Pharmacokinetics of single dose doxycycline in the rectum, vagina, and urethra: implications for prevention of bacterial sexually transmitted infections

Summary

Background

Clinical trials showed a single oral dose of doxycycline taken after sex protects against STIs among men who have sex with men (MSM) but not women. Pharmacokinetic data at vaginal, rectal and penile sites of STI exposure are lacking. We examined vaginal, rectal and urethral doxycycline concentrations in men and women to better inform STI prevention.

Methods

Doxycycline pharmacokinetics in male and female participants 18–59 years of age were evaluated in blood and urine and on rectal and vaginal swabs collected at 1, 2, 4, 8, 24, 48, 72, 96 and 168 h after receiving a 200 mg oral doxycycline dose in a non-randomised single dose open label single centre study in Atlanta, Georgia. Rectal, vaginal, and cervical biopsies and male urethral swabs were collected 24 h after dosing (Trial registration: NCT04860505). Doxycycline was measured by liquid chromatography-mass spectrometry.

Findings

Eleven male and nine female participants participated in the study. Doxycycline concentrations on rectal and vaginal swabs collected up to 96 h after dosing were approximately twice those of plasma and remained above minimum inhibitory concentrations (MICs) for at least four, three, and two days for Chlamydia trachomatis, Treponema pallidum, and tetracycline-sensitive Neisseria gonorrhoeae, respectively. Geometric mean doxycycline concentrations in male urethral secretions (1.166 μg/mL; 95% CI 0.568–2.394 μg/mL), male rectal (0.596 μg/g; 0.442–0.803 μg/g), vaginal (0.261 μg/g; 0.098–0.696 μg/g) and cervical tissue (0.410 μg/g; 0.193–0.870 μg/g) in biopsies collected 24 h after dosing exceeded MICs. Plasma and urine doxycycline levels defined adherence markers up to four and seven days postdosing, respectively. No adverse events were reported in this study.

Interpretation

Doxycycline efficiently distributes to the rectum, vagina and urethra. Findings can help explain efficacy of STI prevention by doxycycline.

Funding

Funded by CDC intramural funds, CDC contract HCVJCG-2020-45044 (to CFK).

Research in context.

Evidence before this study

A search in PubMed on January 15, 2020, using the keywords “doxycycline” “rectum” “vagina” and “pharmacokinetics” was performed. While doxycycline has been used for treatment and prevention of a variety of bacterial and parasitic infections, the distribution of doxycycline at rectal, vaginal or urethral sites of STI exposure has yet to be explored. A single oral dose of doxycycline taken as post-exposure prophylaxis (PEP) within 72 h after sex has been shown to provide protection against bacterial sexually transmitted infections (STIs) in three clinical trials among men who have sex with men (MSM) and transgender women (TGW) with higher clinical efficacy observed against chlamydia and syphilis compared to gonorrhoea. Despite demonstrated efficacy against bacterial STIs among MSM, a similar clinical trial of doxycycline as PEP conducted among heterosexual women was unable to demonstrate efficacy. We examined doxycycline concentrations after a single oral dose in men and women to better interpret doxycycline prevention efficacy results and inform STI prevention.

Added value of this study

We demonstrate that after a single oral dose doxycycline rapidly disseminates to the rectum, vagina, and urethra, achieving concentrations greater than the minimum inhibitory concentration (MIC) for bacterial STIs. Doxycycline concentrations remain above the MICs for bacterial STIs up to 4 days after dosing. We also note persistent detection of doxycycline in plasma and urine up to 4 and 7 days after dosing, respectively, that can provide a marker of recent doxycycline dosing.

Implications of all the available evidence

We provide doxycycline pharmacokinetic data associated with observed clinical efficacy of doxycycline PEP against bacterial STIs among MSM. These data also suggest lack of significant doxycycline PEP efficacy observed among women in one trial is not likely due to inadequate doxycycline concentrations in vaginal tissues. Pharmacokinetic assessments and clinical efficacy can inform new strategies for bacterial STI prevention by doxycycline.

Introduction

The incidence of bacterial sexually transmitted infections (STIs) caused by Neisseria gonorrhoeae (gonorrhoea), Chlamydia trachomatis (chlamydia), and Treponema pallidum (syphilis) continues to increase with an estimated 375 million global cases annually.¹ Increasing STI incidence has been noted particularly among gay, bisexual, and other men who have sex with men (collectively referred to as MSM).² High rates of infection are also documented among heterosexual women. Currently, there are no approved vaccines to prevent bacterial STIs and existing strategies focusing on STI testing and treatment have not prevented increases in STI incidence.³ Recently there has been increased interest in developing pre-exposure (PrEP) or post-exposure prophylaxis (PEP) strategies using antibiotics to prevent bacterial STIs.^4,5

Doxycycline is a broad-spectrum, second-generation tetracycline antibiotic with bacteriostatic effect that is generally well-tolerated and has been used as primary prophylaxis for bacterial and parasitic infections such as Lyme disease and malaria.6, 7, 8 The doxycycline safety and activity profile led to recent clinical studies exploring doxycycline in event driven STI prevention strategies. An open label substudy of 232 MSM enrolled in the IPERGAY on-demand HIV prevention trial in France showed a single 200 mg oral dose of doxycycline given within 24 h, but not more than 72 h, after sex as PEP reduced risk of chlamydia and syphilis infection by 70% and 73%, respectively.⁹ However, risk of gonorrhoea infection was not significantly reduced in the IPERGAY study, likely due to high levels of pre-existing tetracycline resistance among N. gonorrhoeae isolates.⁹ The open label DoxyPEP study also tested 200 mg doxycycline as PEP in 327 MSM taking PrEP to prevent HIV infection and 174 MSM living with HIV in the United States. DoxyPEP was stopped following interim analysis demonstrating doxycycline as PEP reduced risk of chlamydia and syphilis infection by 82% among all study participants and significantly reduced risk of gonorrhoea infection by 55%.¹⁰ The DOXYVAC study conducted among 502 MSM in France was also stopped following interim analysis demonstrating doxycycline PEP reduced the risk of acquiring chlamydia, syphilis, and gonorrhoea by 89%, 79%, and 51%, respectively.¹¹

