# The Metabolism and Transplacental Transfer of Oseltamivir in the Ex Vivo Human Model

Authors: Kevin C. Worley; Scott W. Roberts; Roger E. Bawdon
Journal: Infectious Diseases in Obstetrics and Gynecology
 (2008)

---

## Abstract

Oseltamivir phosphate is extensively metabolized in the ex vivo
human placenta model, and the transplacental passage of the metabolite oseltamivir
carboxylate is incomplete.Objective. To evaluate the metabolism and transplacental transfer of oseltamivir (Tamiflu) in the ex vivo human placental model.
Study Design. Perfusion studies were performed in six placentas from term, uncomplicated deliveries. Concentrations of oseltamivir phosphate (OP) that were 5-6 fold, 20–30 fold, and 600–800 fold above the therapeutic peak were tested, as neither OP nor its active metabolite, oseltamivir carboxylate (OC),
could be detected at near-therapeutic concentrations. The transplacental transfer and accumulation of OC were
assessed using the 14C antipyrine reference method.
Results. OP was extensively metabolized to OC. In the 4 placentas with the highest concentration of OP, OC had a mean clearance index of 0.13±0.08, suggesting that transplacental passage occurs at a relatively low rate. Measurable fetal accumulation occurred in the two placentas with the highest initial concentrations.
Conclusions. Oseltamivir phosphate was extensively metabolized in the ex vivo model. Transplacental transfer of the metabolite was incomplete and accumulation was minimal.


---

## Body

## 1. Introduction

Oseltamivir
phosphate (Tamiflu, Roche), (3R, 4R,
5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1cyclohexene-1carboxylic acid,
ethyl ester, phosphate (1 : 1) is an antiviral agent with activity against both
influenza A and B [1].  Oseltamivir is one
of two members of the class of antivirals known as neuraminidase inhibitors
whose proposed mechanism of action is to interfere with the release of progeny
viral particles from infected host cells, thereby preventing local spread of
infection [2].  Both oseltamivir and
zanamivir (Relenza, GlaxoSmithKline) are approved for the treatment of
influenza A and B; however, only oseltamivir is administered orally and is
approved for postexposure chemoprophylaxis [1].Globally,
influenza remains one of the most significant causes of febrile respiratory
morbidity and mortality, as it affects approximately 20% of the world’s
population annually [2].  In the United
States alone, epidemics of influenza are thought to be responsible for up to
36,000 deaths per year [3]. Immunoprophylaxis remains the cornerstone
of controlling the spread of influenza, and annual vaccination is recommended
for all persons who are at increased risk for complications from influenza
infection [3].  Among those included
within these high-risk groups, pregnant women are of particular interest
because of the implications of maternal illness to her unborn child. The risks
for severe illness and death related to influenza infection are known to be
elevated in pregnant women, a concept historically supported by the
disproportionate number of influenza-related maternal deaths during the
pandemics of 1918 and 1957 [4].  Based on
these risks, the CDC now recommends universal vaccination for pregnant women,
regardless of gestational age. Despite these recommendations, however, the
annual number of hospitalizations for seasonal influenza infection in pregnant
women remains high [5, 6].In addition to
seasonal influenza, significant attention has recently been given to the
particularly pathogenic strain of avian influenza, H5N1.  A number of cases of  human infection with H5N1 have been reported
with a high case fatality rate, including a report of a pregnant woman who
succumbed rapidly to viral pneumonia caused by H5N1 [7]. There is growing
concern that H5N1 will cause the next influenza pandemic, yet  no vaccine exits for this virulent strain of
the virus [8].There exists,
therefore, great clinical interest in antiviral agents that may reduce the
burden of annual clinical disease, and oseltamivir, in particular, may also
have activity against avian influenza [9]. Unfortunately, limited data exist
concerning the use of oseltamivir in pregnancy. The purpose of this study was
to evaluate the metabolism and maternal-fetal transplacental passage of
oseltamivir in the ex vivo
human placental model.


