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Detectable Tenofovir Levels in Breast-Feeding Infants of Mothers Exposed to Topical Tenofovir Lisa M. Noguchi,a Elizabeth T. Montgomery,b Joseph R. Biggio,c Craig W. Hendrix,d Debra L. Bogen,e Sharon L. Hillier,f James Y. Dai,g Jeanna M. Piper,h Mark A. Marzinke,i Charlene S. Dezzutti,f S. Karen Isaacs,j* Jill L. Schwartz,k D. Heather Watts,l* Richard H. Beigif Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAa; Women’s Global Health Imperative, RTI International, San Francisco, California, USAb; Department of Obstetrics and Gynecology, University of Alabama, Birmingham, Alabama, USAc; Department of Medicine (Clinical Pharmacology), Johns Hopkins University, Baltimore, Maryland, USAd; Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania, USAe; Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USAf; Statistical Center for HIV/AIDS Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAg; Division of AIDS/NIAID, U.S. National Institutes of Health, Bethesda, Maryland, USAh; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAi; Science Facilitation, FHI 360, Durham, North Carolina, USAj; CONRAD/Eastern Virginia Medical School, Arlington, Virginia, USAk; Maternal and Pediatric Infectious Disease Branch/NICHD/U.S. National Institutes of Health, Bethesda, Maryland, USAl
Lactation studies are necessary evaluations of medications for reproductive-age women. We evaluated pharmacokinetics (PK), pharmacodynamics, safety, and adherence profiles associated with 7 days of 1% tenofovir (TFV) vaginal gel use during lactation. Tenofovir levels (maternal/infant serum, milk) and anti-HIV activity (milk), adverse events (AEs), and adherence were measured for 17 HIV-1-seronegative breast-feeding mother-infant pairs. Tenofovir use was well-tolerated and detected at low levels in maternal serum, milk, and infant serum but demonstrated no anti-HIV activity in milk.
E
xtended periods of breast-feeding are common in many countries with high HIV-1 incidence (1), and normal physiologic changes in breast-feeding women may impact drug safety and pharmacokinetics (2). Thus, biomedical HIV prevention strategies for women such as preexposure prophylaxis (PrEP) and topical microbicides should include those suitable during breastfeeding, especially considering higher infant transmission risk associated with incident maternal HIV-1 infection (3). However, both oral and topical HIV-1 chemoprophylaxis studies typically exclude breast-feeding women (1). The U.S. Food and Drug Administration (FDA) has released draft guidance recommending lactation studies for drugs expected for use by reproductive-age women (4). Here, we describe the first pharmacokinetic (PK) and safety study of topical antiretroviral (ARV) administration among breast-feeding mother-infant pairs. In CAPRISA 004, participants randomly assigned to topical tenofovir (TFV) treatment had 39% reduction in HIV-1 (5) and halved risk of herpes simplex virus 2 (HSV-2) acquisition (6) compared to participants treated with placebo. Subsequent trials found no similar protection, presumably due to low adherence (7). Recent results from ASPIRE (8) and The Ring Study (A. Nel, S. Kapiga, L. Bekker, et al., Conference on Retroviruses and Opportunistic Infections [CROI], Boston, MA, 22 February 2016) showed protective benefit against HIV-1 infection for women using a dapivirine (DPV) intravaginal ring. Other studies are investigating tenofovir-containing intravaginal rings and drug transfer to breast milk following use of a DPV intravaginal ring (NCT02431273 and NCT02658227) (9). Previous work supports safety of single-dose topical TFV in HIV-1-uninfected pregnant women (10) and oral tenofovir disoproxil fumarate (TDF) treatment for HIV-1-infected pregnant women (11). Oral TDF has not been associated with high serum or milk levels during breast-feeding (12, 13). Low oral bioavailability for TFV has been assumed to preclude or reduce absorption by breast-feeding infants, although previously no data were available (14). While TDF is not Food and Drug Administration approved or recommended for use in neonates or infants under 2 years, it is
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approved for children aged 2 to 12 years old when given as part of an antiretroviral therapy (ART) regimen. Treatment regimens containing TDF have been associated with gastrointestinal side effects (e.g., flatulence, diarrhea), as well as less common but more serious problems, such as bone and renal toxicity (15). MTN-008 (NCT01136759) was conducted in Pittsburgh, PA, and Birmingham, AL (2011 to 2013). The participants were HIV1-negative women and their breast-feeding infants 4 to 26 weeks old. Women received first and last doses of 1% TFV gel intravaginally in the clinic on days 0 and 6 and self-inserted five daily doses between the first and last doses. The formulation of the TFV gel was identical to the product used for CAPRISA 004 and the VOICE study. On days 0 and 6, milk and blood samples were collected predose and 2, 4, and 6 h postdose; infant blood samples were collected 6 h after maternal dosing (1 to 4 h after breastfeeding). Study investigators made judgments about the potential relatedness of an adverse event (AE) to product exposure prior to pharmacokinetic analyses. Institutional review board approval was obtained, and participants provided written informed consent. Blood was centrifuged after collection (ⱕ8 h), with serum frozen at ⫺20°C. Milk was swirled to mix and transferred by pipette
