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Pharmacodynamics impact of etravirine disposition into breast milk in relation to HIV replication in this milk

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Pharmacodynamics impact of etravirine disposition into breast milk in relation to HIV replication in this milk

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Content Page 1 of 70

PHARMACODYNAMICS IMPACT OF ETRAVIRINE DISPOSITION INTO
BREAST MILK IN RELATIONS TO HIV REPLICATION IN THIS MILK

by

Siyu Liu

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2014

Copyright 2014 Siyu Liu

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DEDICATION
I would like to dedicate my thesis to my dear parents, whose love and support
has allowed me to sojourn into an arena that is foreign to them. Despite this, they
have constantly guided and supported me to pursue my dreams. Their love has
inspired me to take bold steps to advance my education and career goals.
I want to thank my supportive friends, who are my family away from home, for
their persistent inspiration. They have encouraged me to strive to pursue my
goals with pinpoint focus. For their inspiration and encourage, I am deeply
indebted to them. This thesis was only possible because of your inspiration.

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ACKNOWLEDGEMENTS
I am grateful to those who have guided and supported me on my quest to
understand the importance of depth of knowledge. I am indebted to advisor Dr.
Stan Louie, for his support and guidance through my graduate school career.
He saw potential in me to become a scientist, and encouraged me to explore and
ask the right questions. Moreover, his trust in my abilities allowed me the
independence to expand my projects and follow my ideas. I would also like to
thank my committee members, Dr. Wei-Chiang Shen and Dr. Julio Camarero, for
allowing me to work under their tutorage.
I would also like to acknowledge laboratory friends and colleagues. They have
provided the environment for me to thrive. Their full support and kindness made
me feel at home. They comforted me when the experiments were not as I had
thought. They cheered me on when things were going the right directions. They
have become my friends and my family. With their love, they have guided and
encouraged through the thick and thin of life. Thanks to their persistent support, I
could not have done this without your support.
Here, I especially appreciate my former lab mates, Drs Jared Russell and
Dezheng Dong, who taught me the essential skills when I first began my
research career. I also want to thank other laboratory members including Drs.
Natasha Sharma and Julie Yoo for teaching me the intricacy of experimental
design and implementation. To Isaac Asante, Sachin Jadhav, and Eugene Zhou,
I am indebted to your comments and questions. You have “Sharpened” my sword
as I advance to the next stage of my career.

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In addition, I would like to thank Drs. LaShonda Spencer and Michael Neely for
entrusting me your precious samples and idea. The project was only successful
with your persistence in recruiting the patience and collecting the samples just at
the right time. You made this experience one that I will always remember.
Finally, and most importantly, I would like to express my deep gratitude to my
advisor, Dr. Stan Louie. He is an insightful and knowledgeable researcher whose
opinions helped me to conquer the troubles that I was faced with during my
research. I have learned and experience a lot of sciences, however it is the
invaluable experience since becoming a member in your laboratory that has
made me who I am today. Without his patience and wise guidance, I would never
be able to accomplishing what I have in this manuscript. Thank you!

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LIST OF TABLES
Table 1: MS Condition for Etravirine and Efavirenz. ........................................... 48
Table 2:Multiple reaction monitoring information: Q1: Parent ion; Q3:
Product ion. ................................................................................... 49
Table 3 : Mass Spectrometer potential parameters: ........................................... 50
Table 4 : Characteristics and Demographics of Patient subjects ........................ 51
Table 5: Etravirine Plasma Curve Accuracy ........................................................ 52
Table 6: Etravirine Breast Milk Curve Accuracy: .................................................. 52
Table 7 : Linearity of Etravirine plasma standard curves .................................... 53
Table 8: Linearity of Etravirine breast milk standard curves ................................ 53
Table 9: HAART ARVs’ plasma standard accuracy of quality controls at
the concentration of 100, 500 and 2500 ng/mL. ............................ 54
Table 10 : HAART ARVs’ breast milk standard accuracy of quality
controls at the concentration of 100, 1000 and 5000 ng/mL. ......... 55
Table 11 : Linearity of interday HAART ARVs’ plasma standard curves. ............ 56
Table 12 : Linearity of interday HAART ARVs’ Bresat Milk standard
curves. ........................................................................................... 57
Table 13: Regimens summary ............................................................................ 58
Table 14: Etravirine concentration median in patients with detectable
HIV-1 RNA versus viral load undetectable patients ....................... 59
Table 15: Etravirine Median Concentrations in BM and Plasma by time
points. ............................................................................................ 60
Table 16: HAART PK parameters are classified by drugs. ................................. 61

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LIST OF FIGURES
Figure 1 : Robustness of Etravirine plasma standard curves. ............................. 66
Figure 2 : Robustness of Etravirine breast milk standard curves. ....................... 66
Figure 3 : ETR Bayesian posterior predicted time concentration profiles
and measured concentrations. ...................................................... 67
Figure 4 : Plasma ETR population prediction and individual posterior
prediction by Bayesian Posterior Statistics.. .................................. 68
Figure 5 : Breast milk ETR population prediction and individual posterior
prediction by Bayesian Posterior Statistics.. .................................. 69

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ABBREVIATIONS
3TC Lamivudine
APV Amprenavir
ATV Atazanavir
AZT Zidovudine
DRV Darunavir
EFV Efavirenz
ETR Etravirine
FTC Emtricitabine
HAART Highly active antiretroviral therapy
KAL Kaletra
LPV Lopinavir
MTCT Mother-to-child transmission
NH
4
Ac Ammonium acetate
NNRTI Non-nucleoside Reverse transcriptase inhibitor
NRTI Nucleoside Reverse transcriptase inhibitor
PI Protease inhibitor
RTV Ritonavir
TDF Tenofovir

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ABSTRACT
Mother-to-child transmission (MTCT) through breastfeeding continues to be a
major concern for the transmission of HIV infection among children in developing
countries. The transmission usually occurs in the early stage during
breastfeeding, where the infant’s gastrointestinal integrity may be compromised.
Highly antiretroviral therapy (HAART) plays an important role in the control of
viral replication in the plasma of infected HIV patients. HIV positive women
patients receiving HAART during pregnancy and postpartum has become a
crucial strategy to suppress the viral load in breast milk in hope to prevent MTCT.
However it is not known whether the level of ARV is adequate to prevent infant
acquisition of HIV. Etravirine (ETR), a NNRTI, is a second generation NNRTI that
has been approved as a second line treatment for HIV infection. It is active
against several common HIV 1 mutants that are resistant to first generation
NNRTIs. In this study, we used a 14-day intensive ETR included HAART on
postpartum women subjects. The results indicated that the disposition of ETR in
breast milk from plasma after the treatment and a relatively low ETR BM
concentration in both viral detectable subjects. Also, the distinct selection of
ARVs for HAART may affect ETR BM concentration. In summary, intensive ETR
included HAART may be a potential effective measure to prevent MTCT in those
resource-limited areas.

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TABLE OF CONTENTS ................................................................................... PAGE
DEDICATION .................................................................................................. 2
ACKNOWLEDGEMENTS ............................................................................... 3
LIST OF TABLES ............................................................................................ 5
LIST OF FIGURES ......................................................................................... 6
ABBREVIATIONS ........................................................................................... 7
ABSTRACT ..................................................................................................... 8
1. INTRODUCTION .................................................................................... 11
2. MATERIALS ........................................................................................... 25
2.1. Chemicals and reagents .................................................................... 25
2.2. Participant Population ........................................................................ 25
2.2.1. Inclusion Criteria .................................................................. 26
2.2.2. Exclusion criteria .................................................................. 26
2.2.3. Duration of Study: ................................................................ 27
2.3. Data Collection .................................................................................. 27
2.4. Patient samples ................................................................................. 29
3. METHODS ............................................................................................. 29
3.1. Liquid chromatography conditions ..................................................... 29
3.1.1. Etravirine analysis ................................................................ 29
3.1.2. Components of HAART Analysis ......................................... 30
3.2. Etravirine analysis .............................................................................. 31
3.2.1. Sample Preparation ............................................................. 31
3.2.1.1. Preparation of Calibration Standards .................... 31
3.2.1.2. Plasma and Breast Milk Sample Preparation ........ 32
3.2.2. Components of HAART Analysis ......................................... 32
3.2.2.1. Preparation of Calibration Standards .................... 32
3.2.3. Plasma and Breast Milk Sample Preparation ....................... 33
3.3. Data Analysis ..................................................................................... 33
4. RESULTS ............................................................................................... 34
4.1. Demographic of patients .................................................................... 34
4.2. Standard Calibrations and Curves ..................................................... 34
4.2.1. ETV Validation Studies ........................................................ 34
4.2.2. HAART Validation Studies ................................................... 35
4.3. Pharmacokinetics Analysis ................................................................ 35
5. DISCUSSION ......................................................................................... 39
6. CONCLUSIONS ..................................................................................... 42

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7. BIBLIOGRAPHY ..................................................................................... 43
TABLES ........................................................................................................ 48
FIGURES ...................................................................................................... 66
APPENDIX .................................................................................................... 70

