Validation of 24-hour Trough Concentration as a Surrogate for Intensive Pharmacokinetic Measurements for a Combined Oral Contraceptive Pill Containing Desogestrel
Phase: PHASE4
Conditions: Contraception
Sponsor: University of Colorado, Denver

Protocol Template 
Page 1 
CF-146, Effective 7/10/11 
 
Official Title: Desogestrel-containing COCP Pharmacokinetic Validation Study 
 
NCT Number: NCT05002738 
 
Document date: 8/13/2021

Protocol Template 
Page 2 
CF-146, Effective 7/10/11 
 
I. Hypotheses and Specific Aims: 
 
Specific Aim: 
• 
To validate prior pharmacokinetic research with combined oral contraceptive pill users that 
supports utilizing a 24-hour trough concentration as an accurate proxy for the intensive 
pharmacokinetic parameter of area under the curve (gold standard pharmacokinetics). 
 
Hypotheses: 
• 
We hypothesize that 24-hour trough concentration measurements of etonogestrel will have 
high correlation (r≥0.9) with the area under the curve measurements of etonogestrel 
among combined oral contraceptive pill users at steady state (day 21). 
• 
We hypothesis that 24-hour trough concentration measurements of ethinyl estradiol will 
have high correlation (r≥0.9) with area under the curve measurements of ethinyl estradiol 
among combined oral contraceptive pill users at steady state (day 21) 
 
II. Background and Significance:  
 
Women taking the exact same formulation of combined oral contraceptive pill (COCP) will 
have wide variability in their pharmacokinetic (PK) measurements of both the estrogen and 
progestin components1. Traditionally, these PK measurements for COCP users consist of an area 
under the curve (AUC), maximum concentration (Cmax), time to Cmax, elimination half-life, and 24-
hour concentration (C24)1-3. These intensive PK measurements may require up to 15 serial blood 
samples over the course of 24-36 hours, often necessitating an overnight stay in a clinical research 
center. Westhoff et al. found that the C24 measurement alone at steady-state (21 days of COCP 
use) was very highly correlated with AUC values for both ethinyl estradiol (r=0.92, p<0.0001) and 
levonorgestrel (LNG) (r=0.95, p<0.0001) among COCP users2,3. They also tested the correlation 
between single-dose C24 measurements and standard AUC values and found significant yet not as 
robust correlation for both ethinyl estradiol (r=0.72, p=0.003) and LNG (r=0.7, p=0.004)2,3.  
Ultimately, the C24 measurement at steady-state represents an ideal proxy for intensive 
COCP PK measurements that could alleviate much of the time demand placed on participants and 
reduce PK study costs without compromising data integrity. However, these PK analyses have all 
been conducted with the same COCP formulation containing ethinyl estradiol and LNG2. LNG, a 
second generation progestin, has distinct disadvantages compared to newer progestins due to its 
greater androgenicity4,5. Desogestrel (DSG), a third generation progestin, has similar progestin 
receptor binding affinity as LNG, but less androgen receptor binding affinity4,5. Formulations of 
COCPs with DSG often lead to improvements in androgen-related conditions, such as acne, due to 
their anti-androgenic properties6. For this reason, we have largely moved away from prescribing 
LNG-containing COCPs in our actual clinical practice. Thus, validation of this prior PK work with a 
DSG-containing COCP would produce more generalizable and pragmatic findings for COCP users 
in the US.  
Furthermore, DSG is a pro-drug of etonogestrel (ENG)4, which is the progestin of focus for 
our ongoing pharmacogenomic research with hormonal contraception through ENG contraceptive 
implant users7,8. As we begin to consider bridging this work to COCP users, we need to validate 
the prior findings of Westhoff and Basaraba et al.2,3 with a DSG-containing COCP to directly inform 
and support the pharmacokinetic methodology for a planned pharmacogenomic study of COCP 
users. This study will directly address this need through testing whether the C24 measurement 
maintains high correlation for the AUC measurements of ENG and ethinyl estradiol among COCP 
users at steady state. 
 
III. Preliminary Studies/Progress Report:  No direct preliminary studies have been 
conducted at our institution. See prior published pertinent literature discussed above. 
 
