Placental transfer of Letermovir & Maribavir in the ex vivo human cotyledon perfusion model. New perspectives for in utero treatment of congenital cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus infection can lead to severe sequelae. When fetal infection is confirmed, we hypothesize that fetal treatment could improve the outcome. Maternal oral administration of an effective drug crossing the placenta could allow fetal treatment. Letermovir (LMV) and Maribavir (MBV) are new CMV antivirals, and potential candidates for fetal treatment.
METHODS: The objective was to investigate the placental transfer of LMV and MBV in the ex vivo method of the human perfused cotyledon. Term placentas were perfused, in an open-circuit model, with LMV or MBV at concentrations in the range of clinical peak plasma concentrations. Concentrations were measured using ultraperformance liquid chromatography coupled with tandem mass spectrometry. Mean fetal transfer rate (FTR) (fetal (FC) /maternal concentration), clearance index (CLI), accumulation index (AI) (retention of each drug in the cotyledon tissue) were measured. Mean FC were compared with half maximal effective concentrations of the drugs (EC50(LMV) and EC50(MBV)).
RESULTS: For LMV, the mean FC was (± standard deviation) 1.1 ± 0.2 mg/L, 1,000-fold above the EC50(LMV). Mean FTR, CLI and AI were 9 ± 1%, 35 ± 6% and 4 ± 2% respectively. For MBV, the mean FC was 1.4 ± 0.2 mg/L, 28-fold above the EC50(MBV). Mean FTR, CLI and AI were 10 ± 1%, 50 ± 7% and 2 ± 1% respectively.
CONCLUSIONS: Drugs' concentrations in the fetal side should be in the range for in utero treatment of fetuses infected with CMV as the mean FC was superior to the EC50 for both molecules.

Introduction

Congenital cytomegalovirus (cCMV) infection is a major cause of neurological and sensory impairment in children. The overall CMV birth prevalence estimate was 0.7%, based on 15 studies with a total of 117,986 infants screened [1]. Vertical transmission occurs either after maternal primary infection or after a secondary infection (reactivation of the endogen CMV strain or reinfection with a new CMV strain). CMV is a viraemic herpes virus reaching to the fetus through the placenta, which acts as a barrier against CMV but also as a reservoir where the virus replicates. All organs of an infected fetus could be affected including the developing brain in the worst cases, leading to a poor prognosis [2].

Currently, cCMV infection is not officially screened for in any country although serological screening is routinely offered to pregnant women in many developed countries. The diagnosis, during pregnancy, can be suspected in women showing seroconversion or in case of compatible features on fetal ultrasound. The diagnosis of fetal infection is made by the detection of viral DNA by polymerase chain reaction (PCR) in the amniotic fluid following amniocentesis. One can hypothesize that prompt initiation of an effective treatment in cases of proven fetal infection could reduce both placental and fetal damages.

To date, antiviral fetal treatment has shown a plausible benefit using valaciclovir, but the use of potent anti-CMV drugs in pregnancy is a debated issue [2,3]. (Val)ganciclovir, cidofovir, and foscarnet are highly genotoxic in vitro and are not approved for use during pregnancy. In addition, maternal toxicities could be significant. Valacyclovir is less effective against CMV, in vitro, but has the best safety profile of the anti-CMV, with no increased risk of birth defects in the offspring of thousands of women exposed during pregnancy [4]. All these drugs inhibit the viral replication by interacting with virally encoded DNA polymerase. Letermovir (LMV) and Maribavir (MBV) are newly announced specific and effective anti-CMV drugs acting through new antiviral mechanisms likely to alleviate the side effects and toxicity described above. Because clinical trials are challenging in pregnant women, preclinical approaches are warranted. Our objective was to investigate the placental transfer of MBV and LMV in the ex vivo human cotyledon perfusion model which is a well validated model to study placental transfer of drugs [5,6].

Materials and methods

Placentas

Placentas were collected in the maternity ward of Necker Hospital (Paris, France) after uncomplicated pregnancies and term deliveries. Women were informed of the use of their placentas and gave written consents. Procedures were approved by the ethics committee (Agence de Biomédecine, approval: PFS 15–009). General clinical characteristics are presented in Table 1. The perfusion experiment was initiated immediately on site and completed within two hours after delivery. Time from delivery to processing was summarized in Table 2.

Table 1

Clinical characteristics.

	Letermovir				Maribavir
	Mean	SD	Range		Mean	SD	Range
			min	max			min	max
Gravidity	2,4	0,9	1	3	1,7	0,6	1	2
Parity	2,2	0,8	1	3	1,3	0,6	1	2
Gestational age (weeks)	38,4	1,1	37	40	39,3	1,5	38	41
Maternal age	32	3	30	37	32,0	1,7	31	34
Birth weight (gram)	3292	207	3085	3605	3205	213	3000	3425
Prenatal medications	None N = 5				None N = 3
Drugs (including tobacco)	None N = 5				None N = 3
Previous prenatal admission	None N = 5				None N = 3
Blood pressures <140/90 mmHg	All N = 5				All N = 3
Gestational diabetes	None N = 5				None N = 3
Antibiotics in labor	None N = 5				None N = 3
Beta strep status	None N = 5				None N = 3
Antenatal steroids	None N = 5				None N = 3
Magnesium sulfate	None N = 5				None N = 3
Anesthesia	Epidural N = 5				Epidural N = 3
Cervical ripening agent	None N = 5				None N = 3
Delivery mode	Vaginal N = 3 hours of labor = 5h +/- 6.1h				Vaginal N = 1 hours of labor = 5h
	C-section, Repeat, no labor: n = 2				C-section, Repeat, no labor: n = 2
Maternal Oxygen at delivery	None N = 5				None N = 3
Cotyledon weight (for accumulation index) mean +/SD	258 g +/- 76 g				250 g +/- 53 g
Baby’s sex	Male N = 1				Male N = 2
	Female N = 4				Female N = 1