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1. INTRODUCTION
First discovered in 1983, human immunodeficiency virus (HIV) was identified as
a lentivirus that infects CD4 positive cells like lymphocytes, monocytes and
macrophages. CD4 cell infection initially thought to promote the HIV entry into
the infected cells. However, it was later found that chemokine receptors such as
CCR5 and CXCR4 also facilitated viral entry [1]. The critical role of chemokine
receptors was demonstrated in 1) the progression from HIV infection to clinical
acquired immunodeficiency, and 2) the importance of mutations in the chemokine
receptors and its relations to resistance towards HIV infection. The finding that
highlighted importance of chemokine receptors was that mutation on the 32
amino acid of the CCR5 sequence could block the entry of HIV into the host cell.
Once the host cell is infected, the virus unloads its genomic cargo into the
cytoplasm, where the RNA template is then converted into viral complement DNA
(cDNA). An antisense viral DNA is then synthesized, and thus forming the double
strained DNA. The viral DNA can integrate into the host cell DNA with the
assistance of viral integrase. Production of the viruses is promoted through the
biosynthesis of viral protein precursors, where the active protein is liberated
using viral protease. Viral assembly is the final step of the viral replication
process, by which the infectious viruses are liberated or “budded off” into the
extracellular space [1-3, 13]. HIV also can establish latency within CD4 positive T
cells, which lead to great challenges to develop completely treatments [4].
According to the data from UNAIDS, there are more than 2.5 million newly
infected worldwide during 2012, where most of the new cases occurred in

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developing countries, especially Africa [5]. No doubt HIV infection has become
severe epidemic, which threatens the global public health.
HIV is a sexually transmitted infection that can be also transmitted through
intimate contact with contaminated bodily fluids. Normally, viral infectious occurs
via percutaneous penetration leading to systemic infection. An alternative
infectious route include oral infection through the ingestion of contaminate breast
plasma or milk since HIV is able to binding to CCR5 and CXCR4 to infect
mucosal epithelial cells [6]. Therefore, maternal transmission to the newborn is a
major problem confronted in Africa, which is a resource-limited environment.
Previously, United Nation (UN) and World Health Organization (WHO) have
already set a goal to be achieved by 2015, emphasizing on the elimination of
newly infected children and reduction of the maternal related death [5, 7, 12].
Most paediatric HIV infections occurring in the children result from HIV-infected
mother due to delivery or breast-feeding. This process is more commonly known
as mother-to-child transmission (MTCT). Several measures have been taken to
reduce the risk of MTCT in developed and developing countries such as using
formula milk instead of breast milk and choosing elective caesarean sections
instead of virginal delivery [8]. However, in current time, MTCT rate continues to
be high in developing countries, especially in resource-limited areas [9]. MTCT
has become a serious public health problem for controlling the spread of HIV
infections. Thus, prevention of MTCT is of great importance to protecting the
children from HIV infections [10]. HAART for pregnant women can effectively

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lower the risk of MTCT, which is a major way to reduce the MTCT rate worldwide
[11, 12].
Highly active antiretroviral therapy (HAART) is a global used combination
treatment of antiretrovirals (ARVs) for HIV-1 infection among patients [13].
HAART has an aggressive potency to suppress HIV activity, lower the viral load
and control the disease progression. Usually, HAART normally contains three or
four different antiretroviral agents that combine either non-nucleoside-reverse
transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) with two nucleoside
reverse transcriptase inhibitors (NRTIs). Most current recommendation has
added integrase inhibitors like raltegravir, elvitegraivr or dolutegravire with two
NRTIs as a potential alternative to either PI- or NNRTI–containing regimens [14].
Nucleoside reverse transcriptase inhibitor (NRTI) like zidovudine (AZT) and
nevirapine (NVP) were the first antiretroviral agents used to treat HIV infections
[15]. These nucleosides were analogues of naturally occurring thymidine or
cytosine. On the other hand, NRTIs can block virus replication by inhibiting the
activities of reverse transcriptase to prevent the production of virus double-
strained DNA [16]. In response to viral resistance mechanisms, a number of
acyclic nucleoside phosphonates were designed and tested in humans. Currently
tenofovir is an important component in Department of Health and Human
Services (DHHS) guidelines for the treatment of HIV infections [17].
Although NNRTIs have been part of the DHHS guidelines, improvements in the
class of compounds have included the development of etravirine (ETR) and
rilpravirine (RPV) to enhance the efficacy found with efavirenz (EFV) [15]. ETR

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binds to the pocket structure of viral polymerase but not the active site. Rather
the binding onto an allosteric site can lead to the conformational change of
reverse transcriptase and thus inhibit viral DNA elongation [18].
In contrast to the other two classes of ARVs, protease inhibitors are substrate
analogues of HIV protease so that they can prevent the production of virus
particles by disturbing the virus replication [13, 19]. ARVs are faced with the
problem of drug resistance because of the mutation. HAART can help to slow
down the resistance progress. Nevertheless, HAART still confronts the problem
of drug resistance and tolerance in the long term.
Etravirine Pharmacology
Etravirine (ETR) is a second generation NNRTI, which is normally employed as
the second line treatment of HIV-1 infection for adult patients who have
developed drug resistance for the first-line antiretroviral treatments like EFV. ETR
is diarylpurimidine (DAPY) based compound that binds onto the allosteric site
found on the viral polymerase. The binding will trigger a conformational change
and thus lead to the inhibition of the synthesis of virus DNA by preventing RNA
and DNA-dependent polymerase activities. ETR has the conformational flexibility
to adapt a wider range of reverse transcriptase mutations that was attributed to
HIV ability to become resistant of other NNRTIs. The chemical flexibility permits
ETR to accommodate these mutational changes and thus continue to be active
despite the presence of these adaptive mutations. In particular, ETR is able to
overcome several common HIV-1 mutations like Tyr181Cys (Y181C) and
Lys103Asn (K103N). ETR-mediated suppression of HIV has been shown to

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enhance immune response as evident by increase number of circulating CD 4
cells found in the blood. These types of clinical responses have led to FDA
approval of this NNRTI, which is orally administered as 200 mg given twice daily
(bid). ETR must be combined with other anti-HIV therapy. [18-22]
ETR Pharmacokinetics Parameters
Due to its high lipophilicity, ETR is recommended to be taken with food. ETR is a
high protein-binding drug, which pharmacokinetics studies showed that ETR has
a plasma Cmax of around 400 ng/mL and a plasma Cmin of around 180 ng/mL
when the dosage is 200 mg twice daily. The half-life in plasma is approximately
30-40 hours and the Tmax occurs at about the 4th hour. Due to its hydrophobicity,
it is expected that ETR is predominately broken down by CYP450 enzymes
(CYP3A, CYP2C9, and CYP2C19) and the excretion of ETR is majorly via faeces
and bile. [19, 23, 24]
ETR Clinical Activity
ETR was found to have potent in vitro antiviral activity. Not only was ETR active
against wild-type HIV-1, but also against strains harboring different resistance-
associated mutations (RAMs) [20]. The 50% effective concentration in cell based
assays (EC50) for wild-type laboratory and primary HIV-1 isolates ranges from
0.9 to 5.5 nM (0.4 to 2.4 ng/mL), with little or no loss of activity against HIV-1
variants with key NNRTI RAMs. In extensive in vitro testing, ETR showed potent
antiviral activity against a panel of > 6000 recombinant clinical isolates resistant
to at least one of the first generation NNRTIs, with EC50 values below 10 nM

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(4.4 ng/mL) for 83.2% of isolates and below 100 nM (43.5 ng/mL) for 98% of
isolates. Overall, ETR has an antiviral profile that is superior to that of first
generation NNRTIs like efavirenz, nevirapine and delavirdine. Non-clinical safety
evaluations demonstrated that ETR was safe for use in clinical testing. Further
details can be found in the investigator brochure. The accumulated safety data
from completed and ongoing trials with ETR indicate that the drug is generally
safe and well tolerated at the doses being tested in clinical trials. [18, 21, 25]
No consistent pattern or clear dose relationship was apparent for any laboratory
parameter evaluated in the single and multiple dose trials with ETR in healthy
subjects [26]. Most of the abnormalities were grade 1 or 2. Analysis of vital signs
and electrocardiogram (ECG) data in the single and multiple dose trials showed
that ETR is not associated with any consistent effect on cardiovascular
parameters, including corrected QT (QTc) interval.
Two Phase III trials with identical design (DUET-1 and DUET-2) were contributed
to the efficacy and safety data of ETR [25]. The DUET-1 and DUET-2 performed
involving a total of 1203 treatment-experienced subjects with documented
resistance to NNRTIs. Subjects were randomized, in a double-blind manner, to
ETR 200 mg or placebo, both b.i.d., with a background regimen of darunavir
(DRV)/ritonavir and investigator-selected NRTI(s), with or without enfuvirtide
(ENF). The primary analyses of these Phase III trials were performed when all
subjects had completed 24 weeks of treatment or had dropped out earlier. The
Week 48 results are available. Both the 24- and 48-week efficacy results were
consistent in the demonstration of a statistically significant superior efficacy for

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ETR versus (vs) placebo. The proportion of subjects in the pooled DUET trials
with plasma viral load < 50 copies/mL at Week 48 (time to loss of virologic
response [TLOVR]) was 61% in the ETR group and 40% in the placebo group.
With the exception of rash, the incidence of AEs and laboratory abnormalities
were generally comparable to placebo. There was no relationship between the
pharmacokinetics of ETR and either efficacy or safety. The results from the
pooled DUET trials also demonstrate that at Week 48, the ETR group was
superior over the placebo group irrespective of whether ENF was used de novo
or not. The high virologic and immunologic benefits with the ETR-based regimen
were translated in significantly fewer clinical endpoints (AIDS defining illnesses or
death) when compared with the placebo group after 48 weeks of treatment. As
for the safety data, the most common AEs reported during ETR treatment with
the selected dose were rash (any type, 19.2%), diarrhea (18.0%), nausea
(14.8%), and anemia (4%). The majority of AEs were grade 1 or 2 in severity. 12
subjects (2.0%) died due to AE(s), which emerged during treatment with ETR;
most frequent fatal events were AEs related to infections and infestations. There
was no consistent pattern for SAEs. The most frequently reported individual
SAEs were pneumonia (0.5% in the ETR selected dose group).
The incidence of AEs and laboratory abnormalities were generally comparable to
placebo. However, severe, potentially life-threatening and fatal skin reactions
have been reported, with an incidence of < 0.1%. This includes cases of
Stevens-Johnson syndrome, hypersensitivity reaction, toxic epidermal necrolysis
and erythema multiforme. There was no relationship between the
pharmacokinetics of ETR and either efficacy or safety. [21, 25]

