Pharmaceutical Science Related PhD only!
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PregnancyandDrugDeliveryFinal.pdf
Wisdom of the Day
“The ability to DISCIPLINE YOURSELF to delay gratification in
the short term in order to enjoy greater success in the long term is the indispensable prerequisite for
success.”
Maxwell Maltz
Placental and Pediatric Drug
Delivery
Human Embryonic Stem Cells.
Panel A shows a human embryo at the blastocyst
stage. Panel B shows colonies of human embryonic
stem cells after enzymatic dissociation.
http://www.eshre.com/ecm/files/madrid/COURSE3_EmbryolGenet.pdf
Placenta-A Multifunctional Organ • Lung-respiration control between the fetus and the
mother
• Liver-metabolizes maternal xenobiotics and
eliminates fetal waste
• Intestinal epithelium-regulates directional nutrient and
drug transfer from the mother to the fetus and vice-
versa
• Endocrine glands-hormonal regulation of pregnancy
• Adipose-can store fat and other nutrients to be feto-
protective (malnourished mothers)
• Capable of adapting to maternal states and
regulating proper fetal development.
Structural Features of the Placenta
• Membranous structure formed by the apposition or
fusion of fetal membrane (chorion frondosum) with
the uterine mucosa (deciduas basalis) and divided
into functional units called cotyledons.
• Structure changes dramatically during pregnancy: – Fully differentiated at the third month of pregnancy
– Thinning and aging:
• 10 µm at early pregnancy to 1-2 µm at late term
– Blood flow and surface area: increase significantly during the late
pregnancy and influenced by pathophysiological conditions
• Presence of influx and efflux transporters
facilitate the transfer of nutrients and the export of
wastes and can mediate the passage of
drugs/xenobiotics
Biological Roles of the Placenta
• Membranous structure formed by the
apposition or fusion of fetal membrane with
the uterine mucosa
• Regulate transport of nutrients and wastes between the mother and the fetus
• Immunological role in preventing fetal rejection
• Endocrine role – Fetus to mother
– Mother to fetus
Placental-Maternal Organization
M. Saunders. (2009) Transplacental transport of nanomaterials. Nanomed Nanobiotechnol. 1:671– 684
(Tortora GJ & Grabowski SR, Principles of Anatomy and Physiology, Seven Edition.
HarperCollins, NY (1993) p. 980)
Organization of the placenta and structure
of the placental membrane
Organization of the placenta (a), and the structure of
the placental membrane (b) composed of the epithelium
of the villous and of the capillary.
Human Placenta
Schematic Representation of
Human Placenta
Regulation exchange of nutrients, wastes and endogenous and foreign molecules, including drugs, between the mother and the fetus.
ST: Syncytial Trophoblast cells
CT: Cytotrophoblast cells.
Rat Model Advantages:
– Reproductive patterns well defined
– Morphogenesis and transport properties can be readily monitored throughout pregnancy.
Placental physiology:
– Invasive/endocrine (junctional zone) and transport functions (labyrinth zone) are physically separated and attributed to distinct cell populations within the rodent placenta
– Syncytial trophoblast cells of the labyrinth zone regulate the transport of nutrients and waste across the rat placenta
Figure 2. Schematic representation of the rat placenta which is functionally
separated into 2 distinct, separated zones ; the junctional
(endocrine and invasive function) and the labyrinth
(transport regulatory function) zones.
Syncytial Trophoblast Layers I & II
TGC-trophoblast giant cells
Junctional Zone
• Endocrine function
• Invasion of the uterine wall
• Trophoblast cell types:
– trophoblast giant cells (TGC)
– spongiotrophoblasts
– glycogen cells
• Cell culture models
– Rcho-1 TGC differentiation dependent model
Labyrinth Zone
• Transport barrier
– regulation of fetal to maternal and maternal to fetal
transfer of nutrients and wastes
• Trophoblast cell types:
– syncytial trophoblasts
• Human placental cells also syncytialize to form
barrier
• Cell culture models for studying placental
transport
– HRP-1 cell model
– BeWo cell line
Table 2. Spatial arrangement of trophoblast cell types in the rat and
human placentas and their ascribed functions. Last column shows the cell
culture models most frequently used to study the function of the different
placental sections.