Table 2

Mean maternal and fetal parameters during the validated procedures.

	Letermovir	Maribavir
Fetal pressure (mmHg)	52 +/- 11	54 +/- 8
Maternal pressure (mmHg)	14 +/- 6	17 +/- 3
Fetal pH	7.22 +/- 0.02	7.23 +/- 0.02
Maternal pH	7.42 +/- 0.02	7.41 +/- 0.03
Fetal temperature (Celsius)	37.3 +/- 0.2	37.1 +/0.3
Maternal temperature (Celsius)	37.2 +/- 0.4	37.2 +/- 0.2
Delivery to processing (mins)	20 +/- 5	15 +/- 5

First the placenta was examined macroscopically, and an isolated cotyledon was chosen for its integrity. In the fetal side of this cotyledon, the chorionic plate artery and its associated vein were identified and cannulated with two needles. Then, the placenta was placed in the perfusion chamber with the maternal side up. The perfused cotyledon was easily distinguishable while starting to be white due to the fetal perfusion. Finally, we perfused the intervillous space on the maternal side, with two needles in the middle of the whitened cotyledon. Maternofetal circulation was then established.

Drugs

Powder form of the drugs (for research use only) were bought from MedChem Express (NJ 08852, USA): LMV (CAS N°917389-32-3, purity 98.87%) and MBV (CAS N°176161-24-3, purity 98.69%).

Cotyledon perfusion

Placentas were perfused in an open circuit model, according to a method described by Schneider et al [7]. Maternal and fetal solutions were prepared with Earle medium (Earle’s Balanced Salt 10 X, US Biological, Salem, MA, USA) plus 2 g/Liter of human serum albumin (Ydralbumin 20%, LFB-Biomedicament, Courtaboeuf, France). In the maternal solution, we added the study drug (LMV or MBV) prepared with 2 mL of DMSO (Merck, Germany) as a solvent. The dilution was calculated with target concentrations for the active drug within the range of clinical peak concentrations in plasma (Cmax). For a 480 mg dose of LMV, Cmax(LMV) = 13 mg/L [8], for a 400 mg dose of MBV, Cmax(MBV) = 16.1 mg/L [9]. We also added 20 mg/L of antipyrine (Merck, Germany) in the maternal compartment. This is an inert molecule that diffuses freely through the placenta, and acts a marker to validate the cotyledon’s viability throughout the experiment [10]. Sodium hydroxide was used to adjust the pH in both compartments to reach a fetal pH of 7.20 and a maternal pH of 7.40. Each circuit, maternal and fetal, included: a warming bath where the solution was maintained at 37°C, a peristaltic pump (running at 6 and 12 mL/min in the fetal and maternal sides respectively), a flowmeter. Two manometers were connected, one in the maternal side and one in the fetal artery side. Perfusion pressure and its stability were then measured during controlled experiment. The mean perfusion pressures values in the fetal and maternal side were, under 65 mmHg and under 20 mmHg respectively during all procedures, (Table 2).

Parameters (temperatures, pressures, pH) were recorded and data were summarized in Table 2. Samples were collected every 5 minutes to determine the antiviral drug and antipyrine concentrations in the fetal vein side and in the maternal compartment. The total perfusion duration was 90 minutes. Samples were then stored at -80°C until analysis. The concentrations were measured using ultraperformance liquid chromatography coupled with tandem mass spectrometry (Waters Xevo UPLC-TQD, Milford, MA, USA).

Four different parameters were looked for, all calculated at steady state: mean fetal concentration (FC), mean maternal concentration (MC), fetal transfer rate (FTR) calculated as the ratio of FC to MC, clearance index (CLI ) which calculated as the ratio of the FTR of the study drug to the FTR of the control (= antipyrine). An antipyrine FTR over 20% was required to validate each experiment [6]. In addition, for LMV and MBV, the results of the mean FC were analyzed in conjunction with their half maximal effective concentrations (EC50), as the EC50 is a marker of the drug's potency. According to the literature, EC50 of LMV = 2.1 nM = 1.2 x10-3 mg/L [8] and EC50 of MBV = 0.12 μM = 0.05 mg/L [11].