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Genotype analyses demonstrated that the presence of specific NNRTI mutations
were predictive for decreased virologic response to ETR [21]. Seventeen ETR
RAMs were identified and a weighted genotypic score was developed. The
relation between this ETR weighted genotypic score and virologic response was
demonstrated.
Mother to Child transmission (MTCT)
Maternal to child transmission (MTCT) of HIV can occur at different time periods
perinatally: in utero, at time of delivery or during breast-feeding. Highly Active
Antiretroviral Therapy (HAART) has decreased the risk of transmission of HIV to
exposed infants considerably in the United States. Women receiving potent
combination antiretroviral therapy over at least the last trimester of pregnancy,
receiving proper obstetric interventions and who are able to avoid breast-feeding,
decrease the risk of having an infected infant to about 1% [27, 28]. However,
despite the limited use of antiretroviral therapy (ART) perinatally, it has been a
challenge to decrease the rate of MTCT in resource-limited countries because of
the necessity to breast-feed.
In resource limited settings where the majority of HIV infected women breastfed
due to cost constraints, cultural norms, stigma, and unsafe water supply, the risk
of MTCT without any intervention is much higher, ranging from 30-45% at 24
months after delivery [29]. Several studies have tried to quantify the absolute
transmission rates through breastfeeding. Nduati et al [30], in a randomized
clinical trial comparing breastfeeding to formula feeding, where HIV-1 infection
rate of 36.7% in the breastfeeding arm as compared to 20.5% in formula fed

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(p=0.001). Breast milk transmission accounts for 44% of all infant infections. In a
critical review of the literature, De Cock et al [29] reported that one third to one
half of perinatal HIV infections in African settings were due to breastfeeding.
Although ART has been effective in reducing perinatal transmission of HIV-1, no
therapeutic regimen has been developed where throughout the lactation period,
significantly reduces transmission via breast-feeding [31]. In resource-limited
countries, many HIV infected women are treated perinatally with short course
antiretroviral therapy such as AZT, AZT/3TC or NVP. Even with this therapy, the
overall risk of transmission at 12-24 month ranges from 16-23% [32].
It is important to understand the risk factors for breast milk transmission of HIV in
order to develop interventions to prevent transmission. Characteristics of the
mother, infant, breast milk, and type and duration of breast-feeding are all
important factors which contribute to breast milk transmission.
Multiple studies have shown that exclusive breast-feeding carries a much lower
risk of HIV transmission than mixed breast-feeding (defined as breast milk along
with complementary food, other milk, and/or infant formula). Studies from South
Africa noted that children who received mixed breast-feeding were more likely to
be infected by 15 months of age compared with those who were exclusively
breast-feed for at least 3 months [33]. Mixed feeding is thought to cause
disruption in an infant’s gut environment resulting in local inflammation and
increased risk of transmission. The duration of breastfeeding is also an important
factor related to viral transmission. Although transmission can occur at anytime
during breast-feeding, the probability increases with duration of breastfeeding.

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Multiple characteristics of both mother and infant contribute to the increased risk
of HIV transmission through breastfeeding. Maternal factors such as age, parity,
HIV disease stage and breast pathology all contribute to this risk. High maternal
viral load and low CD4 count has consistently shown to be an important risk
factor. Breast pathology such as nipple lesions, mastitis and abscess has also
been associated with late postnatal transmission through breastfeeding. Oral
candidiasis in the infant was also associated with late postnatal transmission
(1.53-2.29%) [34].
Breast milk HIV-1 RNA (cell free) viral load is significantly associated with breast
milk transmission. Rousseau et al [31] reported a 2-fold increased risk of
transmission associated with every 10-fold increase in breast milk viral load. In
addition, cell associated virus (HIV DNA) was associated with a significant
increase in risk of transmission independent of the level of cell-free viral RNA [34].
There are several strategies to try and prevent MTCT of HIV through
breastfeeding. Unfortunately the simplest, avoidance of breastfeeding is
sometimes neither safe (increased risk of diarrheal morbidity), affordable, or
feasible in resource limited countries. In this setting, WHO recommends
exclusive breastfeeding during the first months of life with early weaning as
rapidly as possible [35]. Two additional strategies to prevent MTCT include
providing antiretroviral drugs to infants exposed to HIV during breast-feeding or
decreasing HIV viral load in breast milk via maternal treatment with HAART while
breastfeeding. The Kisumu Breastfeeding Study in Kenya provided antenatal
HAART at 34 weeks through 6 months postpartum [38]. The cumulative rate of

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HIV infection in infants uninfected at birth was 4.2% at 6 weeks and 5.0% at 6
months. The Mitra-Plus study in Tanzania also provided HAART antepartum
through 6 months postpartum; the infant infection rate was 4.1% at 6 weeks and
5.0% at 6 months [39]. There are several ongoing trials evaluating maternal
HAART for prevention of MTCT via breastfeeding, however there is currently no
recommendation on which regimen would be most effective for this indication.
A previous study reported on ARV levels in breast milk in mothers receiving
antiretroviral therapy. Shapiro et al [40] reported on ARV concentration of AZT,
3TC, and NVP in 20 breast-feeding women in Botswana. Concentrations of NVP,
3TC and AZT in breast milk were similar to or higher than those found in serum.
The median breast-milk concentrations of NVP, 3TC , and AZT were 0.67, 3.34,
and 3.21 times those in maternal serum. In addition, the concentration of NVP in
the serum of infants who were breastfed by mothers taking the drug was above
the levels necessary for prophylaxis against HIV. Furthermore, the same study
demonstrated that HAART suppresses HIV RNA in breast milk [42]. However, it
did not have a significant effect on HIV DNA (cell-associated virus). Thus,
maternal HAART may reduce breast milk transmission by decreasing HIV viral
load in breast milk and providing HIV inhibitory concentrations in the serum of
breast fed infants sufficient for prophylaxis against transmission. To date, there
are limited studies published, that have looked intensively at the
pharmacokinetics of ARV drugs in breast milk. Limited studies as above have
measured 1 sampling point per woman and infant, which limits the ability to fully
characterize relationships between ARV drug levels in breast milk and serum in
relation to dosing interval. As the cost of antiretroviral therapy decreases, more

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regimens will become available to women in resource-limited areas. ARVs with
good penetration into breast milk and with the ability to decrease local replication
of HIV will have the greatest impact on MTCT transmission through
breastfeeding.
It is difficult to predict how drugs will transfer into breast milk. Nucleoside reverse
transcriptase inhibitors (NRTI) concentrate in breast milk and are present in
breast milk at higher levels than in maternal blood. In animal models, tenofovir
has been shown to penetrate breast milk. Van Rompay et al [43] in a pilot study
on tenofovir (TDF) pharmacokinetics in lactating macaques detected tenofovir in
milk of both animals, but the peak concentrations were 2-4% of those detected in
the serum. In addition, limited studies have demonstrated lower levels of
protease inhibitors in breast milk than in serum. In other studies, compared with
the plasma concentration, the breast milk concentration was about 70% for
nevirapine (NVP), nearly 250% for 3TC, and between 6 and 24% for nelfinavir
(NFV) [44].
The risk of transmission of resistant virus to breastfeeding infants is seen in both
maternal HAART and infant prophylaxis. With compartmentalization of HIV in
breast milk and selective pressure from ARV therapy, local HIV replication in
breast milk may select for a reservoir of resistant virus. Because HIV viral
replication may not be suppressed adequately secondary to low ARV
concentration in breast milk, breast-feeding mothers have the potential to
transmit resistant virus. In fact, in the Kisumu Study, resistant virus was detected
in 67% of the postnatally infected infant. Studies have shown evidence of this

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genotypic resistance in other compartments, such as the female genital tract. In
addition, other studies have looked at the relationship of resistant virus and ARV
concentration in the female genital tract. Si-Mohamed et al [45] investigated the
presence of HIV-1 RNA, DNA and HIV resistance mutations in 58 women, and
demonstrated that free HIV RNA variants harboring genotypic resistance to
NRTIs, NNRTIs, and protease inhibitors were found in the genital tract of 25% of
antiretroviral experienced women. They concluded that the resistant variants
likely originated as a consequence of selected pressure by penetration of
suboptimal ARV concentrations. Thus to better understand the risk of
transmission of resistant HIV virus through breast milk, it is necessary to
determine whether there is compartmentalization of resistant virus in breast milk
by comparing HIV-1 genotype in both breast milk and serum.
Since there is limited data relevant to the pharmacokinetics of ARV drugs in
breast milk, this study will examine ARV concentrations in our HIV positive
pregnant clinic population. The goal of this study is to determine whether
adequate antiretroviral agents found in the postpartum patients’ breast milk may
be able to reduce HIV-1 oral transmission. We also hypothesize that the milk-to-
plasma ratio of ARV may differ depending on the class of ARV. Reduction of oral
transmission risk is measured by the inability to detect viral replication in the
breast milk together with plasma. Additionally, this will be correlated to whether
the disposition of the ARVs and in particular ETR can sterilized breast milk and
thus reduce MTCT. We hypothesize that ARVs with higher concentrations in
breast milk will have lower amounts of cell free (HIV RNA) and cell associated

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(HIV DNA) virus. ARVs’ concentrations were measured by a validated LC/MS/MS
assay. Non-compartmental analysis was used to estimate PK parameters.