Cell type Placental
location
Function Cell culture
models
Rat Placenta
Trophoblast giant cell
Spongiotrophoblast cell
Glycogen cell
Syncitial trophoblast cell
Junctional zone
Labyrinth zone
Junctional zone
Junctional zone
Labyrinth zone
Endocrine,
invasive
Endocrine
Energy storage
Transport barrier
Rcho-1
HRP-1
Human Placenta
Cytotrophoblast cells
Syncitial trophoblast cell
Villous and
extravillous
Vilous and
extravillous
Progenitor cell,
invasive
Transport barrier,
endocrine
BeWo, JEG,
JAR
Placental FA Transporters
Knipp GT, et al. Adv Drug Deliv Rev. 1999 Jun 14;38(1):41-58.
Peroxisome Proliferator-Activated
Receptors (PPARs)
Isoform Function Tissue distribution
PPAR Lipid catabolism,
FA transport
Well expressed in cells with high FA catabolism (liver, heart, kidney, skeletal muscle, intestine, etc.)
Detectable in pancreas, lung, placenta and adipose tissue, etc.
PPAR FA oxidation & utilization,
FA transport
Ubiquitous, with higher expression than PPAR and in most investigated tissues.
Detectable in placenta.
PPAR Adipocyte differentiation,
lipid storage,
FA transport.
Abundantly expressed in adipose tissue and at lower levels in skeletal muscle, liver, heart and bone marrow stromal cells, etc.
Detectable in placenta.
Note: Hepatic PPAR mRNA expression is reported to be lower in human than in rodents.
Summary of placenta fatty acid transport & metabolism pathways
FABPpm
Albumin
Binding
Protein
Fetal
plasma
membrane
Lipoprotein
Receptor
PLACENTAL CELL
Maternal
plasma
membrane
FATP1
FAT/CD36
FATP1
FAT/CD36
Triacylglycerol
Phospholipid
Sphingolipid
Albumin
Complex
Lipoprotein
LPL
Hydrolysis
Free fatty
acid
Disassociation
FABP
Signal transduction
Gene regulation
Other biological
activity
Metabolism
Mitochondria
Peroxisome
Endoreticulum
Others
Lipid resynthesis
Triacylglycerol
Phospholipid
Sphingolipid
Free fatty
acid
Cytoplasm
(Apical) (Basolateral)
Diffusion
Albumin
Complex
Free fatty
acid
Diffusion
Fetal Nutrient Accumulation
Haggarty P. (2004) Eur. J. Clin. Nutr. 58, 1559-1570.
Placental Accumulation
1. Selective uptake/transport of LCPUFA by the trophoblast cells.
1 3
Esterified
NEFA
Fetal
tissues
Maternal Circulation Fetal Circulation
4
?
3 3
2
1
2. Selective metabolism/utilization of individual fatty acid by the placenta.
3. Preferential incorporation of LCPUFA into esterified lipids by the placenta (also
fetal liver) and transfer to fetus
4. Possible selective lipolysis of LCPUFA in maternal circulating triglyceride(TG)
by placental lipase in microvillous membrane
Haggarty P. Placenta. 2002 Apr;23 Suppl A:S28-38.
Maternal pH 7.42
Higher 1-acid glycoprotein
Lower albumin
Fetal pH 7.40
Lower 1-acid glycoprotein
Higher albumin
Simplified scheme of the conversion of the precursor EFAs to their
long-chain polyunsaturated metabolites.
Haggarty P. Eur J Clin Nutr. 58(12):1559-70. (2004)
Docosahexaenoic Acid
[DHA 22:6,n-3]
[EPA 20:5n-3]
Sterol Regulatory Element
Binding Proteins (SREBPs)
• Transcription factors that bind to sterol
regulatory elements to regulate a wide range
of lipid genes
– SREBP-1: genes to make fatty acids
– SREBP-2: genes of cholesterol metabolism
• Control cholesterol and fatty acid synthesis
• Activation depends on sterol levels in cells
• Can DEHP increase the expression of PPAR
isoforms through SREBPs? Shimano, H. Prog in Lipid Res. 2001. 40(6): 439-452.
29
EFAs and Fetal Development • Fatty acids, especially essential fatty acids (EFAs) are critical
for proper fetal development, most importantly, the brain: – Membrane synthesis
– Energy storage
– Precursors of the lipid-derived signaling molecules:
prostaglandins, leukotrienes, and thromboxanes, etc.