Received 20 March 2016 Returned for modification 22 April 2016 Accepted 26 June 2016 Accepted manuscript posted online 11 July 2016 Citation Noguchi LM, Montgomery ET, Biggio JR, Hendrix CW, Bogen DL, Hillier SL, Dai JY, Piper JM, Marzinke MA, Dezzutti CS, Isaacs SK, Schwartz JL, Watts DH, Beigi RH. 2016. Detectable tenofovir levels in breast-feeding infants of mothers exposed to topical tenofovir. Antimicrob Agents Chemother 60:5616 –5619. doi:10.1128/AAC.00645-16. Address correspondence to Lisa M. Noguchi, [email protected]. * Present address: S. Karen Isaacs, Parexel International, Durham, North Carolina, USA; D. Heather Watts, Office of the Global AIDS Coordinator, U.S. Department of State, Washington, DC. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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TABLE 1 Participant characteristics Characteristica Mothers (n ⫽ 17) Age, yr [median (IQR)] Have primary male partner [n (%)] Married [n (%)] Race [n (%)] Black White American Indian/Alaskan native, Asian, and black Asian and white Biracial, unspecified Wt (kg) [median (IQR)] Baseline creatinine clearance (ml/min) [median (IQR)] Infants (n ⫽ 17) Age, wk [median (IQR)] Wt (kg) [median (IQR)] a
Value for characteristic 27.1 (23.0–29.0) 13 (76.5) 8 (47.1) 7 (41.2) 7 (41.2) 1 (5.9) 1 (5.9) 1 (5.9) 75 (63–90) 122 (98–156)
10.0 (8.1–13.3) 6 (5–6)
Abbreviation: IQR, interquartile range.
into cryogenic vials, with aliquots frozen immediately at ⫺20°C or lower. Tenofovir concentrations were measured via liquid chromatography-tandem mass spectrometric analysis by validated methods (16, 17). Lower limits of quantitation (LLOQ) were 0.31 ng/ml (serum) and 1.0 ng/ml (milk). Pharmacodynamic (PD) activity was assessed by the TZM-bl assay, (18) using serial dilutions of milk from baseline and 6 h after last dose. Analyses were conducted using SAS 9.2, IBM SPSS 22.0, and Stata 12.1. Wilcoxon rank sum tests were used to compare TFV levels by matrix on day 0 versus 6. Seventeen pairs of women and infants enrolled in the study (Table 1). Tenofovir was detected in serum samples from all women following the first dose (Table 2). At day 6, serum tenofovir levels were detected for 56.3% of women predose and 100% of women postdose. A minority of milk samples had detectable TFV (25% at postdose day 0, 12.5% at predose day 6, and 37.5% at postdose day 6). The milk-to-maternal serum ratio for 18 samples
FIG 1 Anti-HIV activity in breast milk. Women in MTN-008 provided breast milk before (baseline) and after topical application of tenofovir (TFV) gel. Anti-HIV activity was measured before TFV treatment (baseline) and after TFV treatment (day 6/6 h) in specimens using the TZM-bl assay. Milk was modestly diluted (1:50) in assay medium. No difference in anti-HIV activity was noted between baseline and day 6 specimens (P ⫽ 0.14 by Wilcoxon matched-pair signed-rank test), indicating that no anti-HIV activity could be attributed to TFV gel use.
where both were higher than the LLOQ was 0.91 (median) (interquartile range [IQR] of 0.43 to 1.34). Eight infants were male, and nine were female. Six infants (37.5%) had detectable TFV after maternal dosing on day 0 (two females and four males) and 75% of infants (n ⫽ 12) postdose on day 6 (eight females and four males). Infant TFV concentrations were higher on day 6 than on day 0 (P ⬍ 0.03). Pharmacodynamic activity in milk demonstrated measurable innate anti-HIV activity (baseline specimen) (Fig. 1). Day 6 milk had no additional anti-HIV activity attributable to TFV beyond baseline (not statistically significant). Nine of 17 mothers experienced one or more AEs (total of 20 AEs), of which 9 (47%) were reproductive tract (all mild, 40% deemed related to gel, including genital burning, metrorrhagia, and diarrhea). Four of 17 infants had one or more AEs (total 8
TABLE 2 Tenofovir levels in maternal blood, breast milk, and infant blood Valueb for parameter in: Time and parametera
Maternal serum
Breast milk
Infant serum
Day 0 (n ⫽ 16) TFV detected postdose, n (%, 95% CI) Tmax, h (IQR) Cmax, ng/ml (IQR) AUC (IQR) Heel stick, ng/ml (IQR)
16 (100) 3.0 (1.9–5.4) 7.6 (4.0–62.6)d 40.5 (24.2–174.5)d
4 (25, 7.3–52.4) 5.0 (4.0–6.0)c 0.0 (0.0–0.75) 0.0 (0.0–1.5)
6 (37.5, 15.2–64.6)
Day 6 (n ⫽ 16) TFV detected predose, n (%, 95% CI) TFV detected postdose, n (%, 95% CI) Tmax (h) Cmax (ng/ml) AUC Heel stick (ng/ml)
9 (56.3, 29.9–80.2) 16 (100) 3.9 (1.2–4.0) 5.6 (3.4–22.6) 30.0 (19.8–101.2)
0.0 (0.0–1.1)
2 (12.5, 1.6–38.3) 6 (37.5, 15.2–64.6) 5.8 (2.9–6.0)c 0.0 (0.0–1.6) 0.0 (0.0–2.6)
12 (75.0, 47.6–92.7)
2.4 (0.3–4.5)d
a
Abbreviations: Tmax, time to peak concentration; Cmax, peak concentration; AUC, area under the curve; 95% CI, 95% confidence interval; IQR, interquartile range. b Values are median (IQR) values except for “TFV detected,” which are n (%, 95% CI by binomial exact test). c Tmax values represent those values where at least one concentration was detected. d P ⬍ 0.03 by Wilcoxon rank sum test of the value at day 0 versus the value at day 6.