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2. MATERIALS
2.1. Chemicals and reagents
Methanol (EMD, MX0486-1), ammonium acetate (EMD, 2145), acetonitrile (EMD,
AX0145), deionized water, etravirine, efaverinz, tenofovir, lamivudine,
emtricitabine, zidovudine, ritonavir, darunavir, amprenavir, atazinavir, lopinavir
(AIDS Repository).
2.2. Participant Population
Maternal Child Adolescence (MCA) Clinic HIV+ pregnant patients ages 18 years
and above who are receiving HAART for the prevention of MTCT. The CDC
recommends that infected women in the United States refrain from breastfeeding
to avoid postnatal transmission of HIV-1 to their infants through breast milk.
Since HIV positive women should not breast feed, we will only approach women
whose physician and healthcare team feel adequately understand that
participation in this protocol is not an endorsement of breastfeeding and they will
not breast-feed their infant.
Patients on stable HAART regimen with undetectable viral load (VL <48) at time
of delivery who will add additional ARV on postpartum D #1. Some of these
women will be on Trizivir alone for MTCT. However, the protocol will not exclude
women on other HAART regimens as long as there is no contraindication to the
additional ARV.

Page 26 of 70

Participants will start ETR 200mg bid (with food) while continuing their current
HAART regimen. We enrolled 9 women in this protocol.
2.2.1. Inclusion Criteria
1. HIV-1 + pregnant women on HAART for the prevention of MTCT w/
undetectable viral load at time of delivery (30 days prior to delivery and +/- 3
days of delivery).
2. 18 years and older
3. Only women who are deemed by the physician as being capable of
understanding that HIV positive women should not breastfeed will be
approached.
4. Life expectancy greater than 6 months
5. No known allergies to etravirine
6. Willingness of subject to adhere to protocol requirements.
2.2.2. Exclusion criteria
1) Pregnant women with medical or psychological contraindications to breast
milk expression.
2) Requirements for prohibited medications (Intelence Package Insert):
a) ARV: Tipranavir/ritonavir, fosamprenavir/ritonavir,
atazanavir/ritonavir, and protease inhibitors administered without
ritonavir, NNRTIs.
b) Alternative/CAM: St. John’s wort
c) Anticonvulsants: Phenobarbital, carbazamepine, phenytoin

Page 27 of 70

d) Anti-infectives: Rifampin
3) Laboratory values within 30 days prior to study entry
a) Direct Bilirubin >2.5 x ULN
b) ALT or AST >3x ULN
4) Acute infections or other opportunistic diseases requiring medications within
14 days prior to study entry.
5) Detectable HIV-1 viral load (>48) at time of delivery (+/- 3 days)
2.2.3. Duration of Study:
Participants were followed until postpartum day 14.
2.3. Data Collection
The study was thoroughly explained and the consent document read in its
entirety to the study participant. If she chose to participate in the research study,
she would be given a copy of the signed informed consent document. Personal
health information was collected at that time: age, racial/ethnic composition, how
many pregnancies, current medications, most recent CD4 and viral load. Entry
into the study occurred on postpartum day 1. Once the patient had delivered and
prior to leaving the hospital, she was provided with a breast pump to use at
home. Patients were advised to pump breast milk four times/day to establish and
maintain milk production. Any breast milk pumped at home was discarded.
Patients were advised not to give breast milk to their babies. The patients started
ETR on post partum day 1. ETR was given with food. Baseline labs extracted

Page 28 of 70

from routine labs obtained on admission to hospital for delivery (comprehensive
panel, CBC, HIV-1 RNA PCR) and was within 24-72 hours of first dose of ETR.
Patients may enter the study prior to receiving results of the delivery viral load.
However, if the HIV-1 RNA PCR was found to be >48 copies/mL, then the patient
was no longer eligible for the study and study drug will be discontinued.
On postpartum days #5 and 14, the participant had intensive PK analysis done
over 24 hours at MCA Clinic. Before the PK visit, the participant was asked to
hold their usual morning doses of medications on the day of the appointment until
they arrive in clinic. A pre-dose blood and breast milk sample were collected,
after which they were observed taking their mediations. A standard breakfast was
provided prior to taking their medications. The exact time of the dose was
recorded; the subsequent samples were taken at 2, 4, 8 and 12 hours after the
first dose. Each breast milk collection was 1 ounce and should take less than 15
minutes to collect. Each PK blood sample was no more than 5 mL in a yellow-top
tube. Safety labs, including CBC and comprehensive panel were also obtained
on post-partum day 14. Infants may accompany their mothers during the PK
study visits.
Plasma HIV viral load (5 mL) and breast milk HIV viral load were obtained one
time at each PK study visit. If the participant planed to stop ARV after delivery of
the infant, she may still participate, but was asked to stop her ARV after the post-
partum Day 14 PK visit.

Page 29 of 70

2.4. Patient samples
A total of 9 postpartum patients with an undetectable viral load took part in this
project. Patient was allowed to receive different components of HAART, when
combined with ETR treatment. The type of HAART components is summarized in
Table 3. Serial plasma and breast milk samples were collected on Day 5 and Day
14 at the designated time points: 0, 2, 4, 8, 12 (for ETR and HAART) and 24 (for
HAART only) hours. All the plasma samples and breast milk samples were
stored at -80
o
C until analysis.

3. METHODS
3.1. Liquid chromatography conditions
3.1.1. Etravirine analysis
The level of ETR was measured using a validated LC/MS/MS method that was
operating in negative mode. The analytes were separated using a BDS Hypersil
C18 column (Thermo Scientific, 50 x 2.1 mm, 5μ, Serial # 10068261) to separate
analytes in plasma and breast milk samples by using a gradient program. Mobile
phase A contained 10 mM ammonium acetate dissolved in deionized water while
mobile phase B was 100% acetonitrile. The total running time was 12 minutes
with a flow rate of 300 μL/min. From 0 to 8 minutes, the concentration of mobile
phase B gradually increased from 30% to 90%; and then at 8.01 minutes, the
concentration of mobile phase B immediately dropped to 30% and maintained
the concentration until 12 minutes.

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For each sample, 10 μL was injected into a Shimadzu LC20AD linked onto a
SCIEX AB API3000 Mass Spectrometer for detection. The level of analytes was
quantified by their respective signature, multiple reaction monitoring (MRM) was
used for analytes identification. The signature MRM transition was m/z 434.8→
142.4 and 313.8 → 244.2 for ETR and EFV, respectively.
The Mass spectrometer ion spray parameters were optimized as following: the
temperature was 350
o
C with an ion spray voltage of 4.5 kV; nebulizer gas was 14
U; curtain gas was 14 U; and collision gas was 10 U. Besides, Mass
spectrometer potential conditions are summarized in Table 1.

3.1.2. Components of HAART Analysis
In this study, a simultaneous method to detect the concentration of total seven
PIs, NRTIs and NNRTIs, including TDF, 3TC, FTC, LPV, RTV, DRV, and AZT.
This assay used APV as internal standard. For this simultaneous analysis of
HAART components, a Shimadzu LC20AD linked onto Sciex AB API3000 was
use and operated in positive mode. The analytes were separated using a C18
ACE column (50 x 3.0 mm, 3μ, Serial # A119211). The Mobile phase consisted
of two components where component mobile phase A was deionized water
containing 0.1% formic acid; and mobile phase B was HPLC grade acetonitrile
containing 0.1% formic acid. A gradient program was used to resolve the
analytes that had a total running time of 20 minutes. To be specific, the gradient
program was set in stages from 0 to 8 minutes, the concentration of mobile

Page 31 of 70

phase B increased gradually from 20% to 90% and maintained for 6 minutes.
From 14 to 19 minutes, the concentration of mobile phase B gradually decreased
from 90% to 20% and maintained until 20 minutes. The flow rate was 350 μL/min.
The ion spray voltage of the mass spectrometer was 5.5 kV along with a
temperature of 350
o
C. Collision gas was 3 U; nebulizer gas was 8U; and curtain
gas was 14 U. Multiple reaction monitoring was used for analyte quantification.
Beside, MRM transition information of the analytes and MS potential setting
information is summarized in the Table 2 and Table 3.
3.2. Etravirine analysis
3.2.1. Sample Preparation
3.2.1.1. Preparation of Calibration Standards
The ETR calibration standards for both plasma samples and breast milk samples
range from 25 ng/mL to 5000 ng/mL, where EFV worked as internal standard.
Standard ETR working solutions were prepared by diluting the ETR stock
standard solution (approximately 1000 μg/mL) to make 25, 50, 100, 250, 500,
1000, 2000, and 5000 ng/mL solutions. To prepare working EFV (500 ng/mL), 1
mL of 1000 μg/mL EFV was diluted with methanol. All solutions were made fresh
every day that samples will be run. For the plasma and breast milk standard
curve samples, 25 μL EFV and 25 μL each of the appropriate concentration of
prepared ETR standard were added to 25 μL of blank human plasma or breast
milk and vortex thoroughly. Then the standard samples were extracted with 100
μL methanol. After centrifuging at 13,000 rpm for 10 min, 100 μL supernatant of

