outcome 
measures that are continuous variables (maternal and infant hospital days, maternal and fetal 
plasma concentrations of progesterone, hydroxyprogesterone, 17-OHPC and its metabolite, 
etc.), linear regression models with appropriate covariates will be utilized. 
 
We will also compare preterm birth rates between treatment groups (250 or 500 mg). 
Descriptive statistics will be generated for key characteristics of the treatment groups at 
baseline. For continuous variables, we will report means and standard deviations or medians 
and interquartile ranges (the latter in the case of skewed distributions or large outliers).  Counts 
and percentages will be reported for categorical variables.  We will then construct a Cox model 
and produce adjusted survival curves to assess the magnitude of differences in PTB rate 


33 
 
between groups while adjusting for covariates that may influence treatment effectiveness (e.g., 
BMI, race, gestational age at study entry). 
 
We have previously performed population pharmacokinetic analysis of 17-OHPC in twin and 
singleton pregnancies. In this study, we will also use population pharmacokinetic analysis to 
evaluate the contribution of various covariates to the observed variability in the trough plasma 
concentrations of 17-OHPC, a surrogate measure of drug exposure. 
 
D.2. Sample Size and Power 
A Cox proportional hazards regression model will be used to assess the effect of log-
transformed 17-OHPC concentration on event-free survival, while adjusting for covariates such 
as maternal BMI.   
 
The recruited cohort of women will be sufficiently large to provide a wide range of drug 
exposures and enable evaluation of several potential covariates on plasma concentration of 
17-OHPC and to describe the association between plasma concentration of 17-OHPC and its 
efficacy. Based on the large variation in 17-OHPC concentrations we have reported in clinical 
trials with the standard dose, we are confident that the range of concentrations of 17- OHPC 
that will be achieved with the two doses selected will provide a sufficiently wide range (between 
4 and 112 ng/ml) to enable the performance of a robust concentration response assessment 
(Figure 7). Based on these simulated data, we estimate that 99% of subjects will achieve a 
concentration > 10 ng/ml with the 500 mg/week dose.  
 
Based on the simulated distribution of 17-OHPC plasma concentration at higher doses in this 
study, we expect the variance of log-transformed concentration to be 0.226.  Given the results 
of the prior study, we have estimated the hazard ratio corresponding to a one-unit increase in 
log-transformed 17-OHPC concentration to be 0.49.  We also anticipate that 28% of study 
participants will deliver preterm. Thus, a Cox regression based on a sample of 208 women will 
have 80% power at the a=0.05 significance level to detect a hazard ratio of 0.49.  This sample 
size will be adjusted for the presence of other covariates in the Cox model; assuming that 
additional covariates such as BMI account for 30% of the variation in log-transformed plasma 
concentration, we require a sample size of 296 women to achieve 80% power (Table 1). Our 


34 
 
published data indicate that only maternal BMI affects plasma concentrations of 17-OHPC and 
accounts for no more than 30% of the variation in plasma concentrations (Figure 7). We will 
recruit 300 subjects overall to account for non-compliance or non-collection of plasma for 
analysis of 17-OHPC.  Data from non-compliant subjects will not be used for any analysis 
related to plasma 17-OHPC concentration. In addition, data analysis related to plasma 17-
OHPC concentration in those subjects who stop 17-OHPC treatment because of a drug allergy, 
thrombosis or cholestatic jaundice will not be utilized, but their outcomes will be kept and 
included in the secondary outcomes that are not related to plasma 17-OHPC concentrations. 
The sample size will be sufficient to address secondary outcomes since the variation in the 
variables studied will be far smaller than the variation in plasma 17-OHPC concentration. 
 
All three centers have recruited very well in other studies of 17-OHPC from the MFMU.  The 
pool of subjects annually available for recruitment will exceed 300. Even at a study 
acceptance rate as low as 30%, there are more than enough subjects to enable us to reach 
our recruitment goal. We anticipate an acceptance rate closer to 50%. All power analyses were 
performed using PASS 13. [CITE: PASS 13 Power Analysis and Sample Size Software (2014). 
NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/pass.] 
 
Table 1. Total Sample Size Required for 80% Power 
 
 
% variation accounted for by additional covariates 
 
 
0% 
10% 
20% 
30% 
% 
participants 
delivering 
preterm 
26% 
223 
248 
279 
319 
27% 
215 
239 
269 
307 
28% 
208 
231 
259 
296 
29% 
200 
223 
250 
286 
E. Data Management and Quality Assurance 
 
E.1. Investigational Product Supply 
All the 17-OHPC will be provided by AMAG Pharmaceuticals Inc. the supplier of the FDA 
approved Makena product. The product will contain 17-hydroxyprogesterone caproate  
250 mg/ml with concentrations of benzyl benzoate and castor oil of 46% and 44-54%, 
respectively. To assure the concentration of 17-OHPC is as stated in the product label, we will 


35 
 
save a 1 ml aliquot from one vial of each product lot used in this trial for future analysis of 17-
OHPC content.  
E.2. Clinical Data Management  
Clinical data will be acquired throughout the subject’s participation in the clinical trial and will 
include information related to screening, randomization, enrollment, pregnancy outcome, 
adherence, and adverse outcomes. These data will be collected on case report forms (CRFs) 
specific to this study.  
All key data from case report forms will be entered into the University of Pittsburgh (UPITT) 
REDCap database program, which will be used as a secure web application for research data 
storage and archiving. The password protected database provides system validation, 
metadata, and is supported by UPITT Information Technology with back-up via UPITT servers. 
The database provides features including auto-validation, calculated fields, branching/skip 
logic, and data quality tools such as Data Resolution Workflow which tracks query generation, 
data alterations, and resolution. 
E.3. Laboratory Test Results 
Analyses of samples (e.g. blood, DNA, placenta and cord blood) will be performed at the 
University of Pittsburgh (Dr. Raman Venkataramanan’s lab) or at another NICHD supported 
research center. Analysis of 17-OHPC, its primary monohydroxylated metabolite, 
progesterone and hydroxyprogesterone will be simultaneously measured in human plasma 
by a specific and sensitive validated HPLC-MS-MS assay developed in his lab.39 This assay 
is sensitive down to a plasma concentration of 1 ng/ml for 17-OHPC.39  The assay may be 
further validated to detect lower concentrations of 17-OHPC.  
Analysis of 17-OHPC will be performed using plasma and whole blood samples at the 
Magee-Womens Hospital site. Northwestern University and the University of Texas 
Medical Branch will collect plasma samples only.  
These laboratory results will be sent to the Lead Site PI and merged with the data for this study.  
All data from CRFs and biological fluid analysis will be linked using a participant identification 
number assigned at study entry. 
 
 


36 
 
 
 
 


37 
 
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