Furthermore, at the end of each perfusion, a piece of tissue was collected, weighted (approximately 250 mg by sample, mean weight is in Table 1) and crushed. Two different samples from the same cotyledon were collected by placenta. Then, the drug was extracted from the cotyledon and its concentration was determined using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Retention of each drug in the placental cotyledon tissue was determined, and expressed as an accumulation index (AI), defined by the ratio of the concentration of the study drug in the cotyledon (ng/mg) multiplied by the tissue density (1.04 g/mL) divided by the mean maternal drug concentration of the relevant experiment.

Mean Letermovir concentrations (Panel A) and mean Maribavir concentrations (Panel B) for all placentas. Panel A: Mean (± SD) maternal (full triangle) and fetal (empty triangle) concentrations of Letermovir (mg/L). Panel B: Mean (± SD) maternal (full square) and fetal (empty square) concentrations of Maribavir (mg/L).

Results

Letermovir (Table 3, Fig 1A)

Among the 10 experiments of placental perfusion, 5 were validated with a mean ± standard deviation (SD) antipyrine FTR of 31 ± 2%.

In these validated procedures, the system reached steady state at T30 min. The concentration in the maternal compartment was (mean ± SD) MCLMV = 12.5 ± 0.7 mg/L. The concentration in the fetal vein was: FCLMV = 1.1 ± 0.2 mg/L. As the EC50 of LMV = 1.2 x10-3 mg/L [8], FCLMV was approximately 1,000 fold above the EC50. The mean FTR was FTRLMV = 9 ± 1% and the mean CLI was CLILMV = 35± 6%. The mean AI was AILMV = 4 ± 2%.

Maribavir (Table 3, Fig 1B)

Among the 8 experiments of placental perfusion, 3 were validated with a mean ± standard deviation (SD) antipyrine FTR of 21 ± 1%. The system reached steady state at T20min.

In these validated procedures, the concentration in the maternal compartment was (median ± SD) = 14.3 ± 0.5 mg/L. The concentration in the fetal vein was FCMBV = 1.4 ± 0.2 mg/L. As the EC50 of MBV = 0.05 mg/L [11], FCMBV was approximately 28 fold above the EC50. The mean FTR was FTRMBV = 10 ± 1% and the mean CLI was CLIMBV = 50 ± 7%. The mean AI was AIMBV = 2 ± 1%.

Discussion

In this ex vivo human cotyledon perfusion model, LMV and MBV crossed the placenta at a low to moderate rate (FTRLMV = 9 ± 1% and FTRMBV = 10 ± 1%) and the mean concentration in the fetal compartment was superior to the EC50 for both molecules. Small parts of the perfused concentrations accumulated in the placental tissue. As the CMV replicates in the placenta [2], the accumulation of the drugs in this reservoir might contribute to the antiviral efficacy. This opens new perspectives for in utero treatment of infected fetuses using either of these 2 candidate-drugs.

In vivo, over 24 hours, the concentration of the antiviral will vary in plasma between the clinical peak concentration (Cmax) and the minimal concentration (Cmin). To treat the fetus efficiently, fetal concentrations of the antiviral should be superior to the EC50 of the drug over the whole day. Our results suggest that this would be the case since fetal Cmax and Cmin would be above the EC50 of both drugs. In this study, the drugs were used within the range of the Cmax and we showed that the mean concentration in the fetal compartment was superior to the EC50 for both anti-CMV drugs (FCLMV ≈ 1,000 EC50 and FCMBV ≈ 28 EC50). We did not performed experiments with concentrations in the range of the Cmin but the relation between Cmax and Cmin is available in the literature. For LMV, according to Kropeit et al [12], in healthy subject, for 120 mg of LMV Cmin = 0.1 mg/L and Cmax = 2.5 mg/L so C = Cmax/ 25. As the pharmacokinetic of LMV is linear this relation can be used in our model. So, the minimal plasma concentration should be Cmin(LMV) = FCLMV/25 = 1,000 EC50(LMV) /25 = 40 EC50(LMV). For MBV, according to Wang et al [9], in healthy subject, for 400 mg of MBV Cmin(MBV) = 3 mg/L and Cmax(MBV) = 16.7 mg/L so Cmin(MBV) = Cmax(MBV) / 5.6. Therefore, the theoretical minimal plasma concentration of MBV, in the fetal compartment, in our model, should be Cmin(MBV) = FCMBV/5.6 = 28 EC50(MBV)/5.6 = 5 EC50(MBV). In conclusion, theoretically, the concentration over a day should be, at any time, above the EC50 for both molecules (between 40–1,000 EC50(LMV) and 5–28 EC50(MBV) for LMV and MBV respectively), so within the appropriate range to treat the fetus.

LMV is a moderate molecular weight (572.55 Da) and highly lipophilic (log P oct/wat = 4.58) molecule that acts via the viral terminase complex which cleaves DNA to package the genome into the capsid [13]. In vitro, LMV is a substrate of drug metabolizing enzymes: CYP3A, CYP2D6, UGT1A1, and UGT1A3, and transporters OATP1B1/3 and P-gp. 97% of the molecule is unchanged with no metabolites detected in the plasma [8]. The mean elimination half-life is 12 hours allowing an administration once daily (480 mg/d) [8]. In vitro, LMV is currently the most active molecule against CMV, with a very low EC50 which surpasses the EC50 of the Ganciclovir by more than 400-fold [14]. The safety profile of LMV is good, with minimal adverse effects and no documented hematologic toxicity or nephrotoxicity [8,15]. There are no human data on safety of LMV in pregnancy but no teratogenic effect nor maternal toxicity were reported in animal studies at up to 3-fold the recommended human dose, both in rats and in rabbits [8]. LMV has received approval from the FDA and European Medicines Agency, in November 2017, for the prevention of CMV infection and disease in adult allogeneic stem cell transplant patients [8].