– Variety of other physiological functions…….
• The fetus relies on the placental transfer of EFAs from maternal circulation for development: – Fatty acid transfer is highly directional from the mother to the fetus.
• Two primary processes governing placental EFA supply from mother to fetus: – Placental fatty acid uptake/transport and metabolism.
– Many of fatty acid transporters and metabolizing enzymes are under the regulation of the PPARs.
CH3 3 6
Fetal Imprinting
• The developing fetus undergoes rapid changes during the 9 month gestation period
• Genetics, diet, environment, etc. can all impact the fetus.
• Fetal imprinting has been linked to:
– Cardiovascular disease-Barker Hypothesis
– Neurological disorders
– Obesity/diabetes, as well as others
• Very difficult to predict human effects based on:
– Many factors involved aside from genetics, e.g., diet
– Many exposure studies done in animals.
“Every four years, one trillion pounds of
plastics are made in the world. They are
being thrown away in the landfill …
leaching back into our water. So, to say
all endocrine disrupting chemicals are at
lower levels today...is just ludicrous.”
Environmental Toxicity
-Frederick vom Saal, Professor of Biological
Sciences, University of Missouri 32 http://www.pbs.org/wgbh/pages/frontline/shows/nature/interviews/vomsaal.html
Fetal Imprinting During Pregnancy Can Affect Health
Outcomes
Sites for Xenobiotic Impact
C. Prouillac, S. Lecoeur. The Role of the Placenta in Fetal Exposure to Xenobiotics: Importance of Membrane Transporters and Human Models for Transfer Studies. DMD 38:1623–1635, 2010
37
http://www.cleanandhealthyme.org/Portals/0/
bodyburden/PollutionInMaineGraphic.jpg
• Continuous
exposure to
household
chemicals with
potential to cause
toxic effects
• Food contains
potentially harmful
chemicals such as
pesticides and
antifungals
DEHP: Toxicokinetics Absorption:
Orally administered DEHP is rapidly hydrolyzed in the gut by esterase to MEHP and EHA, however, there are species differences in the rate of hydrolysis.
Distribution and Metabolism: DEHP and its metabolites are widely distributed throughout the body.
MEHP and other primary metabolites can be excreted unchanged or further converted in the liver into numerous secondary metabolites.
Excretion: DEHP is metabolized quickly and the metabolites are rapidly excreted via urine
(90%) and feces (10%).
No significant retention in organs and tissues.
DEHP readily crosses the placenta:
Intra-uterine toxicity?
Di -(2-ethylhexyl)-phthalate (DEHP)
• Most widely used industrial plasticizer of polyvinyl chloride: – Contents vary up to 40 to 50% of
the final weight.
• Ubiquitous environmental contaminant: – water, air, food and soil.
– General population: 3-30g/kg/day
– Medical: exceed general population by up to 2-3 orders of magnitude
• Special higher exposure: – Infants and young children.
– The highest risk: neonates (particularly male) under intensive therapeutic medical interventions.
C24H38O4 MW = 390.65
(117-81-7) Log K ow
= 7.50
Kavlock R, et al. NTP report. Reprod Toxicol. 2002,16 (5):529-653.
Effects of DEHP on the Fatty Acid Placenta
Transfer, Fetal Distribution and Lipid Profile in
Fetal Brain upon Maternal Exposure
• To confirm the in vivo expression data and to elucidate the in vivo functional effect of DEHP on placental/fetal fatty acid homeostasis.
• In the rat model:
– DEHP treatment: oral gavage at 1500 mg/kg (GD 0 to 19).
– At GD 20: • Organ Toxicity
• Fatty acid placental transfer and fetal distribution studies:
– Radiolabeled compound distribution.
• Fetal brain lipid profiling studies:
– Lipomics assay.
Maternal weight changes until GD 20
Weight Record
-20.00
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
0 5 10 15 20
Ge station Days
W e
ig h
i n
c re
a s
e (g
)
DEHP
Control
DEHP Treated rat damns have less weight gain-is this linked DEHP induced
toxicity or local GI distress….No signs of emesis or diarrhea-perhaps nutrient
homeostasis?