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AEs; 7/8 mild; one infant had diarrhea that was deemed related to TFV exposure). Eleven (73%) women reported inserting five doses at home, two (13%) reported inserting four doses, and two (13%) reported inserting six doses. Vaginally administered TFV gel resulted in detectable TFV in milk and infant blood; however, only a small fraction of drug was transferred. While TFV is a small molecule, facilitating passage into milk, it has properties limiting transfer, including limited lipid solubility and protein binding (19). Despite expectations, TFV was detectable in blood at low concentrations for 12/16 infants. While our sample was too small to assess the potential presence of gender differences in pharmacokinetic or safety profiles, we did not observe any trends suggesting this. Breast milk TFV was quantifiable in only four women after dose 1 and in six women after dose 7, but there was no evidence of accumulation between doses. However, the number of infants with detectable serum TFV (n ⫽ 6) exceeded the number of mothers with detectable TFV. Similarly, on day 6, TFV was detected in milk samples from only two mothers predosing and six mothers at postdosing, but 12 infants had detectable TFV in their sera. These findings may be related to differences in assay sensitivity—the LLOQ for the milk (1.0 ng/ml) and serum (0.31 ng/ml) samples differ— or maternal and infant drug clearance and, therefore, accumulation differences. We identified significant increases in infant serum TFV levels on day 6 (with slightly lower serum TFV concentrations in mothers), consistent with modest TFV accumulation in blood samples from breast-feeding infants, though not in their mothers. However, the absolute TFV levels detected were ⱖ100-fold below treatment effective concentrations and unlikely of clinical concern. These findings are supported by no observable pharmacodynamic activity in milk beyond innate activity using the TZM-bl assay. While there was a small 1- to 2-h difference in the median time to peak concentration (Tmax) and the 6 h breast milk sample used for TZM-bl assay, a majority of these samples were coincident and, therefore, very closely representative of peak concentration and antiviral effect within the study population. Previous studies of maternal oral TDF use have not reported significant safety concerns for breast-feeding infants. In HPTN 057, TFV was detected in 3/4 milk samples collected within 2 days of delivery (6.3 to 17.8 ng/ml), and in 1/21 collected 4 to 6 days after delivery following peripartum oral TDF use by HIV-infected mothers (15.7 ng/ml) (13). In ANRS 12109, HIV-infected mothers administered oral TDF in labor and 7 days postpartum had milk TFV concentrations of 5.83 to 8.75 ng/ml (12). Using milk TFV concentrations averaging higher than those here, Benaboud and colleagues simulated plasma TFV concentrations as 0.03% of proposed prophylactic doses for neonates (12). This study had several strengths, including directly observed doses and measurement of multiple matrices over time. Our findings showing few AEs are consistent with other tenofovir studies of mothers and infants. However, our ability to characterize safety is limited if adherence were overreported. We did not observe any correlation between adherence to the self-administration protocol and TFV levels detected in the mothers or infants. However, our sample size was small, and self-reported adherence was high overall. Variability in volume, frequency, and/or timing of infant feeding may have impacted our PK results; the frequency and duration of infant feeding were captured for study days 0 and 6, while participants were on-site for study visits (not on interim study days). Estimating the volume of ingested breast milk by maternal report
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is imprecise, and techniques to approximate intake using infant weight before and after feeding were not used in MTN-008. The interval between topical TFV administration and the next breastfeeding session was controlled for time in the clinic on days 0 and 6, but not for self-administered doses on days 2 through 5. Thus, it is possible that time-dependent variations in TFV in breast milk may be related to differences in TFV ingestion. Medication use during breast-feeding should follow a careful risk/benefit assessment by the woman and her health care provider. Findings suggest low levels of drug transfer to milk and nursing infants associated with topically delivered TFV in mothers. Further study among breast-feeding mother-infant pairs should occur for topically formulated ARVs delivered via sustained release platforms, such as vaginal rings. ACKNOWLEDGMENTS We acknowledge the MTN-008 Lactation Cohort study participants, site staff, Community Advisory Boards, and site-affiliated lactation consultants, as well as the contributions of staff at the MTN Statistical and Data Management Center, FHI 360, the MTN Leadership and Operations Center, and the MTN Laboratory Center. C. W. Hendrix reports a grant from Gilead Science, outside the submitted work and a patent pending. S. L. Hillier reports an advisory relationship with Merck, outside the submitted work. J. L. Schwartz reports that CONRAD has a coexclusive license for tenofovir gel. None of the other authors report a commercial or other association that might pose a conflict of interest. The conclusions and opinions expressed in this article are those of the authors and do not necessarily reflect those of the National Institutes of Health or the U.S. Department of State.