MBV is a low molecular weight (376.23 Da) and lipophilic (log P oct/wat = 2.15) molecule that inhibits the UL97 viral kinase. It therefore opposes to viral DNA elongation, DNA packaging, and nuclear egress of encapsidated viral DNA [16]. MBV is rapidly eliminated, with a mean half-life in plasma of 3 to 5 h so it has to be administered orally twice-a-day [11]. The principal metabolite of MBV is 4469W94 (which is derived by N-dealkylation of MBV via CYP3A4) but this metabolite is pharmacologically inactive [9]. MBV also shows a favorable safety profile in both animals and human studies [9,17]. However, there is a lack of data about teratogenicity of MBV. MBV is being developed for treatment of CMV disease in patients with impaired cell mediated immunity. Two Phase III clinical trials have recently started in this population [18,19]. MBV has not yet been approved by the FDA.

Concentrations in fetal blood of both LMV and MBV are expected to be appropriate in terms of benefit/risk ratio. We have demonstrated that both anti-CMV drugs crossed the placenta at a low to moderate rate. These rates are concordant with the physico-chemical properties of the molecules with low to moderate molecular weight and a highly lipophilic profile. However, they are both highly bound to plasma proteins, mainly albumin (98% and 99% for MBV and LMV respectively) which is a significant obstacle to placental transfer since only the unbound fraction of the drug can cross the placenta membrane[20,21]. In our model we used 2 g/L of human albumin in the maternal and fetal solutions. This concentration is admitted as a standardized condition in this ex vivo model, by many experienced teams [5,22-24]. However, it is likely that fetal levels of free drugs in vivo will be lower than the values that were obtained in our perfusion experiments because most of the drug will remain bound to serum albumin in the maternal circulation. It is difficult and impractical to exactly mimic physiological protein binding during the perfusion experiment. We also think that using a human serum albumin is one of the strengths of our study since others did not add it at all to the perfusion solutions[25]. Optimal concentration is also difficult to define in this placental transfer model because significant changes in serum albumin occur during pregnancy. In vivo, when drugs are highly bound to plasma proteins, the amount of drug can significantly vary during pregnancy in both maternal and fetal compartments. Krauer B et al. showed that, maternal serum albumin concentrations ranged between 25 and 35 g/L and fetal serum albumin was much lower in early gestation, ranging from 7.5 to 16 g/L at 12–15 weeks. With advancing gestation, the authors noticed that there was a linear increase so that at 30 weeks both fetal and maternal serum albumin concentrations were in the same range and after 35 weeks the fetal concentrations exceeded the maternal by some 20% [26].

The issue of fetal toxicity cannot be addressed by our study. However, our results indicate that the fetus would be exposed to the drug, over a day, at concentrations between 40–1,000 EC50(LMV) for LMV and between 5-28EC50(MBV) for MBV. Whether these ranges could be toxic for a fetus is questionable. These data are theoretical, and it is difficult to predict what would exactly be the respective drug disposition in both maternal and fetal compartment in vivo.

The limitations of our model include that it does not account for fetal disposition nor for the accumulation of the drug in the amniotic fluid. Fetal drug metabolism has its own specificities, and these are disregarded in our ex vivo model. First, it has been shown that the zonation of xenobiotic metabolism is not localized in the same hepatic pattern in fetuses and adults [27]. Secondly, the enzyme activities are different especially in CYP activities where CYP3A7 is the predominant CYP enzyme in fetuses, while it is CYP3A4 in adults. Glucuronidation is also impacted with a limited activity of UDP-GT in the fetal liver leading to an immaturity of hepatic glucuronidation in fetuses and neonates. Full maturation may not occur for many years in the child [27,28]. The fetal pharmacology of LMV and MBV will be affected by these differences between fetal and adult hepatic elimination. Further investigations in vivo are required to evaluate the clinical implications.

Another limitation is the relatively small study sample of our study. However, studies published by other experimented teams also evaluated placental transfer of drugs on small studies samples [25,29-31]. We only used pure and titrated new drugs and human serum albumin, so the price of each experiment was very expensive. This high cost was the reason for not doing more experiments.

The issue of maternal toxicity is easier to appraise. LMV has a good safety profile in animal and in human studies [8,12,15]. Its oral administration once daily should facilitate acceptability by pregnant women. The US FDA pregnancy category has not been assigned yet. The French institute working on teratogenicity CRAT (Centre de Référence sur les Agents Tératogènes) [32] gave us a favorable opinion for the prospective use of LMV in pregnant women carrying a CMV infected fetus within a clinical trial. MBV is not available yet as it has not been approved by the FDA. Despite a satisfactory safety profile in the preliminary works, further studies are still needed.