General Toxicology
•
Ave STD Ave STD Significance
Mother
body weight gain
D13(g) 38.00 14.83 18.57 11.44 0.028
D20(g) 128.33 25.66 81.67 12.58 0.047
Liver weight(g) 9.27 0.15 9.99 0.31 0.022
% to body weight 2.35 0.12 2.81 0.14 0.000
Fetus
Fetal weight(g) 2.22 0.18 2.10 0.16 0.030
Liver weight(g) 0.06 0.01 0.06 0.01 0.715
% to body weight 2.77 0.36 3.02 0.70 0.028
Number per litter 11.71 4.89 15.60 3.36 0.158
Resorption per litter 0.60 0.55 2.40 1.52 0.037
Control DEHP(750mg/kg/day)
Results: EFA Transfer across the Placenta
Changes of placental transfer of arachidonic acid (AA) and docosahexaenoic acid (DHA) and
their esterified metabolites at GD20 upon maternal exposure to DEHP (1500 mg/kg). means ± SD, n =3 or 4. *, p<0.05; **, p<0.01, ***, p<0.001.
A. AA B. DHA
In spite of increased transporter expression due to DEHP treatment, there is a
marked decrease in both AA and DHA placental transfer to the fetus
Results: EFA Fetal Distribution
Changes of fetal distribution of arachidonic acid (AA) and docosahexaenoic acid (DHA) and
their esterified metabolites at GD20 upon maternal exposure to DEHP (1500 mg/kg). (means ± SD) n =3 or 4. *, p<0.05; **, p<0.01, ***, p<0.001.
A. AA B. DHA
The changes in EFA placental transfer due to DEHP treatment, result in
dramatic decreases in both AA and DHA fetal brain accumulation
Samples
Multivariate Analysis
Lipomics Assay
http://www.lipomics.com/
Results: Fetal Brain Lipid Concentrations
Changes of fetal brain lipid concentrations at GD 20 upon maternal exposure of DEHP
(1500mg/kg). Data shown are relative arbitrary units (R.A.U.) with a value of 100 for the respective
vehicle controls at each specific lipid class (means ± SD, n = 3 or 4). *, p<0.05.
FA, Free fatty acid
DAG, Diacylglycerol
TAG, Triacylglycerol
FC, Free cholesterol
CE, Cholesterol ester
PC, Phosphatidylcholine
PE, Phosphatidylethanolamine
PS, Phosphatidylserine
LYPC, Lysophosphatidylcholine
CL, Cardiolipin
SM, sphingomyelin.
Results: Fetal Brain lipid Profile of AA
Changes of fetal brain lipid profile of arachidonic acid (AA) at GD 20 upon in utero exposure
of DEHP (1500mg/kg). Data shown are relative arbitrary units (R.A.U.) with a value of 100 for the
respective vehicle controls at each specific lipid class (means ± SD, n = 3 or 4). *, p<0.05.
FA, Free fatty acid
CE, Cholesterol ester
DAG, Diacylglycerol
TAG, Triacylglycerol
PC, Phosphatidylcholine
PE, Phosphatidylethanolamine
PS, Phosphatidylserine
LYPC, Lysophosphatidylcholine
CL, Cardiolipin
SM, Sphingomyelin
Results: Fetal Brain lipid Profile of DHA
Changes of fetal brain lipid profile of docosahexaenoic acid (DHA) at GD 20 upon in utero
exposure of DEHP (1500mg/kg). Data shown are relative arbitrary units (R.A.U.) with a value of
100 for the respective vehicle controls at each specific lipid class (means ± SD, n = 3 or 4). *, p<0.05.
FA, Free fatty acid
CE, Cholesterol ester
DAG, Diacylglycerol
TAG, Triacylglycerol
PC, Phosphatidylcholine
PE, Phosphatidylethanolamine
PS, Phosphatidylserine
LYPC, Lysophosphatidylcholine
CL, Cardiolipin
SM, Sphingomyelin.
Autism Spectrum of Disorders (ASD) • Characterized by impairments in
communication and social interaction as well as restricted and repetitive behaviors and interests.
• Believed to arise from a combination of genetic predisposition and environmental insult.
• Changes in the fatty acid/lipid homeostasis have been implicated in the propensity to develop ASD and other neuro- developmental disorders.