FUNDING INFORMATION This work, including the efforts of Lisa M. Noguchi, Elizabeth T. Montgomery, Joseph R. Biggio, Craig W. Hendrix, Debra L. Bogen, Sharon L. Hillier, James Y. Dai, Jeanna M. Piper, Mark A. Marzinke, Charlene S. Dezzutti, S. Karen Isaacs, Jill Schwartz, D. Heather Watts, and Richard H. Beigi, was funded by HHS | National Institutes of Health (NIH) (UM1AI068633, UM1AI068615, UM1AI106707, and P30AI042855). This work was conducted by the Microbicide Trials Network, which is funded by the National Institute of Allergy and Infectious Diseases, with cofunding from the Eunice Kennedy Shriver National Institute of Child Health and Development and the National Institute of Mental Health (UM1AI068633, UM1AI068615, and UM1AI106707), all components of the National Institutes of Health. Tenofovir gel was provided by CONRAD (Arlington, VA) using funding from USAID. TFV assays were supported, in part, by the Johns Hopkins Center for AIDS Research (P30AI042855).
REFERENCES 1. Beigi RH, Noguchi L, Brown G, Piper J, Watts DH. 29 June 2013. Performing drug safety research during pregnancy and lactation: biomedical HIV prevention research as a template. J Womens Health (Larchmt) http://dx.doi.org/10.1089/jwh.2013.4398. 2. Fleishaker JC, Desai N, McNamara PJ. 1989. Possible effect of lactational period on the milk-to-plasma drug concentration ratio in lactating women: results of an in vitro evaluation. J Pharm Sci 78:137–141. http://dx.doi .org/10.1002/jps.2600780213. 3. Moodley D, Esterhuizen T, Reddy L, Moodley P, Singh B, Ngaleka L, Govender D. 2011. Incident HIV infection in pregnant and lactating women and its effect on mother-to-child transmission in South Africa. J Infect Dis 203:1231–1234. http://dx.doi.org/10.1093/infdis/jir017. 4. US Food and Drug Administration. 2005. Guidance for industry: clinical lactation studies – study design, data analysis, and recommendations for labeling. Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, MD.
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5. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, Mansoor LE, Kharsany AB, Sibeko S, Mlisana KP, Omar Z, Gengiah TN, Maarschalk S, Arulappan N, Mlotshwa M, Morris L, Taylor D, CAPRISA 004 Trial Group. 2010. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 329:1168 –1174. http://dx.doi.org/10.1126/science.1193748. 6. Abdool Karim SS, Abdool Karim Q, Kharsany AB, Baxter C, Grobler AC, Werner L, Kashuba A, Mansoor LE, Samsunder N, Mindel A, Gengiah TN, CAPRISA 004 Trial Group. 2015. Tenofovir gel for the prevention of herpes simplex virus type 2 infection. N Engl J Med 373: 530 –539. http://dx.doi.org/10.1056/NEJMoa1410649. 7. Marrazzo JM, Ramjee G, Richardson BA, Gomez K, Mgodi N, Nair G, Palanee T, Nakabiito C, van der Straten A, Noguchi L, Hendrix CW, Dai JY, Ganesh S, Mkhize B, Taljaard M, Parikh UM, Piper J, Masse B, Grossman C, Rooney J, Schwartz JL, Watts H, Marzinke MA, Hillier SL, McGowan IM, Chirenje ZM, VOICE Study Team. 2015. Tenofovirbased preexposure prophylaxis for HIV infection among African women. N Engl J Med 372:509 –518. http://dx.doi.org/10.1056/NEJMoa1402269. 8. Baeten JM, Palanee-Phillips T, Brown ER, Schwartz K, Soto-Torres LE, Govender V, Mgodi NM, Matovu Kiweewa F, Nair G, Mhlanga F, Siva S, Bekker LG, Jeenarain N, Gaffoor Z, Martinson F, Makanani B, Pather A, Naidoo L, Husnik M, Richardson BA, Parikh UM, Mellors JW, Marzinke MA, Hendrix CW, van der Straten A, Ramjee G, Chirenje ZM, Nakabiito C, Taha TE, Jones J, Mayo A, Scheckter R, Berthiaume J, Livant E, Jacobson C, Ndase P, White R, Patterson K, Germuga D, Galaska B, Bunge K, Singh D, Szydlo DW, Montgomery ET, Mensch BS, Torjesen K, Grossman CI, Chakhtoura N, Nel A, Rosenberg Z, et al. 22 February 2016. Use of a vaginal ring containing dapivirine for HIV-1 prevention in women. N Engl J Med Epub ahead of print. 9. Johnson TJ, Clark MR, Albright TH, Nebeker JS, Tuitupou AL, Clark JT, Fabian J, McCabe RT, Chandra N, Doncel GF, Friend DR, Kiser PF. 2012. A 90-day tenofovir reservoir intravaginal ring for mucosal HIV prophylaxis. Antimicrob Agents Chemother 56:6272– 6283. http://dx.doi.org /10.1128/AAC.01431-12. 10. Beigi R, Noguchi L, Parsons T, Macio I, Kunjara Na Ayudhya RP, Chen J, Hendrix CW, Masse B, Valentine M, Piper J, Watts DH. 2011. Pharmacokinetics and placental transfer of single-dose tenofovir 1% vaginal gel in term pregnancy. J Infect Dis 204:1527–1531. http://dx.doi.org /10.1093/infdis/jir562. 11. Antiretroviral Pregnancy Registry Steering Committee. 2015. Antiretroviral Pregnancy Registry international interim report for 1 January 1989 through 31 July 2015. Registry Coordinating Center, Wilmington, NC.