The ex vivo cotyledon model allows to study the placental transfer of drugs that have not yet been tested in pregnant women, in standardized conditions and without ethical restrictions but it has several limitations. First, there is no guidelines as to respect the ex vivo infusion of placental human cotyledon, but merely one attempt by Mathiesen et al [33]. Second, it is a challenging procedure: half of our experiments were validated by an antipyrine FTR > 20%, which is a standard success rate [34]. Finally, it is technically available only for full-term placentas. As our final objective is to treat fetuses infected with CMV all along the pregnancy, the placental transfer of these drugs, earlier in pregnancy therefore remains somehow speculative.

The placentas used in our study were not infected with CMV. In the pathophysiology of cCMV infection, the placenta shows placentitis [2]. This may affect its architecture and therefore potentially also modify the transfer of molecules.

In conclusion, the new anti-CMV drugs LMV and MBV are promising in terms of efficacy, placental transfer and safety profile to be used to treat symptomatic infected fetuses with CMV. The latter aspect deserves caution, especially for MBV and phase-1 studies in infected pregnancies should be encouraged.

(XLSX)

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7 Feb 2020

PONE-D-20-01568

Placental transfer of Letermovir & Maribavir in the ex vivo human cotyledon perfusion model. New perspectives for in utero treatment of congenital cytomegalovirus infection.

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Reviewer #1: Faure V et al. examine placental transfer of two new CMV medications utilizing the human placenta cotyledon perfusion assay. This system is accepted and appropriate for pharma comparisons and evaluations of placental transfer. This study is of interest to the OB clinical and research community due to the continued danger that CMV poses to otherwise healthy pregnancies. The manuscript is well written (with exceptions cited below), experiments were done accordingly, and measurements appropriate using established modalities and the antipyrine control. However, there are important data elements missing, specifically in regards to clinical characteristics and technical details. The manuscript in its present state is suitable for publication with the following modifications:

1. Please include additional clinical characteristics: fetal sex, anesthesia, use of magnesium sulfate or other drugs/steroids, and if tested positive for Group B strep.

2. Correct inconsistencies in text and labeling. For example, in table one drug names are fully capitalized whereas the remaining tables only the first letter is capitalized. Please change so that only the first letter is capitalized when referencing these drugs. Finally table/figure titles should not be italicized.

3. Consolidate Figures 1 and 2 into a single figure and label the Letermovir graph as panel A and Maribavir as B. Also provide a figure legend that briefly describes the graphs that includes the labels (mean SD, triangle, and square) vs. stating them in the titles.

4. Ensure all words/labels are in English: for example in Table 2, pressure is spelled in French as pressian.

Reviewer #2: This manuscript consists of a study examining the maternal to fetal transfer of new CMV antiviral medications, Letermovir (LMV) and Maribavir (MBV), across the human placenta using a dual perfusion in vitro model. Authors demonstrated that both drugs crossed the placenta at a low to moderate rate. Their results indicate that the fetus would be exposed to the drug at concentrations between 40-1000 EC50 (LMV) and 5-28 EC50 (MBV). They signify clearly and correctly the limitations and strengths of the current study.

However there are several major limitations of the study:

1. Authors noted that both drugs are highly bound to plasma protein. In order to mimic maternal plasma protein concentrations authors should have added into the maternal circulation BSA in a concentration of 30gr/1 liter and not only 2gr/L. Since their main aims were to quantify the transfer of these drugs across the placenta, to my opinion this is an important obstacle of the study.

Although authors quote other study by Bapat P et. al 2015 who did not use at all albumin to their study, there are others who did it, specifically in a case of studying drug that known to be highly bound to plasma protein (Adverse placental effects of valproic acid: Studies in perfused human placentas. Rubinchik-Stern M et. al Epilepsia. 2018 May;59(5):993-1003)

2. This is a very small study sample, only 3 validated experiments for the MBV and 5 validated experiments for the LMV. Keeping in mind that very important conclusions are made based on the findings, it would be important to include a larger sample number for each drug

3. Measurements of placental viability and integrity during the periods of perfusion as HCG production, glucose consumption or oxygen transfer were not presented

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4 Mar 2020

Reviewer #1: Faure V et al. examine placental transfer of two new CMV medications utilizing the human placenta cotyledon perfusion assay. This system is accepted and appropriate for pharma comparisons and evaluations of placental transfer. This study is of interest to the OB clinical and research community due to the continued danger that CMV poses to otherwise healthy pregnancies. The manuscript is well written (with exceptions cited below), experiments were done accordingly, and measurements appropriate using established modalities and the antipyrine control.

We thank the reviewer for this comment.

However, there are important data elements missing, specifically in regards to clinical characteristics and technical details. The manuscript in its present state is suitable for publication with the following modifications:

1. Please include additional clinical characteristics: fetal sex, anesthesia, use of magnesium sulfate or other drugs/steroids, and if tested positive for Group B strep.

General clinical characteristics are now presented in table 1, in accordance with your request.