EFA imbalance has been found in the plasma of autistic children (23% reduction of DHA, 25% reduction of ratio of -3/-6 EFA compared to matched mentally impaired subjects)
Vancassel S, et al. Prostaglandins Leukot Essent Fatty Acids, 2001 Jul;65(1):1-7.
Pharmacotherapy and pregnancy • ~60 million women at
reproductive age
• ~10% of these women become pregnant annually • Many are on meds for treating
pre-existing conditions (e.g., epilepsy, hypertension, depression, asthma, etc.)
• But…PK of drugs in pregnancy is complex • Physiological changes alter
ADME of drugs
• Toxicity is a significant concern
Coles, L.D., Eddington, N.D. AAPS Mag. 2007. 10(4): 18-21.
50
Maternal Blood pH 7.42
Fetal Blood
pH 7.40
Ion Trapping:
Net accumulation
of basic drugs
R-NH3 +
R-NH2
R-NH3 +
R-NH2
Ion Trapping:
Net accumulation
of acidic drugs
R-CO2 -
R-CO2H
R-CO2 -
R-CO2H
Maternal Blood pH 7.42
Breast Milk pH 6.80
Ion Trapping:
Net accumulation
of basic drugs
R-NH3 +
R-NH2
R-NH3 +
R-NH2
Ion Trapping:
Net accumulation
of acidic drugs
R-CO2 -
R-CO2H
R-CO2 -
R-CO2H
Placental Mechanisms of Transport
Mechanisms of transport of substances (Cm = maternal concentration)
across the placental membrane: A, simple diffusion; B, paracellular
transport across water-filled porous; C, facilitated difusion; D,
active transport and E receptor-mediated endocytosis.
FH Morris Jr., R.D.H. Boyd, D. Mahendran, Placental transport. In: The Physiology of
Reproduction, Second Edition. Raven Press, Ltd., NY (1994) 813-861.
Primary factors determining drug permeation
across the placental barrier (continued)
2. Placental characteristics
• Blood flow (intervillous maternal and fetal)
influenced by pathophysiological conditions
and drugs
• Concentration gradient of drug across the barrier
• Hydrostatic gradient – slight movement toward
the fetal compartment
• pH gradient – slightly acidic fetal compartment
• Thinning and aging barrier with advancement of
pregnancy
Primary factors determining drug permeation
across the placental barrier (continued)
2. Placental characteristics (continued)
• Increasing surface area with advancement of
pregnancy
• Developing metabolism that is largely
unappreciated
• Protein gradients – decreasing albumin
concentration in maternal compartment
with advancement of pregnancy, lower α1-acid 1
glycoprotein concentration in fetal compartment
• Nutrient transporters (e.g., amino acid carriers,
glucose carriers, monoamine carriers, etc.) –
in addition to passive diffusion, drugs may
be transported across the placenta by nutrient
carriers
3. Additional maternal and fetal factors:
• Fetal growth and development
• Fetal metabolism
• Fetal tissue binding
• Maternal metabolism
• Maternal health condition
Primary factors determining drug permeation
across the placental barrier (continued)
Drug Therapy for Pregnant
Women • Pregnant women are a special population for drug
therapy. – Treatment for pregnant woman results in potential
treatment of unborn baby, even when the child is not the target.
• 66% of administered drugs have never been tested on pregnant woman.
• Exposure to other environmental xenobiotics: pesticides, herbicides, house hold insecticidal agents, occupational chemicals etc.
Lessons From Past
• 1950s - widely use of pesticide hexachloro-bezene: increased spontaneous abortion rates and congenital malformations.
• 1960s - thalidomide disaster: phocomedia and other embryopathia.
• 1970s - diethyltsbestrol: induced adenocarcinomas among adolescents.
Thalidomide disaster :
Thalidomide, originally developed in
the late 1950s and early 1960s as a
sedative, was sold in 46 countries and
prescribed by many doctors to
pregnant women for morning
sickness. It was soon found that
thalidomide caused a significant
increases in phocomelia, a previously
rare birth defect consisting of no limbs
or tiny flipper-like arms and legs,
serious facial deformities, and
defective organs.
American Journal of Medical Genetics 103:295-301 (2001)
Syncytial Trophoblast Layers I & II
TGC-trophoblast giant cells
Questions?
• When drug administered to pregnant women:
– Are the PK of drug different in pregnant women
compared with men and non-pregnant women?
– What are the mechanisms and extent of drug
transfer across the placenta?