Extending on promising results of doxycycline PEP among MSM, the D-PEP study examined the same doxycycline PEP modality among heterosexual Kenyan women.¹² However, the D-PEP trial was unable to show that doxycycline PEP significantly prevents STIs among women.¹³ The study reported 27% efficacy against chlamydia that was not statistically significant, and no reduction in N. gonorrhoeae acquisition. There were very low rates of syphilis infection among the study population and only a single incident syphilis infection was identified among all participants.¹³ The lack of efficacy against N. gonorrhoeae is likely due to the predominant resistance to tetracycline found in isolates among Kenyan women.¹⁴ The much lower efficacy against chlamydia infection among women in the D-PEP study than among MSM in the DoxyPEP and DOXYVAC studies was reportedly due in large part to suboptimal adherence to doxycycline PEP dosing that was estimated to be approximately 29%.¹³

Despite decades of use in antimicrobial treatment and prevention and interest in doxycycline for STI prevention, there is limited data regarding doxycycline distribution to anatomical sites of STI exposure and infection such as the rectum, vagina, and urethra.^6,15 This pharmacokinetic information is critical for understanding and interpreting clinical efficacy of doxycycline for STI prevention. The objective of this study was to examine doxycycline distribution in blood, rectal, vaginal, and urethral samples collected from male and female participants. We discuss implications of the data for formulating effective doxycycline prophylaxis modalities to prevent bacterial STIs in men and women.

Methods

Study design

This study analysed specimens from cisgender male and female participants enrolled in a non-randomised, single dose open label single centre clinical trial registered at clinicaltrials.gov (NCT04860505) and conducted between June 2021 and May 2022 at the Emory Hope Clinic in Atlanta, Georgia, USA. Inclusion criteria included being generally healthy, HIV-negative and assigned male or female at birth who report sex with another man in the previous year. Eligible participants were 18–59 years of age and not taking tetracycline-derived antibiotics or antiretroviral drugs.

Participants received a single observed oral dose of 200 mg delayed release doxycycline hyclate (Mayne Pharma, Greenville, North Carolina, USA) as used in the DoxyPEP Study without food. Participants also received a single pill of Biktarvy [oral formulation of tenofovir alafenamide (25 mg), emtricitabine (200 mg) and bictegravir (50 mg)] (Gilead Sciences, Foster City, California, USA) to supplement existing antiretroviral pharmacokinetic data of this regimen for possible HIV prevention strategies.¹⁶ Antiretroviral drug concentrations are not included in this analysis as they are unrelated to bacterial STI prevention. The reporting period for participant adverse events began at enrolment and continued until the subject either completed or withdrew from the study. All adverse events and laboratory abnormalities were graded according to the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (DAIDS AE Grading Table), Version 2.1, July 2017, which can be found on the DAIDS RCC Web site: http://rcc.tech-res.com/tox_tables.htm. Case report forms were collected for each study visit and information was transferred to REDCap hosted by Emory University School of Medicine for electronic data capture.

Participants provided a blood and urine sample prior to dosing to ensure participants did not have measurable doxycycline before study dosing. Participants provided blood, urine and rectal or vaginal swabs at nine clinic visits 1, 2, 4, 8, 24, 48, 72, 96 and 168 h after dosing. Participants also provided rectal or vaginal tissue biopsies and male participants provided urethral and glans surface swabs 24 h after dosing. Seven of the nine female participants also provided rectal swabs and five of the nine female participants also provided rectal biopsies in addition to vaginal specimens. Eight male and five female participants completed all post-dose clinic visits. Three male and four female participants missed a total of eight and five clinic visits, respectively. There was no formal sample size calculation for this study; rather the sample size was determined based on the estimated clinic capacity for recruiting and enrolling study participants and completing study visits within 12 months. We aimed for a total of at least 8 male and 8 female participants providing tissue biopsy specimens 24 h after dosing.

The primary endpoint of this study was to compare doxycycline concentrations in rectal, vaginal, and cervical tissue biopsies collected 24 h after a single oral dose of doxycycline. Additional endpoints included comparison of tissue doxycycline concentrations to those in plasma and measuring doxycycline in urine, rectal and vaginal swab samples collected up to 7 days after a single oral dose of doxycycline.

Peripheral blood was collected in sodium citrate cell preparation tubes (CPT) (Becton Dickinson, Franklin Lakes, New Jersey, USA) and centrifuged to collect plasma. First void urine was collected in sterile specimen containers (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Rectal, vaginal, urethral and glans surface samples were collected using a standard 6” polyester Puritan applicator (Puritan Medical Products, Guilford, Maine, USA) as previously described.¹⁷ Rectal secretions were collected by inserting the applicator approximately 5 cm into the rectum and rotating the applicator for 5 to 10 s. An enema was not used prior to collection of rectal secretions. Vaginal secretions were collected by inserting the applicator approximately 5 cm into the vagina and rotating the applicator along the vaginal wall for 10 to 30 s. Urethral secretions were collected by inserting a polyester Puritan miniature applicator (Puritan Medical Products, Guilford, ME, USA) 2 to 4 cm into the urethra and slowly rotating clockwise for 2 to 3 s. Glans surfaces were sampled by pre-wetting a polyester Puritan applicator (Puritan Medical Products) in PBS and rolling the applicator around the head of the penis and underneath the foreskin, if present. Rectal biopsies were collected via rigid sigmoidoscopy (Olympus America, Center Valley, Pennsylvania) as previously described.¹⁶ One cervical and one vaginal biopsy were collected via speculum exam with mini-Tischler forceps. A rectal or vaginal swab and urine were collected prior to dosing to test for N. gonorrhoeae and C. trachomatis (Aptima 2 Combo Assay, Marlborough, Massachusetts, USA). No specimens tested positive for N. gonorrhoeae or C. trachomatis. All specimens were stored at −70 °C prior to analysis.