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12. Benaboud S, Pruvost A, Coffie PA, Ekouevi DK, Urien S, Arrive E, Blanche S, Theodoro F, Avit D, Dabis F, Treluyer JM, Hirt D. 2011. Concentrations of tenofovir and emtricitabine in breast milk of HIV-1infected women in Abidjan, Cote d’Ivoire, in the ANRS 12109 TEmAA Study, Step 2. Antimicrob Agents Chemother 55:1315–1317. http://dx.doi .org/10.1128/AAC.00514-10. 13. Mirochnick M, Taha T, Kreitchmann R, Nielsen-Saines K, Kumwenda N, Joao E, Pinto J, Santos B, Parsons T, Kearney B, Emel L, Herron C, Richardson P, Hudelson SE, Eshleman SH, George K, Fowler MG, Sato P, Mofenson L, HPTN 057 Protocol Team. 2014. Pharmacokinetics and safety of tenofovir in HIV-infected women during labor and their infants during the first week of life. J Acquir Immune Defic Syndr 65:33– 41. http: //dx.doi.org/10.1097/QAI.0b013e3182a921eb. 14. Van Rompay KK, Hamilton M, Kearney B, Bischofberger N. 2005. Pharmacokinetics of tenofovir in breast milk of lactating rhesus macaques. Antimicrob Agents Chemother 49:2093–2094. http://dx.doi.org /10.1128/AAC.49.5.2093-2094.2005. 15. Panel on Antiretroviral Therapy and Medical Management of HIVInfected Children. 2016. Guidelines for the use of antiretroviral agents in pediatric HIV infection. Office of AIDS Research Advisory Council, HHS Panel on Antiretroviral Therapy and Medical Management of HIV-Infected Children, US Department of Health and Human Services, Washington, DC. http://aidsinfo.nih.gov/contentfiles/lvguidelines/pediatricguidelines .pdf. Accessed 15 June 2016. 16. Hendrix CW, Chen BA, Guddera V, Hoesley C, Justman J, Nakabiito C, Salata R, Soto-Torres L, Patterson K, Minnis AM, Gandham S, Gomez K, Richardson BA, Bumpus NN. 2013. MTN-001: randomized pharmacokinetic cross-over study comparing tenofovir vaginal gel and oral tablets in vaginal tissue and other compartments. PLoS One 8:e55013. http://dx .doi.org/10.1371/journal.pone.0055013. 17. US Food and Drug Administration. 2001. Guidance for the industry: bioanalytical method validation. Center for Drug Evaluation and Research, US Food and Drug Administration, Rockville, MD. 18. Dezzutti CS, Rohan LC, Wang L, Uranker K, Shetler C, Cost M, Lynam JD, Friend D. 2012. Reformulated tenofovir gel for use as a dual compartment microbicide. J Antimicrob Chemother 67:2139 –2142. http://dx.doi .org/10.1093/jac/dks173. 19. Kearney BP, Flaherty JF, Shah J. 2004. Tenofovir disoproxil fumarate: clinical pharmacology and pharmacokinetics. Clin Pharmacokinet 43: 595– 612. http://dx.doi.org/10.2165/00003088-200443090-00003.
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Detectable Tenofovir Levels in Breast-Feeding Infants of Mothers Exposed to Topical Tenofovir Lisa M. Noguchi,a Elizabeth T. Montgomery,b Joseph R. Biggio,c Craig W. Hendrix,d Debra L. Bogen,e Sharon L. Hillier,f James Y. Dai,g Jeanna M. Piper,h Mark A. Marzinke,i Charlene S. Dezzutti,f S. Karen Isaacs,j* Jill L. Schwartz,k D. Heather Watts,l* Richard H. Beigif Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAa; Women’s Global Health Imperative, RTI International, San Francisco, California, USAb; Department of Obstetrics and Gynecology, University of Alabama, Birmingham, Alabama, USAc; Department of Medicine (Clinical Pharmacology), Johns Hopkins University, Baltimore, Maryland, USAd; Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania, USAe; Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USAf; Statistical Center for HIV/AIDS Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USAg; Division of AIDS/NIAID, U.S. National Institutes of Health, Bethesda, Maryland, USAh; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USAi; Science Facilitation, FHI 360, Durham, North Carolina, USAj; CONRAD/Eastern Virginia Medical School, Arlington, Virginia, USAk; Maternal and Pediatric Infectious Disease Branch/NICHD/U.S. National Institutes of Health, Bethesda, Maryland, USAl
Lactation studies are necessary evaluations of medications for reproductive-age women. We evaluated pharmacokinetics (PK), pharmacodynamics, safety, and adherence profiles associated with 7 days of 1% tenofovir (TFV) vaginal gel use during lactation. Tenofovir levels (maternal/infant serum, milk) and anti-HIV activity (milk), adverse events (AEs), and adherence were measured for 17 HIV-1-seronegative breast-feeding mother-infant pairs. Tenofovir use was well-tolerated and detected at low levels in maternal serum, milk, and infant serum but demonstrated no anti-HIV activity in milk.