## 2. Materials and Methods

Term placentas(n=6) were collected from either vaginal or otherwise uncomplicated cesarean
deliveries in accordance with the University of Texas Southwestern Medical
Center Institutional Review Board for Human Studies Guidelines, and they were
transported to the laboratory in normal saline immediately after delivery.  Both oseltamivir phosphate and its active
metabolite oseltamivir carboxylate were obtained, with permission, from Roche
(Basel, Switzerland).The single
cotyledon placental perfusion system was used as described
by Schneider and Huch [10]. A
fetal artery and vein on the chorionic plate were cannulated with 3.0 F and 5.0 F catheters, respectively. The fetal circulation was established by gently
perfusing Eagle’s minimal essential media including 3% bovine albumin and 0.5 units/mL of heparin (Sigma Chemical Co, St. Louis, Mo, USA).  The cotyledon was initially examined for
vascular integrity.The isolated
cotyledon and adjoining placental tissue were transferred to a
temperature-controlled chamber (37∘C) where the fetal circulation was perfused with media at
4.5–5.0 mL/min for 20
minutes to stabilize the pressure and determine if the cotyledon had a vascular
leak. In general, we could not detect leaks of less than 2 cc/min; however,
volumes into and out of the system were carefully measured, and when a change
in volume was noted, the system was presumed to have a leak.  The final pressure was generally about
35 mmHg.  If the cotyledon failed to reach
a stable baseline pressure or was found to have a leak, it was discarded.
Finally, the maternal side of the catheterized cotyledon was sealed, and three
18-guage needles were inserted into the intervillous space of the selected
cotyledon to reestablish the maternal circulation.  Maternal flow rate was at 17 mL/min.The maternal
and fetal compartments each contained 150 cc of Eagle’s minimal essential
medium (pH 7.2–7.4), which was
aerated with 95% oxygen and 5% carbon dioxide. Maternal-fetal transfer was
performed with oseltamivir phosphate (OP) by adding the drug to the maternal
circulation at concentrations that were approximately 5-6 fold(n=2), 20–30 fold (n=2),
and 700–800 fold (n=2)
above the therapeutic peak. Supratherapeutic concentrations were studied, as
neither OP nor its active metabolite, oseltamivir carboxylate (OC), could be
detected when near-therapeutic concentrations were used.In each of the
6 placentas that met study criteria, experiments were conducted for the first
hour with both circulations open to determine the clearance index (CI) of the
drug. During this portion of the experiment, 2–5 cc of outflow
samples were collected from the venous sides of both the maternal and fetal
circulations every ten minutes. Then, both systems were closed and recirculated
for one additional hour to determine levels of accumulation.  The collected fluid aliquots were then
analyzed.  The transport fraction ofC14 antipyrine was used as a
reference compound to determine the CI of oseltamivir carboxylate. A transport
fraction of more than 30% for antipyrine was deemed representative of
maternal-fetal circulatory match [10].The high-pressure
liquid chromatography (HPLC) assay of the prodrug oseltamivir phosphate is a
modification of the procedure described by Sweeny et al. [11].  The HPLC analysis was carried out on Waters
Associates instruments (Milford, Mass, USA) and consisted of a preparation of a
standard curve to breach the range of oseltamivir used. All specimens and
standards were extracted in aliquots of 0.5 mL. To each tube was added 0.5 mL of
acetonitrile to precipitate blood and other proteins. The samples and standards
were mixed and centrifuged at 800x gravity for 10 minutes, and the clear
supernatant was injected into a Supelcosil LC-18-B Column (Supelco
Chromatography Products, Bellefonte, Pa, USA). The HPLC conditions consisted of a
mobile phase of 50% acetonitrile and 50% 0.01 M ammonium acetate. The
sensitivity of the instrument was 0.005 AU at a wavelength of 230 nm. The flow
rate of the mobile phase was 1.5 mL/min, and an injection volume of 40 microliters
was used. The retention time of oseltamivir was 7.5 minutes. All concentrations
of the drug were determined by measuring peak height of the extracted standards
and perfusates. The HPLC method was validated; the reproducibility data are not
shown.The HPLC
method for assaying the metabolite oseltamivir carboxylate was as follows. The
drug was dissolved in water and diluted to anticipated therapeutic
concentrations. The placental perfusion samples were aliquoted in 1.0 mL
samples and made alkaline with 20 mL of 5N NaOH. The samples were then added to
theC18 Sep-pack column (Waters Associate, Milford, Mass, USA) that had
been activated by the following procedure: 1 mL of 0.1 M PO4 buffer
(pH 6.1) was added to 0.1 gm/50 cc of 2-(N-morphalino) ethane sulfonic acid
(MES), an ion buffer; this mixture was then washed through the column and the
procedure repeated. One mL of methyl alcohol was added to the column and
washed, followed by two washings of the phosphate buffer containing MES. After
the sample had been pulled through by vacuum, the column was washed with 1 mL
of PO4 buffer containing MES. At this time the active metabolite was
washed off the Sep-pack column with phosphate buffer containing 5%
acetonitrile. The HPLC assay parameter consisted of a 717 autosampler, a 486
detector, a 515 pump (Waters Associate instruments, Milford, Mass, USA), and a 0.1
millivolt Linear 1200 chart recorder (Scientific Marketing, Houston, Tex, USA). The
HPLC column was a Supelcosil LC-18-DB, 25 cm × 4.6 mm, 5 μm column (Supelco,
Bellefonte, Pa, USA).  Seventy-five microliters
of extracted samples were injected into the system with a flow rate of 2.5 mL/min
at detection settings of 0.01 AU, 220 nm wavelength with a metabolite retention
time of about 6.4 minutes. The mobile phase was 5% acetonitrile in 0.1 M PO4 buffer (pH 6.1) containing 0.5 gm of MES per 500 mL.  The standard curve was validated for within
and between batch reproducibility and the lower limit of quantitation
determined.