Laboratory measurements

Doxycycline concentrations were measured using a validated high performance liquid chromatography tandem mass spectrometry method (HPLC-MS/MS) (Sciex, Foster City, California, USA; Shimadzu Scientific Instruments, Durham, North Carolina, USA) based on previously published methodology.¹⁸ For plasma, 500 μL of Buffer B (0.1% trifluoroacetic acid in acetonitrile) containing 5 ng/mL of isotopically labelled doxycycline-d3 hyclate (doxycycline IS) (Toronto Research Chemical, Toronto, Ontario, Canada) was added to 100 μL of plasma and extracted by protein precipitation. The specimen was vortexed briefly and centrifuged for 20 min at 3500g and the supernatant was evaporated to 100 μL. For swabs, 500 μL of 0.1% trifluoroacetic acid in acetonitrile containing doxycycline-IS was added to each swab and centrifuged at 3500g for 15 min. The eluate was centrifuged again at 10,000g for 5 min and 350 μL of the supernatant was transferred to a 96-well plate and evaporated to 100 μL. Tissue biopsies were processed by adding 500 μL of 0.1% trifluoroacetic acid in acetonitrile containing doxycycline-IS, sonicating for 30 min at room temperature and 350 μL of the supernatant was transferred to a 96-well plate and evaporated to 100 μL. After evaporation, 100 μL of Buffer A (0.1% trifluoroacetic acid in water) was added to all plasma, swab and biopsy specimens. Urine specimens were diluted 1:10 in 0.1% trifluoroacetic acid in water containing doxycycline-IS.

Ten μL of resuspended samples was injected onto a 100 × 1 mm UnisonCK C18 column (Imtakt, Portland, OR, USA) attached to a Shimadzu Nexera chromatography system (Shimadzu Scientific Instruments) connected to a Sciex 4500 Triple Quadrupole mass spectrometer (Sciex). Samples were separated on a gradient from 10 to 90% 0.1% trifluoroacetic acid in acetonitrile over 4 min, with a flow rate of 0.2 mL/min. Doxycycline and doxycycline-IS were analysed in positive ion mode (ESI+). Mass transitions (Q1→Q3) were 445.0/428.0 m/z and 445.0/321.0 m/z for doxycycline and 448.0/431.0 m/z for doxycycline-IS. Data were analysed and drug concentrations were estimated from standard curves with a range of 0.0005–2 μg/mL using Analyst 1.7.1 software (Sciex). Doxycycline measures are reported as μg/mL for plasma and urine, μg/swab for rectal, vaginal, urethral and glans surface swabs, and μg/g for tissue biopsies. Doxycycline concentrations in rectal, vaginal and urethral secretions were estimated according to the amount of material collected on swabs. Estimated average material collected on swabs was determined as the difference between post- and pre-collection weight of 25 swabs. Estimated material collected was 50 mg/swab, 100 mg/swab, 5 mg/swab and 2 mg/swab for rectal, vaginal, glans surface and urethral swabs, respectively. A weight density of 1 g/mL was used to convert the weight of material collected on swabs to the estimated volume of secretions collected for conversion of material collected to volume of secretions collected. The lower limit of quantification (LLOQ) was 0.01 μg/mL for plasma and urine and 0.0025 μg/sample for swabs and tissue biopsies. Assay accuracy and precision ranged from 89 to 112% and 4 to 8%, respectively. Measurements below LLOQ were assigned a value of one-half LLOQ for statistical calculations.

Statistical analysis

The 90% minimum inhibitory concentration (MIC₉₀) of doxycycline for C. trachomatis and T. pallidum used in this analysis was 0.064 and 0.1 μg/mL, respectively.^19,20 MIC values for tetracycline determined by standardized methods were used for N. gonorrhea susceptibility to doxycycline because they closely resemble MIC values for doxycycline.²¹ Tetracycline-sensitive N. gonorrhoeae were classified as having an MIC less than 0.25 μg/mL and tetracycline-resistant N. gonorrhoeae were classified as having an MIC greater than 2 μg/mL.²² Concentrations above 4 times the MIC were evaluated in this study as a previous study demonstrated doxycycline at concentrations greater than 4 times the MIC showed bactericidal activity.²³

Pharmacokinetic parameters were calculated using non-compartmental analysis with Phoenix WinNonlin version 8.3 (Certara, Princeton, New Jersey, USA). A noncompartmental approach was used to analyse individual concentration–time data. Maximum doxycycline concentration (C_max) and time to maximum doxycycline concentration (T_max) were taken directly from observed data. Elimination half-life (T_1/2) was calculated from the concentration after T_max until the final measurable concentration for each individual completing all study visits between 1 and 96 h after dosing (7 female and 8 male participants). Area under the curve (AUC) measures were calculated from 0 to 24 h and 0 to 96 h using the sparse sampling function and linear trapezoidal rule. AUC_0–24h and AUC_0–96h calculations were only performed on participants with samples collected at all study time points between 1 and 24 h or 1 and 96 h after dosing, respectively. Statistical calculations were performed using Prism 8 software (GraphPad Software, San Diego, California, USA). Unpaired comparisons between concentrations among male and female participants as well as between plasma and tissue, secretions or urine at each time point were calculated using a Mann–Whitney test with a two-sided α < 0.05 considered significant. All mean values were calculated as geometric mean values. A tissue to fluid density of 1 g/mL was used to compare tissue and plasma drug concentrations.