Page 32 of 70

each sample was transferred to a new polypropylene tube. The samples were
diluted by using 100 μL 10mM ammonium acetate. For each sample, transfer 70
μL to HPLC vial and proceed to LC/MS/MS system where the autosampler was
in a temperature of 15 centigrade.
3.2.1.2. Plasma and Breast Milk Sample Preparation
For the plasma and breast milk patient samples, 25 μL EFV (internal standard,
500 ng/mL) was added to 25 μL patient sample plasma or breast milk and mix
thoroughly. Then the samples were extracted with 125 μL of methanol. After
centrifuging at 13,000 rpm for 10 min, 100 μL supernatant of each sample was
transferred into a new polypropylene centrifuge tube. Dilute the samples by using
100 μL 10 mM ammonium acetate. Vortex and transfer 70 μL per sample to
HPLC microvials and proceed to LC/MS/MS analysis.
3.2.2. Components of HAART Analysis
3.2.2.1. Preparation of Calibration Standards
The calibration standards were designed at the concentration of 10, 50, 100, 250,
500, 1000, 2500, and 5000 ng/mL, where 50 μL internal standard (10 μg/mL
amprenavir) and 50 μL each appropriate concentration of stocking mixed
analytes solution was added to 50 μL blank human clarified plasma or breast
milk. After vortexing thoroughly, 300 μL methanol was used for plasma sample
extraction and 300 μL methanol: deionized water (v:v 1:1) was used for breast
milk sample extraction. Samples were mixed thoroughly and centrifuged at

Page 33 of 70

13,000 rpm for 5 minutes. Then 100 μL supernatant of each sample was
transferred to a HPLC vial, and 30 μL was injected to LC/MS/MS system. Quality
control samples were at 100, 500 and 2500 ng/mL of concentrations for plasma
and at 100, 1000 and 5000 ng/mL for breast milk.
3.2.3. Plasma and Breast Milk Sample Preparation
For each patient sample, 50 μL internal standard (10 μg/mL APV) was added to
50 μL sample plasma or breast milk. After vortexing thoroughly, 350 μL methanol
was used for plasma sample extraction and 350 μL methanol: deionized water
(v:v, 1:1) was used for breast milk sample extraction. Samples were mixed
thoroughly and centrifuged at 13,000 rpm for 5 minutes. Then 100 μL
supernatant of each sample was transferred to a HPLC vial, and 30 μL was
injected to LC/MS/MS where the autosampler was in a temperature of 15
centigrade.
3.3. Data Analysis
All the data were quantified and processed using Analyst 1.5.2 software (Sciex
API, Foster City, CA). The means and standard deviations were calculated using
either Excel (Microsoft, Seattle, WA) or GraphPad Prism 5 (GraphPad Software,
Inc., La Jolla, CA). PK/PD modelling was processed by Pmetrics R package
(USC, LA, CA).

Page 34 of 70

4. RESULTS
4.1. Demographic of patients
A total of 9 HIV+ postpartum women treated with HAART therapy were enrolled
in this study. Clinical and demographic characteristics are summarized in Table 4.
The patient population consisted of mainly Latina, where the rest (2) were
Caucasians. The median age was 28 year of age. The 8/9 (89%) of patients
delivered after 37 weeks of gestation, while only one (11.1%) patient delivered
pre-term. The median CD4 was 332 at study entry, where absolute CD4
increased to median of 437 at the time of delivery.
4.2. Standard Calibrations and Curves
4.2.1. ETV Validation Studies
The standard curves for both plasma and breast milk were evaluated for both
inter and intraday variations. The accuracy information is summarized in the
Table 5 and Table 6 below for plasma and breast milk, respectively. The
accuracy ranged from 94% to104% for plasma curve, while the accuracy range
was from 94% to 105% for breast milk curve. The linearity information was
summarized in Table 7 and Table 8 for plasma and breast milk, respectively. The
robustness of data for the standard curve runs are illustrated in Figure 1 and
Figure 2 for plasma and breast milk, respectively. The linearity was measured
using regression analysis where the r
2
was ≥ 0.999 for both biomatrices.
ETV was found to be linearity across the concentration tested on three different
days. The linearity (Figure 1 and 2) and their accuracy (Table 7 and 8) were

Page 35 of 70

within the prescribed standards of the FDA for good laboratory practice (GLP).
Moreover the accuracy and robustness was confirmed in our assay development.
4.2.2. HAART Validation Studies
Since ETV was used in combination with other components of HAART, we
developed a multiplex assay for components of HAART using both plasma and
breast milk. The ability to evaluate all components of the HAART may help us
further understand the impact of each component of HAART with regard to the
clinical outcomes like viral load reduction. Analyte concentrations ranged from 10
ng/mL to 5000 ng/mL, where in Table 9 to Table 12, the accuracy and linearity of
standard and quality control concentrations of each analytes were summarized.
The R
2
of each analyte was ≥ 0.99.
4.3. Pharmacokinetics Analysis
A total of nine HIV positive postpartum women participated in this project. These
patients received the following ARVs for HIV, which is summarized in Table 13.
For each patient, at least one nucleoside drug, one protease inhibitor drug and
ETR intensification was part of the HAART.
All of patients had VLs that were undetectable (<48 copies/mL) at time of delivery.
On Day 14, subject 002E and subject 014E had undetectable plasma VL, but
detectable HIV-1 in the breast milk. These two patients had received different
types of ARV, where 002E received a combination of AZT+3TC+LPV/RTV+ETR,
while 014E was treated with TDF+FTC+Raltragravir+ETR.

Page 36 of 70

The non-compartmental pharmacokinetics analysis was used in this study for
ETR and components of HAART. The results showed that AUC of ETR in the
breast milk was higher level in both virus load detectable patients when
compared to patients with undetectable HIV in the breast milk, although the ratios
of breast milk to plasma were similar across all the patients. Also, among all the
viral load detectable and undetectable patients, ETR level was obviously
accumulated in breast milk after 14-day treatment.
Table 14 summarized relevant PK parameters of ETR level found in plasma and
breast milk. As expected, the median concentration of ETR was significantly
higher than the IC50 range (0.39-2.4 ng/mL) in both plasma and breast milk. In
Day 5, ETR concentrations are similar in breast milk and plasma. The mean BM
ETR AUC is > 4-fold higher on Day 14 than that of plasma ETR AUC. The ETR
BM AUC is statistically significant higher in Day 14 when compared to Day 5
(p=0.046). Both of the two subjects who had detectable HIV-1 RNA in breast milk
on Day 14 had lower concentrations of ETR in BM and plasma compared to
patients who are undetectable.
According to Table 15, the median concentrations by time point suggest that the
Day 14 BM/plasma ratio is 2.4 to 6.4 fold higher than that of Day 5. This result
may suggest the dynamic changes in the disposition of ETR in postpartum
women.
Moreover, Table 16 summarized the pharmacokinetics analysis of concomitant
ARVs taken by these patients. One of the two viral load detectable patients (02E)
who received AZT and 3TC as part of HAART treatment showed a lower

Page 37 of 70

BM:plasma AUC ratio than other viral load undetectable patients in day 14.
Meanwhile, the ARV concentrations found in the second patient who had
detectable viral load were lower in BM but was about 2-fold higher in plasma
compared with those viral load undetectable patients.
The results also showed that viral load detectable patient (014E) who used FTC
as part of HAART treatment had a 20-fold higher BM:Plasma AUC ratio than
other viral load undetectable patients. At the same time, the FTC level was quite
low in Day 5 plasma, which was about 20-fold lower for FTC than other patients
who received same ARV.
Additionally, for TDF treatment, there is no measurable drug concentration in BM
among all the patients. This result was consistent with the finding from other
study [47]. For the viral load detectable patient, the result showed a delayed
absorption in plasma concentration as well as a nearly 3 fold lower Cmax and
AUC than other patients.
As for the protease inhibitors used in this study, their PK parameters were similar
between viral load detectable and undetectable patient subjects. In this study, the
PI concentrations were about 100-fold higher in plasma than in BM, which is
consistent with what is found in the previous studies.
In addition, comparing HAART PK parameters on day 5 with day 14 of VL
undetectable patients, the average BM AUC ratio (Day 14: Day5) is 1.4 and 3.7
for PIs, 0.8 for 3TC and FTC, 2.5 for AZT and 2.7 for ETR. These data indicated
that the disposition of ETR and PIs in BM from Day 5 to Day 14.

Page 38 of 70

Above results suggest that HAART ARVs may influence the ETR’s effect and
disposition in these postpartum women.
PK/PD modelling was conducted by using Pmetrics R package with NPAG
engine [48]. The model had a single compartment with linear oral absorption and
elimination for plasma ETR, and linear transfer to and from the breast milk
compartment. From the full individual Bayesian posterior time-concentration
profiles, we calculated non-compartmental PK parameters. Figure 3 below
showed the relation of ETR observation over time. This result indicated that ETR
breast milk concentrations were parallel to the plasma concentrations. Also,
Bayesian Posterior Statistics was used to predict the population and individual
ETR observation (Figure 4 and Figure 5). Basically, ETR exhibited linear
absorption into a central compartment. Although the correlation efficient of
population prediction were low in both breast milk and plasma due to different
treatment strategies, the individual predictions statistically significant correlated
to our observed results in both plasma and breast milk (r
2
=0.887 and r
2
=0.976).