E
xtended periods of breast-feeding are common in many countries with high HIV-1 incidence (1), and normal physiologic changes in breast-feeding women may impact drug safety and pharmacokinetics (2). Thus, biomedical HIV prevention strategies for women such as preexposure prophylaxis (PrEP) and topical microbicides should include those suitable during breastfeeding, especially considering higher infant transmission risk associated with incident maternal HIV-1 infection (3). However, both oral and topical HIV-1 chemoprophylaxis studies typically exclude breast-feeding women (1). The U.S. Food and Drug Administration (FDA) has released draft guidance recommending lactation studies for drugs expected for use by reproductive-age women (4). Here, we describe the first pharmacokinetic (PK) and safety study of topical antiretroviral (ARV) administration among breast-feeding mother-infant pairs. In CAPRISA 004, participants randomly assigned to topical tenofovir (TFV) treatment had 39% reduction in HIV-1 (5) and halved risk of herpes simplex virus 2 (HSV-2) acquisition (6) compared to participants treated with placebo. Subsequent trials found no similar protection, presumably due to low adherence (7). Recent results from ASPIRE (8) and The Ring Study (A. Nel, S. Kapiga, L. Bekker, et al., Conference on Retroviruses and Opportunistic Infections [CROI], Boston, MA, 22 February 2016) showed protective benefit against HIV-1 infection for women using a dapivirine (DPV) intravaginal ring. Other studies are investigating tenofovir-containing intravaginal rings and drug transfer to breast milk following use of a DPV intravaginal ring (NCT02431273 and NCT02658227) (9). Previous work supports safety of single-dose topical TFV in HIV-1-uninfected pregnant women (10) and oral tenofovir disoproxil fumarate (TDF) treatment for HIV-1-infected pregnant women (11). Oral TDF has not been associated with high serum or milk levels during breast-feeding (12, 13). Low oral bioavailability for TFV has been assumed to preclude or reduce absorption by breast-feeding infants, although previously no data were available (14). While TDF is not Food and Drug Administration approved or recommended for use in neonates or infants under 2 years, it is
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approved for children aged 2 to 12 years old when given as part of an antiretroviral therapy (ART) regimen. Treatment regimens containing TDF have been associated with gastrointestinal side effects (e.g., flatulence, diarrhea), as well as less common but more serious problems, such as bone and renal toxicity (15). MTN-008 (NCT01136759) was conducted in Pittsburgh, PA, and Birmingham, AL (2011 to 2013). The participants were HIV1-negative women and their breast-feeding infants 4 to 26 weeks old. Women received first and last doses of 1% TFV gel intravaginally in the clinic on days 0 and 6 and self-inserted five daily doses between the first and last doses. The formulation of the TFV gel was identical to the product used for CAPRISA 004 and the VOICE study. On days 0 and 6, milk and blood samples were collected predose and 2, 4, and 6 h postdose; infant blood samples were collected 6 h after maternal dosing (1 to 4 h after breastfeeding). Study investigators made judgments about the potential relatedness of an adverse event (AE) to product exposure prior to pharmacokinetic analyses. Institutional review board approval was obtained, and participants provided written informed consent. Blood was centrifuged after collection (ⱕ8 h), with serum frozen at ⫺20°C. Milk was swirled to mix and transferred by pipette
Received 20 March 2016 Returned for modification 22 April 2016 Accepted 26 June 2016 Accepted manuscript posted online 11 July 2016 Citation Noguchi LM, Montgomery ET, Biggio JR, Hendrix CW, Bogen DL, Hillier SL, Dai JY, Piper JM, Marzinke MA, Dezzutti CS, Isaacs SK, Schwartz JL, Watts DH, Beigi RH. 2016. Detectable tenofovir levels in breast-feeding infants of mothers exposed to topical tenofovir. Antimicrob Agents Chemother 60:5616 –5619. doi:10.1128/AAC.00645-16. Address correspondence to Lisa M. Noguchi, [email protected]. * Present address: S. Karen Isaacs, Parexel International, Durham, North Carolina, USA; D. Heather Watts, Office of the Global AIDS Coordinator, U.S. Department of State, Washington, DC. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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TABLE 1 Participant characteristics Characteristica Mothers (n ⫽ 17) Age, yr [median (IQR)] Have primary male partner [n (%)] Married [n (%)] Race [n (%)] Black White American Indian/Alaskan native, Asian, and black Asian and white Biracial, unspecified Wt (kg) [median (IQR)] Baseline creatinine clearance (ml/min) [median (IQR)] Infants (n ⫽ 17) Age, wk [median (IQR)] Wt (kg) [median (IQR)] a
Value for characteristic 27.1 (23.0–29.0) 13 (76.5) 8 (47.1) 7 (41.2) 7 (41.2) 1 (5.9) 1 (5.9) 1 (5.9) 75 (63–90) 122 (98–156)
10.0 (8.1–13.3) 6 (5–6)
Abbreviation: IQR, interquartile range.