Ethics

This study was approved by Emory University and CDC Institutional Review Boards with approval numbers STUDY00002242 and 0900f3eb81cd28a6, respectively. The study protocol is provided as Supplemental Data. There were no major changes to the trial conduct or analysis after study commencement. All participants were recruited from existing Emory University study databases and the Atlanta community and gave written informed consent. The trial conforms to the US Federal Policy for the Protection of Human Subjects.

Role of funders

This work was funded by CDC intramural funds, CDC contract HCVJCG-2020-45044 (to CFK). The Funders had no role in study design, data collection, data analyses, interpretation, or writing of the report. Study design, data collection, analysis, interpretation and writing of the manuscript, as well as the decision to submit the manuscript for publication were done solely by the authors.

Results

Study participants

Thirty-one prospective participants were screened for this study with 11 male and nine female participants enrolling in the study (Fig. 1). Male participants had a median age of 38 years (interquartile range: 24–48 years) and a body-mass index of 25 kg/m² (22–41 kg/m²) (Table 1). Female participants had a median age of 34 years (26–44 years) and a median body-mass index of 24 kg/m² (24–36 kg/m²). Most participants were Black or African American (55%). There were no adverse events reported during this study.

Fig. 1.

Participant flow for single oral dose doxycycline.

Table 1.

Demographics of male and female participants receiving a single oral dose of doxycycline.

	Male (n = 11)	Female (n = 9)
Age	38 (24–48)	34 (26–44)
Weight (kg)	77 (70–122)	70 (60–98)
Height (cm)	178 (175–183)	165 (165–169)
Body-mass index (kg/m2)	25 (22–41)	24 (24–36)
Race/Ethnicity
Black	6	5
White	5	4

Data are presented as median (interquartile range).

Doxycycline concentrations in plasma, rectal and vaginal secretions

Doxycycline concentrations in plasma, rectal, and vaginal specimens following dosing are depicted together in Fig. 2, and individually in Supplemental Figures S1, S2a and b, respectively. Mean plasma doxycycline concentrations among male and female participants were highest at 4 and 2 h after dosing for male and female participants, respectively, and remained measurable for at least 96 h in all participants with a T_1/2 of 22 and 18 h for male and female participants, respectively (Supplemental Figure S1). Doxycycline was measurable in 17 of 17 (100%) and three of 16 (19%) plasma specimens collected four and seven days after dosing, respectively. Plasma C_max, T_1/2 and AUC_0–96h appeared similar between male and female participants (Table 2).

Fig. 2.

Mean plasma doxycycline concentrations and estimated mean rectal and vaginal secretion concentrations collected following a single oral dose in 11 male and 9 female participants. Geometric mean concentrations and 95% confidence intervals are presented from 1 to 96 h following dosing for male (a) and female (b) participants. Plasma, rectal secretions and vaginal secretions are represented by circles, squares and triangles, respectively. The dotted line indicates the lower limit of quantification (LLOQ) for doxycycline measurements (0.01 μg/mL).

Table 2.

Mean plasma pharmacokinetic parameters among male and female participants receiving a single dose of doxycycline.

	Cmax (μg/mL)a	Tmax (hours)b	T1/2 (hours)a	AUC0–96h (μg∗h/mL)a
Male (n = 11)	0.925 [0.768–1.112]	4 [1–4]	22 [19–25]	31.295 [25.231–38.815]
Female (n = 9)	1.319 [1.028–1.692]	2 [1–4]	18 [16–21]	37.264 [30.662–45.288]
Combined (n = 20)	1.042 [0.889–1.222]	2 [1–4]	20 [19–22]	33.951 [29.632–38.899]

^aValues presented as geometric mean [95% confidence interval].

^bValues presented as median [interquartile range].

Rectal swab doxycycline measurements peaked later than in plasma at 48 and 24 h after dosing in male and female participants, respectively, with a mean measurement across all rectal swabs of 0.036 μg/swab (95% confidence interval (CI): 0.014–0.098 μg/swab) (Supplemental Figure S2a). Twelve of 16 (75%) and five of 15 (33%) rectal swabs collected four and seven days after dosing, respectively, contained measurable doxycycline and rectal swab doxycycline measurements were similar between male and female participants (Supplemental Figure S2a). Vaginal swab doxycycline measurements peaked 8 h after dosing, earlier than on rectal swabs, with a mean measurement of 0.128 μg/swab (95% CI: 0.074–0.222 μg/swab) (Supplemental Figure S2b). Eight of eight (100%) and one of seven (14%) vaginal swabs collected four and seven days after dosing, respectively, contained measurable doxycycline.

Estimated greatest doxycycline concentrations in rectal secretions were similar to those in plasma for male participants (Fig. 2a) but cumulative drug exposure over four days in rectal secretions was greater than in plasma with an AUC_0–96h ratio of 3.03 (Table 3). Estimated greatest doxycycline concentrations in vaginal secretions were similar to those in plasma for female participants and cumulative doxycycline exposure in vaginal secretions appeared greater than in plasma with an AUC_0–96h ratio of 3.14 (Table 3).

Table 3.

Estimated pharmacokinetic parameters in plasma, rectal and vaginal secretions among male and female participants receiving a single dose of doxycycline.