Page 39 of 70

5. DISCUSSION
MTCT continues to be a major issue in resource-limited environment where there
is no other source of formula for babies born from HIV+ patients. Currently, there
are several effective measures to prevent MTCT in developed countries. These
include using milk formulation in place of HIV contaminated breast milk. However,
in developing countries, especially those resource-limited areas, MTCT is still a
severe problem that requires intense understanding of the problem confronting
us.
One of the possible routes of MTCT may be through breastfeeding of HIV
contaminated breast milk. An intensive combination of several ARVs for those
postpartum women is a feasible and affordable method to diminish the risk of
MTCT through breast-feeding. In this work, we used an intensive ETR together
with traditional HAART as a treatment. The results indicated that the disposition
and the concentration of ETR were related to the detection of HIV viral load.
In this study, the detected concentration of ETR exhibited its disposition in a 14-
day treatment period, during which ETR had an accumulation in breast milk
rather than plasma among the postpartum subjects. The ability of ETR to diffuse
into breast milk (BM) from plasma may attributable to its lipophilicity and its small
molecular weight. These results are similar results were found in cervicovaginal
fluid disposition [46]. Also, the ETR PK parameters in this study are similar to
published ETR PK parameters found in plasma. ETR concentration exceeded
plasma HIV-1 IC50 at all time points. On the other hand, the high ETR BM

Page 40 of 70

concentration can suppress the viral load progress, which might also relate to the
ARVs selection of the HAART that patient subjects received and the individual
pharmacodynamics. The composition of breast milk changed with the time in
postpartum period, colostrums and transitional milk (Day 5) versus mature milk
(Day 14), which probably resulted in the accumulation of ETR in breast milk after
14-day intensive ETR treatment.
Although the ETR BM AUC remained high in both of the viral load detectable
patients, their median BM concentrations in Day 14 were lower than the other
viral load undetectable patients. Thus, intensive ETR dosage may play an
essential role in the control of viral load. Yet, consider there are other ARVs
included in HAART for those two patients, the efficacy of ETR still need to be
further verified. It is conceivable that the total ETR may not reflect the free ETR
levels which are the free fraction that may exert antiviral activity.
HAART is a current widely used combination therapy to suppress the HIV load
among patients. The combination of ARVs from distinct classification such as
NRTIs, NNRTIs and PIs has been proved to be a potent treatment owing to its
cost and the effectiveness to adapt to HIV mutation. According to our HAART
medication results, the viral load detectable patient 002E had an about 2-fold
higher plasma concentration of AZT and 3TC in Day 14, but a lower breast milk
concentration of AZT and 3TC compared with other patients who received the
same ARV as part of the HAART. This result indicated that the AZT and 3TC
may influence the viral load control besides ETR. On the other hand, subject
014E, the other viral load detectable patient, who received FTC and TDF as part

Page 41 of 70

of the HAART, had obvious lower concentrations in plasma compared with other
patients with same HAART. This evidence may explain the detectable viral load
in Day 14 of subject 014E. Therefore, the nucleoside agents’ concentrations may
also contribute to the control of BM viral load together with ETR. As a result, the
level of HAART ARVs especially the nucleosides in vivo may affect the HIV
activity as well as ETR.
As for the protease inhibitors included in our study (LPV, RTV and DRV), their
concentrations remained extremely high in plasma in contrast to that in BM,
which was consistent with other published data [49, 50]. What’s more, the drug
level of each PI was about the same among all the subjects. Therefore, protease
inhibitors may not exert important influence on the difference of patient viral load
in Day 14.
The non-compartment PK/PD modelling suggested there is a significant
correlation between plasma ETR and breast milk ETR. Particularly, the individual
posterior statistical prediction statistically significantly correlated to the observed
ETR level either in plasma or in breast milk. Yet, owing to distinct HAART ARVs
selection, the population prediction hardly correlated to our observed ETR results
in neither plasma nor breast milk.
In summary, the combination treatment of intensive ETR along with different
HAART may impact the control of viral load amount among postpartum women.
Further research may focus on the optimal ARV selection for HAART plus
intensive ETR treatment on the prevention of MTCT through breast-feeding.

Page 42 of 70

6. CONCLUSIONS
ETR penetration into BM is largely driven by plasma PK and exceeds the plasma
HIV inhibitory concentration at all time points. This study suggests that unbound
ETR readily diffuses from blood to the breast compartment likely because of its
lipophilicity. However, differences in concomitant ARVs and individual
pharmacodynamics may allow for compartmental viral replication. HAART
combinations that include ETR may prove to be useful for prevention of BM
MTCT in resource-limited countries where exclusive breastfeeding is
recommended.

Page 43 of 70

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17(8):1511-1519.

Page 48 of 70

TABLES
Table 1: MS Condition for Etravirine and Efavirenz.
MS Settings Etravirine Efavirenz
DT (msec) 200 200
DP -100 -60
FP -340 -200
EP -10 -9
CC -40 -25
CXP -9 -9
DT=Dwell time
DP=Declustering Potential
FP=Focusing Potential
EP=Entrance Potential
CC=Collision Cell
CXP=Collision Cell Exit Potential

Page 49 of 70

Table 2: Multiple reaction monitoring information: Q1: Parent ion; Q3:
Product ion.
Drug Name MRM (Q1→Q3)
TDF 288.2→ 176.2
3TC 230.2 → 112.0
FTC 248.2→ 130.1
AZT 268.0 → 127.0
RTV 721.5→ 296.2
DRV 565.7→ 284.5
APV 506.5→ 245.2
LPV 629.5→ 447.4

Page 50 of 70

Table 3 : Mass Spectrometer potential parameters:
MS Settings TDF 3TC FTC AZT RTV DRV APV LPV
DT 250 250 250 250 250 250 250 250
DP 100 86 76 51 68 62 100 80
FP 320 290 300 280 300 250 350 350
EP 10 4 5 4 12 8 8 11
CC 37 19 16 17 32 25 26 24
CXP 30 7 7 9 17 20 15 29
DT=Dwell time
DP=Declustering Potential
FP=Focusing Potential
EP=Entrance Potential
CC=Collision Cell
CXP=Collision Cell Exit Potential

Page 51 of 70

Table 4 : Characteristics and Demographics of Patient subjects
Parameter N=9 Parameter N=9
Median Age (Years) 28 Timing of Delivery
Ethnicity-n Term(≧ 37 weeks) 8
White 2 Pre-term 1
Latina 7 History of Previous Breast Feeding
Previous
Pregnancies
(Median)
Yes 2
G 3 No 7
P 2 CD4 Nadir(Median)
A 1 %CD4 18
Type of Delivery Absolute CD4 (NO.) 332
Vaginal 7 CD4 at Delivery(Median)
C-section 2 %CD4 29
Absolute CD4 (NO.) 437

Detectable HIV RNA Viral Load in
Breast Milk
2

Page 52 of 70

Table 5: Etravirine Plasma Curve Accuracy

Table 6: Etravirine Breast Milk Curve Accuracy:

Etravirine(ng/mL) Accuracy (%) Average
Day 3 Day 2 Day 1
25 100% 101% 100% 100%
50 99% 99% 99% 99%
100 102% 100% 103% 102%
250 104% 97% 104% 102%
500 94% 99% 97% 97%
1000 102% 100% 100% 101%
2000 96% 99% 96% 97%
5000 103% 104% 102% 103%
Etravirine(ng/ml)
Accuracy (%)
Average
Day 3 Day 2 Day 1
25 100% 101% 100% 100%
50 101% 99% 101% 100%
100 101% 96% 98% 98%
250 99% 103% 100% 101%
500 97% 99% 94% 97%
1000 105% 98% 100% 101%
2000 96% 99% 104% 100%
5000 102% 104% 101% 102%

Page 53 of 70

Table 7 : Linearity of Etravirine plasma standard curves

Table 8: Linearity of Etravirine breast milk standard curves
Parameters Day 1 Day 2 Day 3
Slope 0.00241 0.00240 0.00241
Y intercept 0.0183 0.0375 0.0065
R
2
0.999 0.999 0.999

Parameters Day 1 Day 2 Day 3
Slope 0.00171 0.00168 0.00175
Y intercept 0.0561 0.0302 0.0989
R
2
0.999 0.999 0.999

Page 54 of 70

Table 9 : HAART ARVs’ plasma standard accuracy of quality controls at the
concentration of 100, 500 and 2500 ng/mL.
DRUG
Actual
Conc
(ng/mL)
Day1
Calculated
Conc
(ng/mL)
%
Accuracy
Day 2
Calculated
Conc
(ng/mL)
%
Accuracy
Day 3
Calculated
Conc
(ng/mL)
%
Accuracy
TDF
100 109 109 96.4 96.4 103 103
500 487 97.5 513 103 386 77.1
2500 2480 99.2 2730 109 2720 109
3TC
100 106 106 95.3 95.3 105 105
500 544 109 520 104 515 103
2500 2580 103 2540 102 2600 104
FTC
100 107 107 102 102 109 109
500 544 109 534 107 535 107
2500 2470 98.9 2650 106 2610 104
AZT
100 98.3 98.3 102 102 97.1 97.1
500 525 105 508 102 528 106
2500 2380 95.2 2610 104 2630 105
RTV
100 102 102 105 105 101 101
500 521 104 542 108 489 97.8
2500 2670 107 2580 103 2680 107
DRV
100 --- --- 110 110 91.4 91.4
500 524 105 547 109 530 106
2500 2580 103 2710 108 2610 105
LPV
100 107 107 98.4 98.4 95.6 95.6
500 501 100 530 106 544 109
2500 2710 108 2240 90 2520 101

Page 55 of 70

Table 10 : HAART ARVs’ breast milk standard accuracy of quality controls at the
concentration of 100, 1000 and 5000 ng/mL.
Drug
Actual
Conc
(ng/mL)
Day1
Calculated
Conc
(ng/mL)
%
Accuracy
Day 2
Calculated
Conc
(ng/mL)
%
Accuracy
Day 3
Calculated
Conc
(ng/mL)
%
Accuracy
TDF
100 105 105 103 103 105 105
1000 1090 109 946 94.6 934 93.4
5000 5140 103 5130 103 5390 108
3TC
100 108 108 105 105 101 101
1000 1050 105 863 86.3 1030 103
5000 5020 100 5280 106 5150 103
FTC
100 106 106 102 102 107 107
1000 1080 108 933 93.3 1030 103
5000 5430 109 5220 104 5160 103
AZT
100 104 104 107 107 104 104
1000 1000 100 931 93.1 983 98.3
5000 5240 105 5240 105 5160 103
RTV
100 108 108 96.6 96.6 98.5 98.5
1000 1040 104 1070 107 1050 105
5000 5170 103 4870 97.4 5190 104
DRV
100 101 101 104 104 97 97
1000 1020 102 1030 103 1070 107
5000 4910 98.2 5260 105 5360 107
LPV
100 104 104 99.2 99.2 102 102
1000 998 99.8 1060 106 942 94.2
5000 4800 96.1 5060 101 5260 105