into cryogenic vials, with aliquots frozen immediately at ⫺20°C or lower. Tenofovir concentrations were measured via liquid chromatography-tandem mass spectrometric analysis by validated methods (16, 17). Lower limits of quantitation (LLOQ) were 0.31 ng/ml (serum) and 1.0 ng/ml (milk). Pharmacodynamic (PD) activity was assessed by the TZM-bl assay, (18) using serial dilutions of milk from baseline and 6 h after last dose. Analyses were conducted using SAS 9.2, IBM SPSS 22.0, and Stata 12.1. Wilcoxon rank sum tests were used to compare TFV levels by matrix on day 0 versus 6. Seventeen pairs of women and infants enrolled in the study (Table 1). Tenofovir was detected in serum samples from all women following the first dose (Table 2). At day 6, serum tenofovir levels were detected for 56.3% of women predose and 100% of women postdose. A minority of milk samples had detectable TFV (25% at postdose day 0, 12.5% at predose day 6, and 37.5% at postdose day 6). The milk-to-maternal serum ratio for 18 samples
FIG 1 Anti-HIV activity in breast milk. Women in MTN-008 provided breast milk before (baseline) and after topical application of tenofovir (TFV) gel. Anti-HIV activity was measured before TFV treatment (baseline) and after TFV treatment (day 6/6 h) in specimens using the TZM-bl assay. Milk was modestly diluted (1:50) in assay medium. No difference in anti-HIV activity was noted between baseline and day 6 specimens (P ⫽ 0.14 by Wilcoxon matched-pair signed-rank test), indicating that no anti-HIV activity could be attributed to TFV gel use.
where both were higher than the LLOQ was 0.91 (median) (interquartile range [IQR] of 0.43 to 1.34). Eight infants were male, and nine were female. Six infants (37.5%) had detectable TFV after maternal dosing on day 0 (two females and four males) and 75% of infants (n ⫽ 12) postdose on day 6 (eight females and four males). Infant TFV concentrations were higher on day 6 than on day 0 (P ⬍ 0.03). Pharmacodynamic activity in milk demonstrated measurable innate anti-HIV activity (baseline specimen) (Fig. 1). Day 6 milk had no additional anti-HIV activity attributable to TFV beyond baseline (not statistically significant). Nine of 17 mothers experienced one or more AEs (total of 20 AEs), of which 9 (47%) were reproductive tract (all mild, 40% deemed related to gel, including genital burning, metrorrhagia, and diarrhea). Four of 17 infants had one or more AEs (total 8
TABLE 2 Tenofovir levels in maternal blood, breast milk, and infant blood Valueb for parameter in: Time and parametera
Maternal serum
Breast milk
Infant serum
Day 0 (n ⫽ 16) TFV detected postdose, n (%, 95% CI) Tmax, h (IQR) Cmax, ng/ml (IQR) AUC (IQR) Heel stick, ng/ml (IQR)
16 (100) 3.0 (1.9–5.4) 7.6 (4.0–62.6)d 40.5 (24.2–174.5)d
4 (25, 7.3–52.4) 5.0 (4.0–6.0)c 0.0 (0.0–0.75) 0.0 (0.0–1.5)
6 (37.5, 15.2–64.6)
Day 6 (n ⫽ 16) TFV detected predose, n (%, 95% CI) TFV detected postdose, n (%, 95% CI) Tmax (h) Cmax (ng/ml) AUC Heel stick (ng/ml)
9 (56.3, 29.9–80.2) 16 (100) 3.9 (1.2–4.0) 5.6 (3.4–22.6) 30.0 (19.8–101.2)
0.0 (0.0–1.1)
2 (12.5, 1.6–38.3) 6 (37.5, 15.2–64.6) 5.8 (2.9–6.0)c 0.0 (0.0–1.6) 0.0 (0.0–2.6)
12 (75.0, 47.6–92.7)
2.4 (0.3–4.5)d
a
Abbreviations: Tmax, time to peak concentration; Cmax, peak concentration; AUC, area under the curve; 95% CI, 95% confidence interval; IQR, interquartile range. b Values are median (IQR) values except for “TFV detected,” which are n (%, 95% CI by binomial exact test). c Tmax values represent those values where at least one concentration was detected. d P ⬍ 0.03 by Wilcoxon rank sum test of the value at day 0 versus the value at day 6.