	Cmax (μg/mL)a	Tmax (hours)b	T1/2 (hours)a	AUC0-96 (μg∗h/mL)a	Secretion: plasma ratio
Male (n = 11)
Plasma	0.925 [0.768–1.112]	4 [1–8]	22 [19–25]	33.295 [25.231–38.815]
Rectal secretions	0.882 [0.233–3.334]	48 [2–72]	11 [6–19]	94.936 [29.875–301.692]	3.03 [1.03–8.93]
Female (n = 9)
Plasma	1.319 [1.028–1.692]	2 [1–4]	18 [16–21]	37.264 [30.662–45.288]
Vaginal secretions	1.284 [742–2223]	8 [1–8]	20 [16–24]	58.562 [32.719–104.816]	3.14 [1.62–4.77]
Rectal secretions	0.614 [0.077–4.846]	24 [2–48]	19 [8–42]	51.915 [14.004–192.462]	1.46 [0.35–6.14]
Combined (n = 18)
Plasma	1.041 [0.902–1.202]	4 [2–8]	20 [19–22]	33.020 [28.927–37.692]
Rectal secretions	0.704 [0.311–1.596]	48 [2–72]	14 [9–23]	73.511 [34.513–156.572]	2.22 [1.03–6.09]

^aValues presented as geometric mean [95% confidence interval].

^bValues presented as median [interquartile range].

Mean plasma doxycycline concentrations remained at least four times the MIC₉₀ for T. pallidum 30 and 34 h after dosing among male and female participants, respectively, and above the MIC₉₀ for at least 70 h (Table 4). While mean plasma doxycycline concentrations remained above the MIC for tetracycline-sensitive N. gonorrhoeae for 46 and 45 h after dosing among male and female participants, respectively, plasma doxycycline reached four times the MIC for less than 8 h. Mean plasma doxycycline AUC_0–24h/MIC ratios for T. pallidum were 163 and 209 among male and female participants, respectively, yet was less than 100 for tetracycline-sensitive N. gonorrhoeae (Table 5). Estimated mean doxycycline in rectal secretions attained concentrations greater than the MIC₉₀ for C. trachomatis and T. pallidum less than 4 h after dosing among male and female participants. Estimated mean doxycycline concentrations in rectal secretions among male participants remained at least four times the MIC₉₀ for 61 h after dosing and above the MIC₉₀ for 91 h (Table 4). Estimated mean doxycycline concentrations in rectal secretions among female participants remained above 4 times the MIC₉₀ and above the MIC₉₀ for 50 and 69 h, respectively. Additionally, estimated rectal secretion doxycycline concentrations reached the MIC for tetracycline-sensitive N. gonorrhoeae less than 8 h after dosing and remained above that concentration for 75 and 60 h among male and female participants, respectively. Estimated doxycycline concentrations in rectal secretions did not reach four times the MIC for tetracycline-sensitive N. gonorrhoeae. Estimated doxycycline AUC_0–24h/MIC ratios in rectal secretions were greater than 150 for C. trachomatis and T. pallidum yet were less than 75 for tetracycline-sensitive N. gonorrhoeae (Table 5).

Table 4.

Time estimated geometric mean concentrations remain above minimum inhibitory concentrations in hours in plasma, rectal and vaginal secretions among male and female participants receiving a single dose of doxycycline.

	C. trachomatisa		T. palliduma		N. gonorrhoeaeb
	1× MIC	4× MIC	1× MIC	4× MIC	1× MIC	4× MIC
Male (n = 11)
Plasma	91 [82–98]	45 [38–54]	76 [67–79]	30 [21–39]	46 [38–55]	<0 [<0–8]
Rectal secretions	95 [63–103]	74 [<0–99]	91 [52–102]	61 [<0–96]	75 [<0–99]	<0 [<0–84]
Female (n = 9)
Plasma	84 [75–91]	45 [40–48]	70 [65–76]	34 [28–39]	45 [41–49]	7 [ 1–15]
Vaginal secretions	101 [49–108]	46 [34–70]	70 [44–99]	38 [ 25–64]	45 [34–70]	11 [<0–37]
Rectal secretionsc	102 [61–108]	59 [<0–92]	69 [56–105]	50 [<0–81]	60 [<0–93]	<0 [<0–54]
Combined (n = 20)
Plasma	87 [82–93]	45 [41–48]	71 [68–79]	32 [27–37]	45 [42–50]	4 [<0–8]
Rectal secretionsc	97 [66–105]	63 [20–92]	88 [61–102]	52 [<0–82]	62 [22–92]	<0 [<0–54]

^aValues presented as time geometric mean concentrations remain above 90% minimum inhibitory concentration (MIC) or 4 times MIC (hours) [95% confidence interval].

^bValues presented as time geometric mean concentrations remain above minimum inhibitory concentration (MIC) or 4 times MIC (hours) [95% confidence interval].

^cSeven female participants provided rectal secretions.

Table 5.

Area under the curve from 0 to 24 h to minimum inhibitory concentration ratios in plasma, rectal and vaginal secretions among male and female participants receiving a single dose of doxycycline.

	C. trachomatisa	T. palliduma	N. gonorrhoeaeb
Male (n = 11)
Plasma	254 [206–313]	163 [132–200]	65 [53–80]
Rectal secretions	222 [69–714 ]	142 [44–457]	57 [18–183]
Female (n = 9)
Plasma	326 [253–420]	209 [162–269]	83 [65–107]
Vaginal secretions	797 [519–1225]	510 [332–784]	204 [133–314]
Rectal secretionsc	268 [72–991]	171 [46–634]	69 [19–254]
Combined (n = 20)
Plasma	286 [244–334]	183 [156–214]	73 [62–86]
Rectal secretionsc	240 [112–512]	153 [72–328]	61 [29–111]

^aValues presented as ratio of area under the curve from 0 to 24 h to 90% minimum inhibitory concentration (AUC_0–24h/MIC) [95% confidence interval].

^bValues presented as ratio of area under the curve from 0 to 24 h to minimum inhibitory concentration (AUC_0–24h/MIC) [95% confidence interval].