Page 56 of 70

Table 11 : Linearity of interday HAART ARVs’ plasma standard curves.
Drug Name Parameters Day 1 Day 2 Day 3
TDF
Slope 1.17E-5 1.66E-5 1.22E-5
Y intercept 8.37E-5 -0.000572 0.000114
R
2
0.998 0.999 0.997
3TC
Slope 0.000159 0.000181 0.000174
Y intercept -0.000415 -0.00422 -0.000782
R
2
0.996 0.997 0.997
FTC
Slope 0.000278 0.000361 0.000283
Y intercept -0.00121 -0.000984 -0.000882
R
2
0.998 0.999 0.998
AZT
Slope 9.56E-5 0.000118 9.49E-5
Y intercept 0.00356 0.00414 0.000942
R
2
0.998 0.997 0.999
RTV
Slope 0.0028 0.00311 0.00268
Y intercept -0.00414 0.00388 0.00278
R
2
0.998 0.999 0.997
DRV
Slope 0.000496 0.000317 0.000373
Y intercept 0.796 0.49 0.0436
R
2
0.998 0.997 0.998
LPV
Slope 0.00119 0.00209 0.00172
Y intercept -0.00291 0.00242 -0.00195
R
2
0.997 0.998 0.997

Page 57 of 70

Table 12 : Linearity of interday HAART ARVs’ Bresat Milk standard curves.
Drug Name Parameters Day 1 Day 2 Day 3
TDF
Slope 3.39E-5 3.00E-5 2.20E-5
Y intercept -0.000911 -0.000809 -0.000834
R
2
0.997 0.999 0.998
3TC
Slope 9.27E-5 8.01E-5 8.33E-5
Y intercept -0.0025 -0.0085 -0.0093
R
2
0.998 0.997 0.998
FTC
Slope 0.000241 0.000251 0.000235
Y intercept -0.00428 -0.00399 -0.00679
R
2
0.998 0.999 0.999
AZT
Slope 9.53E-5 7.23E-5 9.71E-5
Y intercept -0.00155 0.000479 -0.00271
R
2
0.998 0.997 0.999
RTV
Slope 0.00268 0.00274 0.00265
Y intercept -0.00364 0.0643 0.057
R
2
0.999 0.997 0.998
DRV
Slope 7.04E-5 8.29E-5 9.67E-5
Y intercept 0.34 0.13 0.171
R
2
0.998 0.999 0.998
LPV
Slope 0.00156 0.0018 0.00125
Y intercept -0.00143 0.00771 0.0302
R
2
0.997 0.997 0.998

Page 58 of 70

Table 13: Regimens summary
Regimen Total number of
patients
Nucleoside
Combivir (AZT/3TC) 4
Truvada (TDF/FTC) 5
HIV Protease Inhibitors
Kaletra(Loprinavir/ritonavir) 6
Darunavir/ritonavir 2
Raltragravir 1

Page 59 of 70

Table 14: Etravirine concentration median in patients with detectable HIV-1
RNA versus viral load undetectable patients
Detectable HIV-1 RNA in BM
Yes (n=2) No (n=6)
Day 5(n=8)
Cmedian Breast Milk (ng/mL) 222.58 248.66
Cmedian Plasma (ng/mL) 299.9 247.52
BM/Plasma 0.82 1.14
AUC
0-12
Breast Milk (ng*hr/mL) 5737.9 7431.1
AUC
0-12
Plasma (ng*hr/mL) 6194.5 5816.8
AUC
0-12
BM/Plasma 0.94 1.31
Day 14(n=8)
Cmedian Breast Milk (ng/mL) 272.47 1010
Cmedian Plasma (ng/mL) 141.75 298.8
BM/Plasma 1.99 4.21
AUC
0-12
Breast Milk (ng*hr/mL) 7593.65 27118.5
AUC
0-12
Plasma (ng*hr/mL) 3508.15 6100.5
AUC
0-12
BM/Plasma 0.1 0.18

Page 60 of 70

Table 15: Etravirine Median Concentrations in BM and Plasma by time
points.

Time
point
Day 5
Median, ng/mL (n=8)
Day 14
Median, ng/mL (n=8)
(hr) BM Plasma BM/Plasma
Ratio
BM Plasma BM/Plasma
Ratio
0 65.5 117 0.56 344.5 94.3 3.61
2 120 270 0.44 631.5 385 1.64
4 525.5 501 1.04 1570 368.5 4.26
8 339.5 213 1.59 1148 220 5.21
12 151 156.5 0.96 232 98.9 2.34

Page 61 of 70

Table 16: HAART PK parameters are classified by drugs.
a.
AZT BLD
DAY 5 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 727.00 1220.00 1350.00 1099.00 1220.00
Tmax(hr) 2.00 2.00 2.00 2.00 2.00
t1/2(hr) 4.85 5.99 3.56 4.80 4.85
AUC
0-24
(ng*hr/mL) 4551.50 4332.20 3798.60 4227.43 4332.20
DAY 14
Cmax (ng/mL) 1090.00 2130.00 987.00 1390.00 1399.25 1240.00
Tmax(hr) 2.00 2.00 4.00 2.00 2.50 2.00
t1/2(hr) 4.24 3.76 3.89 4.76 4.16 4.07
AUC
0-24
(ng*hr/mL) 5250.20 11414.70 4007.94 4286.15 6239.75 4768.18

b.
AZT BMK
DAY5 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 89.2 28.3 150.0 89.2 89.2
Tmax(hr) 2.0 4.0 2.0 2.7 2.0
t1/2(hr) 17.2 N/A 2.2 9.7 9.7
AUC
0-24
(ng*hr/mL) 425.3 148.4 524.9 366.2 425.3
DAY14 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 161.0 147.0 292.0 278.0 219.5 219.5
Tmax(hr) 4.0 2.0 4.0 2.0 3.0 3.0
t1/2(hr) N/A 2.9 1.2 3.4 2.5 2.9
AUC
0-24
(ng*hr/mL) 836.9 567.3 1170.0 1148.5 930.7 992.7

c.
3TC BLD
DAY5 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 1280.00 2120.00 819.00 1406.33 1280.00
Tmax(hr) 4.00 2.00 4.00 3.33 4.00
t1/2(hr) 5.89 6.74 18.55 10.39 6.74
AUC
0-24
(ng*hr/mL) 10977.00 13246.00 9806.00 11343.00 10977.00
DAY14 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 1360.00 2000.00 505.00 1020.00 1221.25 1190.00
Tmax(hr) 4.00 4.00 4.00 2.00 3.50 4.00
t1/2(hr) 8.04 4.23 9.81 13.03 8.78 8.93
AUC
0-24
(ng*hr/mL) 7713.25 12550.80 3451.80 15869.00 9896.21 10132.03

Page 62 of 70

d.
3TC BMK
DAY5 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 1080.0 513.0 445.0 679.3 513.0
Tmax(hr) 4.0 4.0 4.0 4.0 4.0
t1/2(hr) 12.1 22.7 21.0 18.6 21.0
AUC
0-24
(ng*hr/mL) 13059.8 7974.0 9462.0 10165.3 9462.0
DAY14 01E 02E 04E 09E AVERAGE MEDIAN
Cmax (ng/mL) 1030.0 245.0 246.0 914.0 608.8 580.0
Tmax(hr) 4.0 8.0 4.0 4.0 5.0 4.0
t1/2(hr) 38.6 7.0 7.6 16.9 17.5 12.2
AUC
0-24
(ng*hr/mL) 12191.0 3249.8 3455.7 13961.0 8214.4 7823.4

e.
LPV BLD
DAY5 01E 02E 04E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 20900 57200 15500 25200 20300 27820 20900
Tmax(hr) 4.00 4.00 0.00 4.00 0.00 2.40 4.00
t1/2(hr) 12.92 15.12 64.23 12.86 13.03 13.48 13.03
AUC
0-24
(ng*hr/mL) 255340 907300 294980 351760 194040 400684 294980
DAY14 01E 02E 04E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 16500.0 24400.0 27600.0 13700.0 18600.0 25300.0 21016.7 21500.0
Tmax(hr) 4.00 4.00 4.00 8.00 4.00 4.00 4.67 4.00
t1/2(hr) 26.30 3.89 6.69 20.10 14.12 7.80 13.15 10.96
AUC
0-24
(ng*hr/mL) 307140.0 230683.7 244406.0 245500.0
25043
0.0 395840.0 279000.0 247965.0

f.
LPV BMK
DAY5 01E 02E 04E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 286.0 296.0 339.0 362.0 287.0 314.0 296.0
Tmax(hr) 4.0 4.0 8.0 4.0 2.0 4.4 4.0
t1/2(hr) 17.1 10.0 14.3 7.4 4.2 10.6 10.0
AUC
0-24
(ng*hr/mL) 3354.0 4378.8 6126.0 3712.8 2954.8 4105.3 3712.8
DAY14 01E 02E 04E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 505.0 465.0 194.0 611.0 316.0 520 435.2 485.0
Tmax(hr) 4.0 8.0 8.0 4.0 8 2 5.7 6.0
t1/2(hr) 16.7 3.6 3.8 8.8 21.1 11.37 10.9 10.1
AUC
0-24
(ng*hr/mL) 4897.0 5541.2 2475.4 8972.0 5638.6 6381.5 5651.0 5589.9