## 3. Results

The
recovery of OP and OC by HPLC analysis was greater than 80%.  The minimum
sensitivities for detection were
30 ng/mL and 2.4 ng/mL for OP and OC, respectively.Previous
pharmacokinetic studies of oseltamivir have determined that the peak plasma
concentrations after a 75 mg twice-daily dosing regimen are 65.2 ng/mL for OP
and 348 ng/mL for OC [1]. In our study, we were unable to detect either the
prodrug or its active metabolite when levels in the perfusate were either in
the therapeutic range or 5-6 fold above the
therapeutic range. At higher concentrations (20- to 830-fold above the
therapeutic range), we were able to detect OC in both the maternal and fetal
circulation (Table 1). The results presented in the table represent the mean
concentrations from the samples assayed in the open circulation. Mean
concentrations were used to ensure that the values were representative of a
steady state. The mean clearance index in these supratherapeutic concentrations
was 0.13±0.08. In a
recirculated (i.e., closed-closed) system, OC accumulated in the fetal
circulation only in the two placentas with the highest initial concentrations
of OP; the mean accumulation in these cases was
17.0±14.6 ng.Table 1
Mean maternal and fetal concentrations
of oseltamivir phosphate and oseltamivir carboxylate from 6 term placental
cotyledons perfused with supratherapeutic concentrations of oseltamivir
phosphate.
Maternal (ng/ml)Fetal (ng/ml)PlacentaOP*OCOPOC1320
(5x)NDNDND2400
(6x)NDNDND31 300
(20x)86ND4.342 160
(30x)140305551 000
(780x)9073009654 000
(830x)9633002.4*concentration relative to therapeutic
dose in parentheses.ND = nondetectable.