^cSeven female participants provided rectal secretions.

Doxycycline in vaginal secretions achieved estimated concentrations more than four times above the MIC₉₀ for C. trachomatis and T. pallidum less than 2 h after dosing and remained above four times the MIC₉₀ values for 38 h and greater than the MIC₉₀ values for more than 70 h (Table 4). Estimated vaginal secretion doxycycline concentrations also reached the MIC for tetracycline-sensitive N. gonorrhoeae less than 2 h after dosing but remained above the MIC for only 45 h. Estimated mean rectal and vaginal secretion doxycycline concentrations were unable to achieve concentrations above the MIC for tetracycline-resistant N. gonorrhoeae. Estimated doxycycline AUC_0–24h/MIC ratios in vaginal secretions were greater than 500 for C. trachomatis and T. pallidum yet were less than 250 for tetracycline-sensitive N. gonorrhoeae (Table 5).

Doxycycline concentrations in vaginal and rectal tissues

Doxycycline concentrations in vaginal and cervical tissues 24 h after dosing were more variable than those measured in rectal tissues (percent coefficient of variation: rectal tissue = 39%, vaginal tissue = 103%, cervical tissue = 71%) (Fig. 3), yet mean doxycycline concentrations appeared similar among rectal, vaginal, and cervical tissues. Mean male rectal tissue doxycycline concentrations (0.596 μg/g; 0.442–0.803 μg/g) appeared similar to female rectal tissue doxycycline concentrations (0.658 μg/g; 0.411–1.054 μg/g) (Fig. 3). Mean rectal tissue doxycycline concentrations 24 h after dosing were similar to corresponding plasma concentrations (male: 0.466 μg/mL; 0.378–0.573 μg/mL, female: 0.491 μg/mL; 0.373–0.645 μg/mL) and were more than five times the MIC₉₀ values for C. trachomatis and T. pallidum, and more than twice the MIC for tetracycline-sensitive N. gonorrhoeae but remained below the MIC for tetracycline-resistance N. gonorrhoeae. Mean vaginal (0.261 μg/g; 0.098–0.696 μg/g) and cervical tissue (0.410 μg/g; 0.193–0.870 μg/g) doxycycline concentrations 24 h after dosing were more than three times greater than the MIC₉₀ for C. trachomatis and T. pallidum and less than two-fold greater than the MIC for tetracycline-sensitive N. gonorrhoeae, yet lower than the MIC for tetracycline-resistant N. gonorrhoeae.

Fig. 3.

Doxycycline concentrations in plasma, rectal, vaginal and cervical tissues, and estimated rectal and vaginal secretions concentrations collected 24 h after a single oral dose from 11 male and 9 female participants. Shaded and open symbols represent male and female participant specimens, respectively. Horizontal dotted line indicates the lower limit of quantification (LLOQ) for doxycycline measurements (0.01 μg/mL, 0.025 μg/swab or 0.025 μg/tissue sample).

Doxycycline concentrations in penile specimens

Doxycycline was measurable on 9/11 and 3/11 male urethral and glans surface swabs collected 24 h after dosing, respectively (Fig. 4). The estimated mean doxycycline concentration in urethral secretions was estimated at 1.166 μg/mL (95% CI: 0.568–2.394 μg/mL), more than ten times the MIC₉₀ for C. trachomatis, T. pallidum and more than four times the MIC for tetracycline-sensitive N. gonorrhoeae. However, urethral secretion doxycycline concentrations were below the MIC for tetracycline-resistant N. gonorrhoeae. Mean urine doxycycline concentrations among male participants 24 h after dosing were 13.549 μg/mL (7.047–26.050 μg/mL), more than ten times the estimated concentration of doxycycline in urethral secretions (Fig. 4).

Fig. 4.

Doxycycline measurements in plasma and urine, and estimated concentrations in urethral and glans surface secretions in samples collected 24 h after a single oral dose from 11 male participants. The dotted line indicates the lower limit of quantification (LLOQ) for doxycycline measurements (0.01 μg/mL or 0.025 μg/swab).

Plasma and urine doxycycline concentrations as adherence markers

Plasma doxycycline concentrations among male and female participants remained above 0.300 and 0.025 μg/mL one and four days after dosing, respectively (Fig. 5). Urine doxycycline concentrations were consistently higher than those in plasma with a urine to plasma ratio of 38:1 (9:1–103:1) and remained measurable longer than in plasma. Mean urine doxycycline concentrations peaked 4 h after dosing for male participants (14.042 μg/mL; 95% CI: 9.825–33.944 μg/mL) and 8 h after dosing for female participants (26.051 μg/mL; 95% CI: 13.974–48.566 μg/mL) (Fig. 5) and remained greater than 1.200 μg/mL and 0.100 μg/mL for all participants two and four days after dosing, respectively. Additionally, 14/15 urine specimens (93%) collected seven days after dosing contained doxycycline concentrations greater than the LLOQ of 0.010 μg/mL. Therefore, plasma doxycycline adherence markers were determined to be 0.300 and 0.025 μg/mL at one and four days after dosing, respectively, while urine doxycycline adherence markers were determined to be 3.000, 0.350 and 0.010 μg/mL at one, four and seven days after dosing, respectively.

Fig. 5.

Mean doxycycline concentrations in urine and plasma collected following a single oral dose in 11 male and 9 female participants. Geometric mean concentrations and 95% confidence intervals are presented from 1 to 168 h after dosing for male (a) and female (b) participants. Urine and plasma are represented by triangles and circles, respectively. Shaded and open symbols represent male and female participant specimens, respectively. The dotted line indicates the lower limit of quantification (LLOQ) for doxycycline measurements (0.01 μg/mL).