Page 63 of 70

g.
RTV BLD
DAY5 01E 02E 04E 07E 08E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 1130 4580 1170 792 633 812 729 1407 812
Tmax(hr) 4.00 4.00 24.00 2.00 4.00 4.00 4.00 3.67 4.00
t1/2(hr) 12.22 11.87 5.45 7.96 15.69 12.85 6.33 10.34 11.87
AUC
0-24
(ng*hr/mL) 15570 56940 16851 14467 7939 10180 7650 18514 14467
DAY14 01E 02E 04E 07E 08E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 1310 2030 2150 1 1060 484 1340 837 1151 1185
Tmax(hr) 4.00 4.00 4.00 0.00 0.00 8.00 4.00 4.00 3.50 4.00
t1/2(hr) 17.69 4.08 7.31 0.84 21.42 15.81 9.87 4.70 10.21 8.59
AUC
0-24
(ng*hr/mL) 18024 14254 20510 2 13792 8600 13248 11194 14232 13520

h.
RTV BMK
DAY5 01E 02E 04E 07E 08E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 20.5 47.0 0.0 24.0 19.7 26.2 4.3 20.2 20.5
Tmax(hr) 4.0 0.0 0.0 0.0 8.0 4.0 4.0 2.9 4.0
t1/2(hr) 8.8 1.9 0.0 2.4 N/A 1.3 N/A 2.9 1.9
AUC
0-24
(ng*hr/mL) 164.7 302.3 0.0 221.8 254.4 111.9 13.0 152.6 164.7
DAY14 01E 02E 04E 07E 08E 09E 10E 12E AVERAGE MEDIAN
Cmax (ng/mL) 51.6 92.2 29.7 0.0 67.8 76.7 35.0 60.2 51.7 55.9
Tmax(hr) 4.0 8.0 8.0 0.0 0 8.0 8 8 5.5 8.0
t1/2(hr) 15.6 N/A N/A 0.0 11.4 N/A 6.8 N/A 8.5 9.1
AUC
0-24
(ng*hr/mL) 456.0 941.4 297.0 0.0 558.2 1100.1 459.5 694.7 563.4 508.9

i.
FTC BLD
DAY5 07E 08E 10E 12E 14E AVERAGE MEDIAN
Cmax (ng/mL) 347.00 527.00 6200.00 35.70 21.00 1777.43 347.00
Tmax(hr) 24.00 0.00 2.00 4.00 8.00 7.50 4.00
t1/2(hr) 4.39 2.69 5.22 6.40 110.81 4.67 5.22
AUC
0-24
(ng*hr/mL) 6022.00 6357.00 32654.00 361.00 382.00 11348.50 6022.00
DAY14 07E 08E 10E 12E 14E AVERAGE MEDIAN
Cmax (ng/mL) 63.40 710.00 2670.00 21.10 866.13 386.70
Tmax(hr) 24.00 0.00 4.00 24.00 13.00 14.00
t1/2(hr) 2.72 2.61 3.79 11.56 5.17 3.25
AUC
0-24
(ng*hr/mL) 741.12 8489.00 14337.30 236.00 5950.86 4615.06

Page 64 of 70

j.

FTC BMK
DAY5 07E 08E 10E 12E 14E AVERAGE MEDIAN
Cmax (ng/mL) 211.0 475.0 1410.0 27.0 543.0 533.2 475.0
Tmax(hr) 24.0 0.0 4.0 2.0 0.0 6.0 2.0
t1/2(hr) 9.0 3.9 5.0 56.9 4.8 5.7 5.0
AUC
0-24
(ng*hr/mL) 3023.1 5785.0 12742.7 541.8 8173.0 6053.1 5785.0
DAY14 07E 08E 10E 12E 14E AVERAGE MEDIAN
Cmax (ng/mL) 17.1 503.0 612.0 227 339.8 365.0
Tmax(hr) 0.0 24.0 8 24 14.0 16.0
t1/2(hr) 51.3 4.5 6.0 N/A 5.2 6.0
AUC
0-24
(ng*hr/mL) 396.1 6737.0 8553.3 2082.8 4442.3 4409.9

k.
DRV BLD
DAY5 07E 08E AVERAGE
Cmax (ng/mL) 10500.00 19300.00 14900.00
Tmax(hr) 8.00 8.00 8.00
t1/2(hr) 27.07 18.22 22.64
AUC
0-24
(ng*hr/mL) 195120.00 358360.00 276740.00
DAY14 07E 08E AVERAGE
Cmax (ng/mL) 6820.00 26300.00 16560.00
Tmax(hr) 8.00 24.00 16.00
t1/2(hr) 37.15 37.15
AUC
0-24
(ng*hr/mL) 144330.00 593000.00 368665.00

l.
DRV BMK
DAY5 07E 08E AVERAGE
Cmax (ng/mL) 2060.0 5500.0 3780.0
Tmax(hr) 8.0 2.0 5.0
t1/2(hr) 18.2 69.7 44.0
AUC
0-24
(ng*hr/mL) 30394.0 112430.0 71412.0
DAY14 07E 08E AVERAGE
Cmax (ng/mL) 1900.0 80700 41300.0
Tmax(hr) 24.0 2 13.0
t1/2(hr) N/A 6.0 6.0
AUC
0-24
(ng*hr/mL) 21963.0 225630.0 123796.5

Page 65 of 70

m.
TDF BLD
DAY5 07E 08E 10E 12E 14E AVERAGE MEDIAN
Cmax (ng/mL) 83.30 56.10 292.00 226.00 50.30 141.54 83.30
Tmax(hr) 2.00 2.00 4.00 2.00 8.00 3.60 2.00
t1/2(hr) 6.03 24.41 10.21 7.07 14.02 12.35 10.21
AUC
0-24
(ng*hr/mL) 920.74 797.70 2722.00 2040.60 794.80 1455.17 920.74
DAY14 07E 08E 10E 12E 14E AVERAGE MEDIAN
Cmax (ng/mL) 27.40 96.60 725.00 52.10 225.28 74.35
Tmax(hr) 2.00 2.00 2.00 8.00 3.50 2.00
t1/2(hr) 54.81 6.86 7.07 407.15 22.91 30.94
AUC
0-24
(ng*hr/mL) 355.44 1062.62 5106.41 1115.00 1909.87 1088.81

P.S. BLD refers to plasma sample; BMK refers to breast milk sample. The red marked
data is excluded due to the variation.

Page 66 of 70

FIGURES
Figure 1 : Robustness of Etravirine plasma standard curves.

Figure 2 : Robustness of Etravirine breast milk standard curves.

Page 67 of 70

Figure 3 : ETR Bayesian posterior predicted time concentration profiles and
measured concentrations.

Page 68 of 70

Figure 4 : Plasma ETR population prediction and individual posterior prediction by
Bayesian Posterior Statistics. Predictions are based on the median
of each subject's Bayesian posterior parameter distribution.
a.

Page 69 of 70

Figure 5 : Breast milk ETR population prediction and individual posterior
prediction by Bayesian Posterior Statistics. Predictions are based
on the median of each subject's Bayesian posterior parameter
distribution.

Page 70 of 70

APPENDIX
Table S1. Evalution of Subjects

Evaluation Screening
Post
Pregnancy (Days)

Entry/
PPD 1
5
± 2
6
± 2
14
± 2
15
± 2
Documentation of HIV X
Medical History/Medication History X
Clinical Assessments X X X X X
Adherence Assessment X X
Hematology X X
Complete Metabolic Panel X X
CD4 and CD8 X
HIV-1 RNA X
1
X
2
X X
Etravirine PK Sample Collection
4
X X X X
Etravirine 200 mg bid start X
1. Within 30 days prior to delivery.
2. Within 24-72 hours of delivery (admission labs to hospital for delivery)

Abstract (if available)

Abstract Mother‐to‐child transmission (MTCT) through breastfeeding continues to be a major concern for the transmission of HIV infection among children in developing countries. The transmission usually occurs in the early stage during breastfeeding, where the infant’s gastrointestinal integrity may be compromised. Highly antiretroviral therapy (HAART) plays an important role in the control of viral replication in the plasma of infected HIV patients. HIV positive women patients receiving HAART during pregnancy and postpartum has become a crucial strategy to suppress the viral load in breast milk in hope to prevent MTCT. However it is not known whether the level of ARV is adequate to prevent infant acquisition of HIV. Etravirine (ETR), a NNRTI, is a second generation NNRTI that has been approved as a second line treatment for HIV infection. It is active against several common HIV 1 mutants that are resistant to first generation NNRTIs. In this study, we used a 14-day intensive ETR included HAART on postpartum women subjects. The results indicated that the disposition of ETR in breast milk from plasma after the treatment and a relatively low ETR BM concentration in both viral detectable subjects. Also, the distinct selection of ARVs for HAART may affect ETR BM concentration. In summary, intensive ETR included HAART may be a potential effective measure to prevent MTCT in those resource‐limited areas.

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Asset Metadata

Creator Liu, Siyu (author)

Core Title Pharmacodynamics impact of etravirine disposition into breast milk in relation to HIV replication in this milk

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/21/2016

Defense Date 03/20/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag breast milk,etravirine,HAART,HIV,MTCT,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Louie, Stan G. (committee chair), Camarero, Julio A. (committee member), Shen, Wei-Chiang (committee member)

Creator Email lindsay.liusiyu@gmail.com,siyuliu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-381738

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Legacy Identifier etd-LiuSiyu-2385.pdf

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Format application/pdf (imt)

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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