IV. Research Methods 
 

Protocol Template 
Page 3 
CF-146, Effective 7/10/11 
 
A.  Outcome Measure(s):   
Primary Outcome Measures: 
• 
24-hour AUC measurement for serum etonogestrel 
• 
24-hour AUC measurement for serum ethinyl estradiol 
• 
C24 measurement for serum etonogestrel 
• 
C24 measurement for serum ethinyl estradiol 
Secondary Outcome Measures: 
• 
Cmax for serum etonogestrel and ethinyl estradiol 
• 
Time to Cmax for serum etonogestrel and ethinyl estradiol 
• 
Elimination half-life for serum etonogestrel and ethinyl estradiol 
 
B. Description of Population to be Enrolled:   
We aim to enroll up to 20 reproductive age (18-45 years) females in this study based on the 
following criteria: 
 
Inclusion Criteria 
• 
Healthy females aged 18-45 years 
• 
Body-mass index ≥18.5kg/m2 
o 
No upper body-mass index 
• 
Willing to abstain from medications and supplements known to induce/inhibit CYP3A4 
during the study9 
• 
Normal blood pressure measurement at screening (systolic <140mmHg, diastolic 
<90mmHg) 
• 
Negative urine pregnancy test at screening 
 
Exclusion Criteria 
• 
Currently taking any known CYP3A4 inducers/inhibitors9 
• 
Medical conditions that affect liver function (e.g. hepatitis, cirrhosis; assessed via 
participant self-report) 
• 
Any contraindications to estrogen-containing contraception (based on any category 3 or 4 
recommendations from the CDC MEC guidelines10) 
• 
Use of an injectable contraceptive method within the last 6 months or current use of an 
ENG contraceptive implant 
• 
Childbirth within the last 6 months 
• 
Known allergy or insensitivity to combined oral contraceptive pills 
• 
Currently taking a combined oral contraceptive pill formulation and not willing to undergo a 
7-day washout period and switch to the study specific COCP formulation for the duration of 
the study. The prior COCP formulation (e.g. 21/7, 24/4, and continuous) and the type of 
progestin are not exclusion criteria for this study given the washout period. 
 
C. Study Design and Research Methods   
We propose a prospective, pharmacokinetic validation study to conduct intensive 
pharmacokinetic measurements on females using a COCP containing desogestrel (DSG) and 
ethinyl estradiol (EE). We will enroll healthy, reproductive-age females (18-45 years) for this PK 
validation study. We will exclude women with contraindications to the use of estrogen-containing 
contraception based on the CDC Medical Eligibility Criteria (MEC) guidelines for any conditions 
with a category 3 or 4 recommendation10. We will also exclude women who have used an 
injectable contraceptive method (e.g. depot medroxyprogesterone acetate) within the last 6 months 
or currently using an ENG contraceptive implant. Our remaining inclusions and exclusions criteria 
are listed in the section above. For body-mass index (BMI), we will utilize a lower-limit cut-off of 
18.5kg/m2 to exclude potentially underweight participants who may have altered baseline 
metabolism. As participants will serve as their own comparator for this study’s analyses, we will not 
have an upper BMI cut-off. For medical conditions that affect liver function, we will assess for these 

Protocol Template 
Page 4 
CF-146, Effective 7/10/11 
 
medical conditions during the screening process and confirm the lack of this exclusion criteria via 
self-report from interested participants. Females currently using a COCP will qualify for enrollment 
into the study after completing a 7-day washout from their current COCP formulation. If a female is 
currently on a different formulation of COCP (e.g. 24/4, continuous), then they will still qualify for 
enrollment into this study after completing a 7-day washout period from their current COCP 
formulation. Interested females will come to our CU Medicine Family Planning clinic 
(Comprehensive Women’s Health Center [CWHC]) for a screening visit where will we confirm study 
eligibility including measuring height, weight, blood pressure, and a urine pregnancy test.  
Eligible participants will then start a COCP formulation containing 30mcg of EE and 
0.15mg of DSG. We will instruct participants to take their COCP first thing in the morning to 
prepare for their PK visit. After taking 20 days of the COCP, participants will return to CWHC for 
their steady-state PK study visit. Participants will present to CWHC fasting, take a urine pregnancy 
test, and then have an intravenous (IV) catheter inserted. We will then provide participants with a 
standard breakfast consisting of no more than 34% fat, during which they will be given their day 21 
COCP to be taken with their food. Participants will then remain in our clinic for at least 8 hours, 
during which we will serially draw blood from the IV catheter at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 
8 hours from the administration of the COCP. Participants will be provided a standard lunch 
consisting of 600-700 calories and no more than 34% fat content during their PK study visit. After 
the 8-hour blood draw, the IV catheter will be removed and the participants will return to CWHC 
again at 24 hours from COCP administration for one additional blood draw. Each participant will 
undergo a total of 12 blood samplings for intensive PK analyses (summarized in Figure 1).  
For sample processing, we will allow samples to clot at room temperature for at least 10 
minutes and then centrifuge all blood samples on site to obtain serum aliquots, which we will then 
store in our -80° freezer until analyzed. Oral DSG undergoes rapid and complete first-pass 
metabolism into ENG, and so we will only measure serum ENG as the systemic progestin 
component of this COCP4. 
At the conclusion of enrollment, all stored serum samples will be de-identified and shipped 
to an outside laboratory that contains validated assays for measurement of EE and ENG. We will 
measure EE and ENG serum concentrations using validated liquid chromatography-tandem mass 
spectrometry (LC-MS/MS) assays through the Biomarkers Core Laboratory at Columbia University 
Irving Institute3,11. Samples will be batched for analysis to reduce inter-assay variability. 
 