2. Correct inconsistencies in text and labeling. For example, in table one drug names are fully capitalized whereas the remaining tables only the first letter is capitalized. Please change so that only the first letter is capitalized when referencing these drugs. Finally table/figure titles should not be italicized.

It was done.

3. Consolidate Figures 1 and 2 into a single figure and label the Letermovir graph as panel A and Maribavir as B. Also provide a figure legend that briefly describes the graphs that includes the labels (mean SD, triangle, and square) vs. stating them in the titles.

It was done.

4. Ensure all words/labels are in English: for example in Table 2, pressure is spelled in French as pressian.

It was done.

Reviewer #2: This manuscript consists of a study examining the maternal to fetal transfer of new CMV antiviral medications, Letermovir (LMV) and Maribavir (MBV), across the human placenta using a dual perfusion in vitro model. Authors demonstrated that both drugs crossed the placenta at a low to moderate rate. Their results indicate that the fetus would be exposed to the drug at concentrations between 40-1000 EC50 (LMV) and 5-28 EC50 (MBV). They signify clearly and correctly the limitations and strengths of the current study.

We thank the reviewer for this comment.

However there are several major limitations of the study:

1. Authors noted that both drugs are highly bound to plasma protein. In order to mimic maternal plasma protein concentrations authors should have added into the maternal circulation BSA in a concentration of 30gr/1 liter and not only 2gr/L. Since their main aims were to quantify the transfer of these drugs across the placenta, to my opinion this is an important obstacle of the study. Although authors quote other study by Bapat P et. al 2015 who did not use at all albumin to their study, there are others who did it, specifically in a case of studying drug that known to be highly bound to plasma protein (Adverse placental effects of valproic acid: Studies in perfused human placentas. Rubinchik-Stern M et. al Epilepsia. 2018 May;59(5):993-1003)

We agree with all comments concerning the concentration of albumin.

In our model we used 2 g/L of human albumin in the maternal and fetal solutions like others experimented teams who work on the placenta perfusion. This concentration is admitted as a standardized condition in this ex vivo model, by many experienced teams. (1–4).

Mandelbrot el al, when they analyzed the placental transfer of Rilpivirine, also a highly protein-bound (>99% ) molecule, used this concentration of 2g/L (2). Furthermore, he also used this concentration of albumin in a study published in Plos One last year about placental transfer of Dolutegravir “ 99% bound to plasma proteins, mainly albumin”(5).

As previously mentioned, we think that using, even in a low concentration, human serum albumin is one of the strengths of our study since others did not add it at all to the perfusion solutions (6). Indeed, Bapat et al. mentioned that : “Perfusion experiments that include proteins in the perfusion medium are technically challenging because of the high viscosity of the perfusate. »

We agree that, it is likely that fetal levels of free drugs in vivo will be lower than the values that were obtained in our perfusion experiments, because most of the drug will remain bound to serum albumin in the maternal circulation. However, the human cotyledon perfusion model remains an ex vivo model with validated and standardized conditions but sometimes also far from the physiologically reality. Indeed, the procedure was carefully monitored and standardized to approach mimicking normal physiology and the FTR of antipyrine was monitored in order to control the integrity of the placental barrier. Differential protein binding between the fetal and maternal circulations is known to be a critical factor in the placental transfer of drugs. However, it is difficult and impractical to exactly mimic physiological protein binding during the perfusion experiment.. (7).

However, even in low albumin conditions, the results obtained with this ex vivo placental perfusion model have been found to be consistent with in vivo findings for a number of compounds. (2,7).

Finally, on a technical point of view, the increase of albumin concentrations in fetal solution might have some limitations:

- The dynamic agitation with magnetic rod or bubbling with gas in the respective vessels might frothing the maternal or fetal solutions, leading to risk of dismantling the peristaltic pumping

- the cost of human albumin is really expensive

- the increase of albumin concentrations in maternal or fetal solutions to 5 or 10 g/L might be theoretically possible but the reservations expressed by the reviewer for 2 g/L are still arise regarding this point

2. This is a very small study sample, only 3 validated experiments for the MBV and 5 validated experiments for the LMV. Keeping in mind that very important conclusions are made based on the findings, it would be important to include a larger sample number for each drug

We agree with these comments however perfusion of a human cotyledon model is a challenging procedure. Only half of our experiments were validated by an FTR of antipyrine > 20 % which is a standard success rate(8). We only used pure and titrated new drugs and human serum albumin, so the price of each experiment was very expensive (approximatively 500 euros per placenta …) . This high-cost was one of the reason for not doing more experiments.

Furthermore, studies published by other experimented teams also evaluated placental transfer of drugs on small studies samples :

• raltegravir : maternal to fetal direction (n=3 placentas) (9)

• dolutegravir (n=5 placentas) (5)

• darunavir (n=5 placentas) (10)

• rivaroxaban: maternal to fetal direction (n=5 placentas) fetal to maternal direction ( n= 2 placentas) (6)

• oseltamivir (n=5 placentas) (11)

3. Measurements of placental viability and integrity during the periods of perfusion as HCG production, glucose consumption or oxygen transfer were not presented

Antipyrine transfer is a control of cotyledon’s viability. If Antipyrine FTR is < 20% the placenta is not validated. Most of laboratories use this antipyrine cut off of 20% to check on cotyledon’s viability (2,4,9,12–16) .