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AEs; 7/8 mild; one infant had diarrhea that was deemed related to TFV exposure). Eleven (73%) women reported inserting five doses at home, two (13%) reported inserting four doses, and two (13%) reported inserting six doses. Vaginally administered TFV gel resulted in detectable TFV in milk and infant blood; however, only a small fraction of drug was transferred. While TFV is a small molecule, facilitating passage into milk, it has properties limiting transfer, including limited lipid solubility and protein binding (19). Despite expectations, TFV was detectable in blood at low concentrations for 12/16 infants. While our sample was too small to assess the potential presence of gender differences in pharmacokinetic or safety profiles, we did not observe any trends suggesting this. Breast milk TFV was quantifiable in only four women after dose 1 and in six women after dose 7, but there was no evidence of accumulation between doses. However, the number of infants with detectable serum TFV (n ⫽ 6) exceeded the number of mothers with detectable TFV. Similarly, on day 6, TFV was detected in milk samples from only two mothers predosing and six mothers at postdosing, but 12 infants had detectable TFV in their sera. These findings may be related to differences in assay sensitivity—the LLOQ for the milk (1.0 ng/ml) and serum (0.31 ng/ml) samples differ— or maternal and infant drug clearance and, therefore, accumulation differences. We identified significant increases in infant serum TFV levels on day 6 (with slightly lower serum TFV concentrations in mothers), consistent with modest TFV accumulation in blood samples from breast-feeding infants, though not in their mothers. However, the absolute TFV levels detected were ⱖ100-fold below treatment effective concentrations and unlikely of clinical concern. These findings are supported by no observable pharmacodynamic activity in milk beyond innate activity using the TZM-bl assay. While there was a small 1- to 2-h difference in the median time to peak concentration (Tmax) and the 6 h breast milk sample used for TZM-bl assay, a majority of these samples were coincident and, therefore, very closely representative of peak concentration and antiviral effect within the study population. Previous studies of maternal oral TDF use have not reported significant safety concerns for breast-feeding infants. In HPTN 057, TFV was detected in 3/4 milk samples collected within 2 days of delivery (6.3 to 17.8 ng/ml), and in 1/21 collected 4 to 6 days after delivery following peripartum oral TDF use by HIV-infected mothers (15.7 ng/ml) (13). In ANRS 12109, HIV-infected mothers administered oral TDF in labor and 7 days postpartum had milk TFV concentrations of 5.83 to 8.75 ng/ml (12). Using milk TFV concentrations averaging higher than those here, Benaboud and colleagues simulated plasma TFV concentrations as 0.03% of proposed prophylactic doses for neonates (12). This study had several strengths, including directly observed doses and measurement of multiple matrices over time. Our findings showing few AEs are consistent with other tenofovir studies of mothers and infants. However, our ability to characterize safety is limited if adherence were overreported. We did not observe any correlation between adherence to the self-administration protocol and TFV levels detected in the mothers or infants. However, our sample size was small, and self-reported adherence was high overall. Variability in volume, frequency, and/or timing of infant feeding may have impacted our PK results; the frequency and duration of infant feeding were captured for study days 0 and 6, while participants were on-site for study visits (not on interim study days). Estimating the volume of ingested breast milk by maternal report
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is imprecise, and techniques to approximate intake using infant weight before and after feeding were not used in MTN-008. The interval between topical TFV administration and the next breastfeeding session was controlled for time in the clinic on days 0 and 6, but not for self-administered doses on days 2 through 5. Thus, it is possible that time-dependent variations in TFV in breast milk may be related to differences in TFV ingestion. Medication use during breast-feeding should follow a careful risk/benefit assessment by the woman and her health care provider. Findings suggest low levels of drug transfer to milk and nursing infants associated with topically delivered TFV in mothers. Further study among breast-feeding mother-infant pairs should occur for topically formulated ARVs delivered via sustained release platforms, such as vaginal rings. ACKNOWLEDGMENTS We acknowledge the MTN-008 Lactation Cohort study participants, site staff, Community Advisory Boards, and site-affiliated lactation consultants, as well as the contributions of staff at the MTN Statistical and Data Management Center, FHI 360, the MTN Leadership and Operations Center, and the MTN Laboratory Center. C. W. Hendrix reports a grant from Gilead Science, outside the submitted work and a patent pending. S. L. Hillier reports an advisory relationship with Merck, outside the submitted work. J. L. Schwartz reports that CONRAD has a coexclusive license for tenofovir gel. None of the other authors report a commercial or other association that might pose a conflict of interest. The conclusions and opinions expressed in this article are those of the authors and do not necessarily reflect those of the National Institutes of Health or the U.S. Department of State.
FUNDING INFORMATION This work, including the efforts of Lisa M. Noguchi, Elizabeth T. Montgomery, Joseph R. Biggio, Craig W. Hendrix, Debra L. Bogen, Sharon L. Hillier, James Y. Dai, Jeanna M. Piper, Mark A. Marzinke, Charlene S. Dezzutti, S. Karen Isaacs, Jill Schwartz, D. Heather Watts, and Richard H. Beigi, was funded by HHS | National Institutes of Health (NIH) (UM1AI068633, UM1AI068615, UM1AI106707, and P30AI042855). This work was conducted by the Microbicide Trials Network, which is funded by the National Institute of Allergy and Infectious Diseases, with cofunding from the Eunice Kennedy Shriver National Institute of Child Health and Development and the National Institute of Mental Health (UM1AI068633, UM1AI068615, and UM1AI106707), all components of the National Institutes of Health. Tenofovir gel was provided by CONRAD (Arlington, VA) using funding from USAID. TFV assays were supported, in part, by the Johns Hopkins Center for AIDS Research (P30AI042855).
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