Discussion

Event-driven PEP for bacterial STIs requires antibiotics to rapidly reach anatomical sites of STI exposure and persist at effective concentrations long enough to prevent establishment of infection. We examined doxycycline exposures in the rectum, vagina and urethra following a single oral dose of doxycycline. Because a pharmacologic correlate of STI prevention is currently undefined, we related doxycycline exposures to the MIC values of doxycycline-sensitive C. trachomatis, T. pallidum and N. gonorrhoeae to gain insights into relative pharmacodynamic effects of doxycycline. We found doxycycline efficiently doses rectal, vaginal, and urethral secretions attaining concentrations between three and 20 times reported MIC₉₀ values for bacterial STIs. We also observed a single dose maintains doxycycline concentrations above MIC₉₀ values in rectal and vaginal secretions for up to four days resulting in doxycycline exposures approximately twice those in plasma. Persistent inhibitory doxycycline concentrations likely explain the STI protection as PEP observed among MSM in clinical trials and suggest lack of demonstrated efficacy against chlamydia among women in one trial is likely not due to inadequate doxycycline concentrations in vaginal tissues compared to blood.

Both the level and duration of doxycycline exposures likely contribute to the pharmacologic basis for the high clinical effectiveness (70–89%) of event-driven doxycycline PEP against chlamydia and syphilis among MSM.9, 10, 11 It is also possible bacterial-related factors contribute to the high clinical efficacy. Both C. trachomatis and T. pallidum have slow replication kinetics between 40 to 48 and 40 to 60 h, respectively, which is within the PEP dosing window of 72 h recommended during clinical efficacy trials resulting in low bacterial burden at the time of dosing that may be easier to inhibit.^20,24 While C. trachomatis infection generally remains restricted to the genital tract and may be inhibited by high doxycycline concentrations at sites of exposure, T. pallidum rapidly disseminates systemically within 24 h following exposure and potentially prior to likely doxycycline PEP dosing.25, 26, 27 Thus, systemic doxycycline concentrations achieved with oral dosing may play a role in inhibiting systemic T. pallidum replication. Our data documenting plasma doxycycline concentrations greater than four times the T. pallidum MIC₉₀ for more than a day and greater than the MIC₉₀ for nearly three days also support a potential role in systemic inhibition of T. pallidum infection. Because syphilis diagnosis depends on seroconversion or increase in antibody titres, it is unclear if doxycycline will affect syphilis diagnostic testing in some individuals through delays in seroconversion or reductions in antibody titers.²⁸

The effectiveness of on-demand doxycycline PEP against gonorrhoea (up to 55%) among MSM was lower than against chlamydia and syphilis in three independent clinical trials.9, 10, 11 One reason for diminished efficacy of doxycycline against gonorrhoea is the transmission of tetracycline-resistant N. gonorrhoeae which varies dramatically by geographic area.²⁹ For example, reported tetracycline-resistant N. gonorrhoeae prevalence was at least 56% in France, the setting of the IPERGAY and DOXYVAC trials, and 28% in the United States, the setting of the DoxyPEP trial.^30,31 Our pharmacologic data show rectal, vaginal, and penile doxycycline concentrations do not attain MIC values of tetracycline-resistant N. gonorrhoeae isolates (2 μg/mL) suggesting limited protection against resistant isolates consistent with single dose doxycycline treatment data.³² In contrast, rectal doxycycline concentrations remain above the MIC value for tetracycline-sensitive N. gonorrhoeae isolates (0.25 μg/mL) for two to three days which may inhibit replication and potentially contribute to protection against tetracycline-sensitive N. gonorrhoeae. However, N. gonorrhoeae has rapid replication kinetics with a reported 60-min doubling time; therefore, it is unclear how timing of PEP dosing in relation to exposure may affect PEP efficacy.³³ The finding that 62% of breakthrough N. gonorrhoeae infections in the DoxyPEP study were tetracycline-sensitive suggests clinical efficacy against tetracycline-sensitive N. gonorrhoeae remains lower than against C. trachomatis and T. pallidum arguing for improved doxycycline modalities for gonorrhoea prevention.¹⁰

The lack of doxycycline PEP efficacy against chlamydia reported in a trial among women in Kenya was extremely surprising in light of demonstrated efficacy among MSM. While low adherence to the PEP regimen offers a primary explanation for the lack of efficacy in the D-PEP study, our pharmacologic data in women predict efficacy in STI prevention.¹³ Our results show doxycycline rapidly reached vaginal tissues, reached 20 times the MIC₉₀ value for C. trachomatis, and persisted at concentrations above the MIC₉₀ values for C. trachomatis and T. pallidum for at least three days and the MIC for tetracycline-sensitive N. gonorrhoeae for at least two days thus predicting similar clinical efficacy against vaginal exposure to bacterial STIs for women as observed in MSM. Our findings showing that vaginal doxycycline concentrations do not attain MIC values of tetracycline-resistant N. gonorrhoeae isolates helps explain the lack of any efficacy against the predominant isolates documented in this trial. We noted higher variability in vaginal and cervical tissue doxycycline concentrations compared to rectal tissues collected 24 h after dosing including 3 of 8 and 1 of 8 vaginal and cervical tissue samples, respectively, that contained undetectable doxycycline concentrations despite high doxycycline concentrations in paired plasma and vaginal secretions. Interindividual variability in doxycycline penetration into the female reproductive tract or genital foci with low doxycycline concentrations may also contribute to incomplete protection by doxycycline against vaginal STI exposure. Biological factors such as differences in STI pathogenesis and pH in the rectum and vagina may differentially affect the ability of doxycycline to prevent STI infection in the vagina compared to the rectum. Additional studies with adherence monitoring are needed to define efficacy of doxycycline PEP in women.