## 4. Discussion

The
pharmacokinetics of oseltamivir have been studied in geriatric and pediatric
populations, and treatment trials have demonstrated the safety and efficacy of
the drug in these populations, both of which are considered high-risk for
complicated influenza-related illness [2].
Little is known, however, regarding the pharmacokinetics or safety of
oseltamivir in pregnancy. Oseltamivir is classified as Pregnancy Category C, as
animal studies have suggested that an adverse effect on the fetus may exist,
but there have been no well-controlled studies conducted in humans.  Animal studies [12] for effects on
embryo-fetal development have been conducted with rats and rabbits who were
administered oseltamivir by the oral route. Relative doses were up to 100 times
the human exposure in the rat and up to 50 times in the rabbit. Fetal exposure
was seen in both species, and there was a dose-dependant increase in the
incidence of minor skeletal deformities. The individual incidences of each
anomaly, however, remained within the background rates of occurrence of the
species studied.  Although we are unable
to examine the fetal effects of exposure to oseltamivir in the ex vivo model, we are not aware of
any other study evaluating the metabolism and transplacental passage of
oseltamivir in the human placenta.Oseltamivir
phosphate is administered orally as an ethyl ester prodrug which is extensively
converted by hepatic esterases to the active compound oseltamivir carboxylate
[1]. Approximately 80% of an orally administered dose reaches the circulation
as OC, and the metabolite has excellent penetration into tissues such as the
lung, nasal mucosa, and middle ear [13]. The peak plasma concentrations are
lower and the half-life is
shorter for OP than for OC, respectively, so the placenta of a pregnant woman
would presumably be exposed to higher sustained levels of OC than the prodrug.
Since, however, no pharmacokinetic studies have been conducted in pregnant
subjects, we chose to examine the prodrug in our placental perfusion model.
Subsequently measuring the concentrations of the metabolite in the maternal and
fetal circulations enabled us to estimate both the extent to which the drug was
metabolized and the efficiency with which the metabolite transfers from one
compartment to another.  Oseltamivir carboxylate
does not undergo further metabolism in
vivo and is excreted unchanged in urine [13]; therefore, the clearance
index obtained from our model is likely a valid estimate of maternal-fetal
transfer.Our results
suggest that OP is extensively metabolized to OC in the ex vivo human placental model. Although hepatic esterases are
primarily responsible for the hydrolysis of
OP to OC in vivo, serum
esterases can also effect significant conversion of the prodrug to the
metabolite. Lindegardh et al. [14] examined the ex vivo metabolism of oseltamivir and found that up to 31.8% of
the prodrug was converted into the metabolite after 4 hours of exposure to
human serum. This conversion was not completely arrested even when the samples
were placed on ice, but it was inhibited by the addition of the esterase
inhibitor dichlorvos. In our model, most residual fetal blood is removed by
perfusing the fetal artery and vein after initial cannulation; however, the
“flushing” of the intervillous space is less complete in the maternal
circulation, and it is possible that the perfusate was exposed to residual
maternal serum and the proteins contained therein. The placenta itself,
however, also contains esterase activity. Histochemical analysis of the human
placenta has demonstrated a high concentration of “nonspecific” esterase in the
trophoblast of the villi and a variable concentration in the villous cores [15].  We are unable to determine in our study
whether OP was metabolized to OC by serum esterases, placental esterases or
both, but our results suggest that this conversion was extensive.The
transplacental transfer of a substance is regulated by a number of variables,
including molecular weight, ionic charge, concentration in the maternal plasma,
maternal blood flow rate, protein binding, and placental metabolism [16].  Most compounds with a molecular weight less
than 500 daltons diffuse readily through the placenta [16].  Given that oseltamivir carboxylate has a
molecular weight of 312.4 daltons and has low protein binding (3%), [1] it was
somewhat surprising that the transplacental passage of this compound was
incomplete in the ex vivo
model. Although OC is not thought to be further metabolized, another metabolite
of OP has been identified by Sweeny et al. [11]. They described a novel
metabolite which was recovered from the urine, plasma, and tissue samples of
rats who were administered OP by the oral route. The (R)-ω-carboxyilic acid metabolite was identified by
HPLC and NMR analysis; and it was the second most abundant metabolite in these
specimens after the active neuraminidase inhibitor. In the present study, we
did not evaluate for the presence of  ω-hydroxylated
products, and if placental metabolism of OP yielded these metabolites, it is
possible—depending on the
distribution of the products of hydrolysis—that our
estimated clearance index of OC is falsely low.
Importantly, however, very high concentrations of OP had to be studied
before either the prodrug or the active metabolite could be detected, and
accumulation in the fetal compartment was minimal.Oseltamivir is
the only orally bioavailable antiviral medication that is recommended for
chemoprophylaxis and treatment of influenza in the United States, and the
demand for this drug is likely to increase. The adamantane class of antivirals,
including amantadine and rimantadine, has activity against influenza A, but
sufficient resistance has developed in contemporary strains of influenza that
the CDC has recommended avoiding the use of these agents until susceptibility
can be reestablished [3].  Resistance to
oseltamivir, conversely, has been observed infrequently, and the resistant
strains identified in clinical samples appear to have reduced infectivity and
potential for transmission [9].
Oseltamivir has also demonstrated efficacy—both in vitro and in animal models—against the H5N1
and H9N2 strains of avian influenza for which no effective vaccine exists [9].  To date, the World Health Organization has
reported 329 confirmed cases of avian influenza A/(H5N1) with 201 deaths  (61% case-fatality rate), and concern that a
mutated H5N1 could result in the next pandemic has caused a number of nations
and international agencies to  generate a
stockpile of oseltamivir [17].  The
potential global importance of this medication is quite evident, but it is
unfortunate that such a paucity of data exists concerning its use in pregnant
women, especially since they are more likely to develop serious complications
from influenza infection than their nonpregnant counterparts.


## 5. Conclusions

Our study
suggests that oseltamivir phosphate is extensively metabolized in the ex vivo human placental model.  The active metabolite oseltamivir carboxylate
was identified in the maternal and fetal circulations when very high doses of
the prodrug were studied, and the transplacental transfer of the metabolite was
incomplete in our model.  Additional
studies are needed to better characterize the pharmacokinetic behavior of
oseltamivir in pregnancy to assess the need for dosing adjustments and the effects
of the medication on the developing fetus.



---

*Source: 927574-2008-06-04.xml*