Figure 1: Timeframe of study procedures 
 
  
F.   Data Analysis Plan:   
We will use noncompartmental analysis procedures with the trapezoidal rule to calculate 
the 24-hour AUC for both serum EE and ENG. We will also determine the C24, Cmax, time to Cmax, 
and elimination half-life for EE and ENG for each participant. For our primary analysis, we will 
calculate the Pearson correlation coefficients (r values) between the C24 serum measurements and 
the steady-state 24-hour AUC measurements for both EE and ENG. This will also include 
calculating 95% confidence intervals and p-values for these correlation coefficients. We have 
determined that duplicating a correlation coefficient of at least 0.9 will provide reassurance that C24 
remains a valid proxy PK measurement for a DSG-containing COCP. To validate previous findings 
of at least an r=0.92,3, we would need only 7 participants with a power of 0.8 and significance cut-

Protocol Template 
Page 5 
CF-146, Effective 7/10/11 
 
off of 0.05. With 20 participants, we will have only a 5.0x10-7 chance of a type II error with 
duplicating an r≥0.9. 
 
 H. References: 
 
 
 
References 
 
1. 
Westhoff CL, Torgal AH, Mayeda ER, Pike MC, Stanczyk FZ. Pharmacokinetics of a 
combined oral contraceptive in obese and normal-weight women. Contraception. 
2010;81(6):474-480. 
2. 
Basaraba CN, Westhoff CL, Pike MC, Nandakumar R, Cremers S. Estimating systemic 
exposure to levonorgestrel from an oral contraceptive. Contraception. 2017;95(4):398-404. 
3. 
Westhoff CL, Pike MC, Tang R, DiNapoli MN, Sull M, Cremers S. Estimating systemic 
exposure to ethinyl estradiol from an oral contraceptive. American journal of obstetrics and 
gynecology. 2015;212(5):614.e611-617. 
4. 
Kuhl H. Pharmacology of estrogens and progestogens: influence of different routes of 
administration. Climacteric : the journal of the International Menopause Society. 2005;8 
Suppl 1:3-63. 
5. 
Louw-du Toit R, Perkins MS, Hapgood JP, Africander D. Comparing the androgenic and 
estrogenic properties of progestins used in contraception and hormone therapy. Biochem 
Biophys Res Commun. 2017;491(1):140-146. 
6. 
Hatcher RA, Trussell J, Stewart F, et al. Contraceptive technology. New York: Ardent 
Media. Inc; 2011. 
7. 
Lazorwitz A, Aquilante CL, Oreschak K, Sheeder J, Guiahi M, Teal S. Influence of Genetic 
Variants on Steady-State Etonogestrel Concentrations Among Contraceptive Implant 
Users. Obstetrics and gynecology. 2019;133(4):783-794. 
8. 
Lazorwitz A, Dindinger E, Harrison M, Aquilante CL, Sheeder J, Teal S. An exploratory 
analysis on the influence of genetic variants on weight gain among etonogestrel 
contraceptive implant users. Contraception. 2020;102(3):180-185. 
9. 
U.S. Food and Drug Administration. Drug Development and Drug Interactions: Table of 
Substrates, Inhibitors, and Inducers. 
https://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/drugintera
ctionslabeling/ucm093664.htm. Updated Updated on 03/10/2020. Accessed 01/17/2017. 
10. 
Curtis KM, Tepper NK, Jatlaoui TC, et al. U.S. Medical Eligibility Criteria for Contraceptive 
Use, 2016. MMWR Recommendations and reports : Morbidity and mortality weekly report 
Recommendations and reports. 2016;65(3):1-103. 
11. 
Thomas T, Petrie K, Shim J, Abildskov KM, Westhoff CL, Cremers S. A UPLC-MS/MS 
method for therapeutic drug monitoring of etonogestrel. Therapeutic drug monitoring. 
2013;35(6):844-848. 
12. 
Hatcher R, Nelson A, Trussell J, et al. Contraceptive Technology. 21st ed. New York: 
Managing Contraception; 2018.