Doxycycline concentrations in urethral secretions were far greater than MIC values for C. trachomatis, T. pallidum, and tetracycline-sensitive N. gonorrhoeae 24 h after dosing and suggest doxycycline penetration into the urethra provides additional protection for men against bacterial STIs. We observed high concentrations of doxycycline in urine consistent with previously published data regarding elimination of doxycycline.³⁴ With urine concentrations being more than ten times greater than those observed in urethral secretions, it is unclear to what extent urinary excretion of doxycycline contributes to urethral STI protection.

The data presented here provides pharmacokinetic data that is associated with clinical efficacy in protection against STIs for MSM. The relationship between pharmacokinetics and pharmacodynamics for on-demand doxycycline PEP dosing to prevent STIs is particularly challenging to define given a 72-h post-exposure window for dosing and irregular dosing patterns among study participants. While several pharmacologic parameters and criteria exist for predicting antimicrobial efficacy including C_max to MIC ratio, time above MIC and AUC to MIC ratio, these have yet to be defined for on-demand STI prevention.³⁵ The ratio of free-drug AUC to MIC has been shown to be the parameter most predictive of doxycycline treatment efficacy for bacterial infections; however further studies will be needed to elucidate the predictive value of AUC_0–24h/MIC ratios for STI prevention and define the most protective ratios for STI prevention.

Persistent doxycycline concentrations following a single oral dose likely contribute to the high protection of doxycycline PEP against bacterial STIs but may have additional implications particularly for persons who frequently use this intervention. For example, the IPERGAY and DoxyPEP studies each reported participants took a median of four doses per month, but 25% of participants in each study took at least seven and ten doses per month, respectively.^9,10 Thus, a substantial portion of doxycycline PEP users will likely dose frequently enough to consistently maintain measurable doxycycline concentrations in the rectum, vagina or urethra. Therefore, frequency of doxycycline PEP use may need to be considered when evaluating the impact of doxycycline PEP on the microbiome or the emergence of antimicrobial resistance.

Objective measures of adherence to event-driven dosing strategies are challenging given irregular dosing patterns; however, persistence of doxycycline concentrations in plasma and urine could provide markers of recent drug dosing. Based on our data, the long half-life of doxycycline in plasma allows for plasma concentrations greater than 0.025 μg/mL to indicate dosing within the previous four days. Urine doxycycline concentrations were greater than those found in plasma and urine concentrations remained measurable up to a week after dosing. Persistent doxycycline excretion in urine allows urine concentrations greater than 0.300 and 0.010 μg/mL to indicate dosing within the previous four and seven days, respectively. Additionally, hair testing provides a marker of cumulative doxycycline exposures over time.¹³ Incorporating these adherence markers in future clinical trials can help further refine efficacy measurements.

This study is limited in that we only examined drug distribution following a single 200 mg oral dose of doxycycline and did not measure drug accumulation after multiple doses. This study was conducted using delayed release doxycycline hyclate that reduces gastrointestinal issues often related to doxycycline. While different formulations may produce pharmacokinetic differences, the IPERGAY and DOXYVAC studies used doxycycline monohydrate and showed similar efficacy against STIs among MSM compared to the DoxyPEP study that used delayed release doxycycline hyclate.9, 10, 11 Additionally, we did not collect fluids from the oral pharynx which is another site of STI exposure and the IPERGAY and DoxyPEP studies reported between 40 and 50% of N. gonorrhoeae positive test results were from pharyngeal swabs.^9,10 Doxycycline is known to be highly protein-bound in plasma and possibly to a lesser degree in vaginal secretions as is observed with other drugs.^36,37 We correlated doxycycline concentrations with commonly used MIC values that have not been adjusted to protein binding in plasma, rectal or vaginal secretions because such values are not available. The lack of standardized antimicrobial susceptibility methods for C. trachomatis and T. pallidum may limit the accuracy of determining the pharmacokinetic correlates of STI prevention.³² These limitations should be considered when interpreting pharmacokinetic parameters and MIC values. This study is also limited as a small single site study that may not be applicable to all populations. Rectal and vaginal specimens are highly heterogeneous specimens collecting a mixture of cells, tissues, mucus, secretions and other material. Rectal, vaginal and urethral secretion doxycycline concentrations were estimated using the weight of material collected as a proxy for secretion volume which may limit calculated doxycycline concentrations in these specimens. The small study size and high level of variability in intra- and inter-individual vaginal and rectal doxycycline measurements suggests additional studies may be necessary to further refine pharmacokinetic estimates for the rectum and vagina.

In conclusion, this study provides pharmacokinetic data demonstrating efficient distribution of doxycycline to the rectum, vagina, and urethra following a single oral dose in men and women. These findings will help inform doxycycline prophylaxis recommendations for bacterial STI prevention.

Contributors

R.E.H.: study design, data acquisition and analysis, interpretation of results and manuscript preparation. J.F.: study design, data acquisition, data analysis. C.D.: data acquisition and analysis. T.E.E.: data acquisition and analysis. A.M.: data acquisition and analysis. D.O.: study design, data acquisition. C.C.W.: study design, data acquisition. C.F.K.: study design, data acquisition and analysis, interpretation of results. W.H.: study design, interpretation of results and manuscript preparation. R.E.H., J.F., C.C.W and C.F.K. have verified the data associated with this manuscript. All authors have read and approved the final version of the manuscript.

Data sharing statement

The deidentified datasets generated and analysed during this study are available from the corresponding author upon reasonable request after publication.

Declaration of interests

C.F.K. reports grants to her institution from Moderna, Novavax, Gilead, ViiV and Humanigen for work not related to this manuscript. All other authors have no competing interests to declare. The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official position of the United States Centers for Disease Control and Prevention or the Department of Health and Human Services.