On a technical point of view, we observed that the dynamic agitation with gas, in the respective vessels, might frothing the maternal or fetal solutions, leading to risk of dismantling the peristaltic pumping. This is why, like others experimented teams who work on the placenta perfusion, we have chosen not to use gas (1–3,5). In the same teams, HCG production and glucose consumption were also not recorded, Antipyrine transfer was sufficient to validate cotyledon’s viability.

One of the limits of the placenta perfusion is that there are no standardized criteria between laboratories. Avoid toxicity associated with hyperoxia is an alternative procedure known and accepted (7).

Furthermore, our parameters (fetal and maternal pressures and pH) were correctly maintained at physiological levels as mentioned in table 2.

References

1. Ceccaldi P-F, Ferreira C, Gavard L, Gil S, Peytavin G, Mandelbrot L. Placental transfer of enfuvirtide in the ex vivo human placenta perfusion model. Am J Obstet Gynecol. avr 2008;198(4):433.e1-2.

2. Mandelbrot L, Duro D, Belissa E, Peytavin G. Placental Transfer of Rilpivirine in an Ex Vivo Human Cotyledon Perfusion Model. Antimicrob Agents Chemother [Internet]. 5 janv 2015 [cité 21 nov 2015];59(5):2901‑3.

3. Gavard L, Gil S, Peytavin G, Ceccaldi P-F, Ferreira C, Farinotti R, et al. Placental transfer of lopinavir/ritonavir in the ex vivo human cotyledon perfusion model. Am J Obstet Gynecol. juill 2006;195(1):296‑301.

4. Gavard L, Beghin D, Forestier F, Cayre Y, Peytavin G, Mandelbrot L, et al. Contribution and limit of the model of perfused cotyledon to the study of placental transfer of drugs. Example of a protease inhibitor of HIV: nelfinavir. Eur J Obstet Gynecol Reprod Biol. déc 2009;147(2):157‑60.

5. Mandelbrot L, Ceccaldi P-F, Duro D, Lê M, Pencolé L, Peytavin G. Placental transfer and tissue accumulation of dolutegravir in the ex vivo human cotyledon perfusion model. PLOS ONE [Internet]. 13 août 2019 14(8):e0220323.

6. Bapat P, Pinto LSR, Lubetsky A, Berger H, Koren G. Rivaroxaban transfer across the dually perfused isolated human placental cotyledon. Am J Obstet Gynecol. nov 2015;213(5):710.e1-6.

7. Hutson JR, Garcia-Bournissen F, Davis A, Koren G. The Human Placental Perfusion Model: A Systematic Review and Development of a Model to Predict In Vivo Transfer of Therapeutic Drugs. Clin Pharmacol Ther [Internet]. juill 2011 [

8. Berveiller P, Gil S, Vialard F. Placental perfusion: interest and limits. J Matern Fetal Neonatal Med [Internet]. 3 juin 2017 ;30(11):1347‑8.

9. Vinot C, Tréluyer J-M, Giraud C, Gavard L, Peytavin G, Mandelbrot L. Bidirectional Transfer of Raltegravir in an Ex Vivo Human Cotyledon Perfusion Model. Antimicrob Agents Chemother 1 mai 2016;60(5):3112‑4.

10. Mandelbrot L, Duro D, Belissa E, Peytavin G. Placental transfer of darunavir in an ex vivo human cotyledon perfusion model. Antimicrob Agents Chemother. sept 2014;58(9):5617‑20.

11. Berveiller P, Mir O, Vinot C, Bonati C, Duchene P, Giraud C, et al. Transplacental transfer of oseltamivir and its metabolite using the human perfused placental cotyledon model. Am J Obstet Gynecol 1 janv 2012 ;206(1):92.e1-92.e6.

12. Vinot C, Gavard L, Tréluyer JM, Manceau S, Courbon E, Scherrmann JM, et al. Placental transfer of maraviroc in an ex vivo human cotyledon perfusion model and influence of ABC transporter expression. Antimicrob Agents Chemother. mars 2013;57(3):1415‑20.

13. Huang H, Wang J, Li Q, Duan J, Yao Q, Zheng Q, et al. Transplacental transfer of oseltamivir phosphate and its metabolite oseltamivir carboxylate using the ex vivo human placenta perfusion model in Chinese Hans population. J Matern Fetal Neonatal Med [Internet]. 3 juin 2017;30(11):1288‑92.

14. Ceccaldi P-F, Mandelbrot L, Farinotti R, Forestier F, Gil S. Apports de la perfusion ex vivo du cotylédon humain dans l’étude du passage placentaire des médicaments. J Gynécologie Obstétrique Biol Reprod [Internet]. déc 2010 ;39(8):601‑5.

15. Evain-Brion D, Berveiller P, Gil S. [Placental transfer of drugs]. Therapie. févr 2014;69(1):3‑11.

16. Forestier F, de Renty P, Peytavin G, Dohin E, Farinotti R, Mandelbrot L. Maternal-fetal transfer of saquinavir studied in the ex vivo placental perfusion model. Am J Obstet Gynecol. juill 2001;185(1):178‑81.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

18 Mar 2020

PONE-D-20-01568R1

Placental transfer of Letermovir & Maribavir in the ex vivo human cotyledon perfusion model. New perspectives for in utero treatment of congenital cytomegalovirus infection.

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for addressing the previous comments. The manuscript is improved and nearly suitable for publication. The only changes I request regard Figure 1. In my previous review I had asked that Figure 1 (Letermovir) and Figure 2 (Maribarvir) be merged into a single figure denoting panel A and B respectively, which the authors have done. However, the graphs were also merged (not requested), which creates confusion as a single graph represents only one panel but is labeled A and B. Please replace with the original two graphs and label Letermovir as A and the Marbarvir as B in vertical order. Also annotate the results section according, line 139 should read "Letermovir (Table 3, Fig 1A)" and line "Maribavir (Table 3, Fig 1B". Finally, please remove the abbreviations from the titles as indicated in my previous sentence. Once these minor changes are made, it is this reviewer’s opinion that the manuscript is ready for publication.

Reviewer #2: 1. Regarding author's response concerning the concentration of albumin. I accept their response. Yet a comment about this specific issue should be added, as they commented in here in their own wards: "It is likely that fetal levels of free drugs in vivo will be lower than the values that were obtained in our perfusion experiments, because most of the drug will remain bound to serum albumin in the maternal circulation. It is difficult and impractical to exactly mimic physiological protein binding during the perfusion experiment".

2. Regarding author's response about the small study sample. I accept their response. Please add a comment in the

study's limitations part, about the relative small study sample of the study.

3. Regarding the comment about measurements of placental viability and integrity during the periods of perfusion. I accept their response

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Mar 2020

Reviewer #1: Thank you for addressing the previous comments. The manuscript is improved and nearly suitable for publication. The only changes I request regard Figure 1. In my previous review I had asked that Figure 1 (Letermovir) and Figure 2 (Maribarvir) be merged into a single figure denoting panel A and B respectively, which the authors have done. However, the graphs were also merged (not requested), which creates confusion as a single graph represents only one panel but is labeled A and B. Please replace with the original two graphs and label Letermovir as A and the Marbarvir as B in vertical order.

It was done.

Also annotate the results section according, line 139 should read "Letermovir (Table 3, Fig 1A)" and line "Maribavir (Table 3, Fig 1B".

It was done.

Finally, please remove the abbreviations from the titles as indicated in my previous sentence.

It was done.

Once these minor changes are made, it is this reviewer’s opinion that the manuscript is ready for publication.

We thank the reviewer for this comment.

Reviewer #2: 1. Regarding author's response concerning the concentration of albumin. I accept their response. Yet a comment about this specific issue should be added, as they commented in here in their own wards: "It is likely that fetal levels of free drugs in vivo will be lower than the values that were obtained in our perfusion experiments, because most of the drug will remain bound to serum albumin in the maternal circulation. It is difficult and impractical to exactly mimic physiological protein binding during the perfusion experiment".

It was done.

2. Regarding author's response about the small study sample. I accept their response.

We thank the reviewer for this comment.

Please add a comment in the study's limitations part, about the relative small study sample of the study.

We added in the limitation part : “Another limitation is the relative small study sample of our study. However, studies published by other experimented teams also evaluated placental transfer of drugs on small studies samples(25,29–31). We only used pure and titrated new drugs and human serum albumin, so the price of each experiment was very expensive. This high-cost was the reason for not doing more experiments.”

3. Regarding the comment about measurements of placental viability and integrity during the periods of perfusion. I accept their response

We thank the reviewer for this comment.

Submitted filename: Response 2 to reviewers.docx

Click here for additional data file.

8 Apr 2020

Placental transfer of Letermovir & Maribavir in the ex vivo human cotyledon perfusion model. New perspectives for in utero treatment of congenital cytomegalovirus infection.

PONE-D-20-01568R2

Dear Dr. Faure Bardon,

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Frank T. Spradley

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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13 Apr 2020

PONE-D-20-01568R2

Placental transfer of Letermovir & Maribavir in the ex vivo human cotyledon perfusion model. New perspectives for in utero treatment of congenital cytomegalovirus infection

Dear Dr. Faure Bardon:

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Table 3

Mean maternal and fetal parameters for Letermovir and Maribavir at steady state.

	Mean FC ± SD (mg/L)	Mean MC ± SD (mg/L)	Mean FTR ± SD	Mean CLI ± SD	Mean concentration of the study drug in the cotyledon (ng/mg)	Mean AI ± SD
LMV	1.1 ± 0.2	12.5 ± 0.7	0.09 ± 0.01	0.35 ± 0.06	0.62 ± 0.32	0.04 ± 0.02
MBV	1.4 ± 0.2	14.3 ± 0.5	0.1 ± 0.01	0.5 ± 0.07	0.25 ± 0.10	0.04 ± 0.01

LMV: Letermovir, MBV: Maribavir, FC: fetal concentration, MC: maternal concentration, FTR: fetal transfer rate, CLI: clearance index, AI: accumulation index, SD: standard deviation