– Is the drug safe for the mother and/or her fetus?
– Is the drug effective for the mother and/or her
fetus?
Drug Transport Across the Placenta • Physical, chemical characteristic of molecule: MW and size, fat solubility,
ionization
– Hydrophobic, non-ionized compounds:
• Simple diffusion governed by Fick’s law
• Permeable to molecular of less than 600 Da
– Hydrophilic, polar compounds:
• Influx and efflux transporters
• Facilitate or active
• Degree of protein binding
• Ability of placental and fetal liver to metabolize the drugs
• The ratio of placental-fetal vs. maternal-fetal blood flow velocities, PH difference.
C.W. Mason, C.P. Weiner. CHAPTER 40 Placental Drug Transport. The Placenta: From Development to Disease, First Edition. Edited by Helen H. Kay, D. Michael Nelson and Yuping Wang, Published 2011 by Blackwell Publishing Ltd., pp 310-317
C.W. Mason, C.P. Weiner. Chapter 40: Placental Drug Transport. The Placenta: From Development to Disease, First Edition. Edited by H.H. Kay, D.M. Nelson, Y. Wang, (2011) Blackwell Publishing Ltd., pp 310-317
Representative Therapeutic
Agents and Abused Drugs
Transferred by Placenta
Category Substance
Therapeutic
agents
Amoxicillin, Chloroquine,
Digoxin, Phenobartital,
Sulfamethoxazole,
Theophylline, Methohexitol,
Fentanyl, Bupivacaine etc.
Abused drugs Morphine, Cocaine etc.
Sastry BV. (1999) Adv. Drug Deliv. Rev. 38, 17-39.
Structural Features of the
Placenta Relevant to Drug
Transport • Membranous structure formed by the apposition or fusion of
fetal membrane ( chorion frondosum) with the uterine mucosa ( deciduas basalis) and divided into functional units called cotyledons.
• Structure changes dramatically during pregnancy: – Fully differentiated at the third month of pregnancy
– Thinning and aging:
• 10um at early pregnancy to 1-2 um at late term
– Blood flow and surface area: increase significantly during the late pregnancy and influenced by pathophysiological conditions
• Endowed with influx and efflux transporter to facilitate the transfer of nutrients and the export of wastes
– Some of them mediate the passages of drugs/xenobiotics
Placental Mechanisms of Transport
A: Simple diffusion
B: Paracellular
transport
C: Facilitated diffusion
D: Active transport
E: Receptor-mediated
endocytosis.
FH Morris Jr., R.D.H. Boyd, D (1994) Mahendran, In: The Physiology of Reproduction, Second Edition. Raven Press, Ltd., NY,813-861.
Intravillous spaces
Microvillous membrane
Syncytial trophoblast
Basalateral membrane
Basal lamina
Fetal endothelial cells
Fetal capillary (Cm = maternal concentration)
Methods to Study Placental Drug Transport – Cultured Placenta Villous Tissues and Cells
Types Origin Characteristics Activity
Placental
villous
trophoblasts
Term placenta 10%
cytotrophoblasts
Preserved at
least for 3 days
First-trimester
placenta
40%
cytotrophoblasts
Useable up to
33-45 days of
culture
Malignant
trophoblast
cell lines:
BeWo, JAR &
JEG
Gestational
choriocarcinoma
Cytotrophoblast
cells and able to
differentiate into
syncytio-
trophoblast
Identical to
common human
trophoblasts;
Used for
polarized
transport study
Sastry BV. (1999) Adv. Drug Deliv. Rev. 38, 17-39.
Methods to Study Placental Drug
Transport – Dually Perfused Placenta Cotyledon
Sastry BV. (1999) Adv. Drug Deliv. Rev. 38, 17-39.
Schematic figure of a section of
human placenta in an inverted
position showing umbilical vessels
entering the cotyledon from below.
Schematic presentation of the placental
cotyledon perfusion chamber, illustrating
the use of blunt needles to simulate
maternal spiral arterioles for perfusion of
the intervillous space.
CLmf and CLfm: Drug clearances
Cfaand Cfv: Drug conc. in the umbilical
vein effluent/ fetal reservoir
Qf: Umbilical flow rate
Wp: Wet weight of placenta
Methods to Study Placental Drug
Transport – Dually Perfused Placenta Cotyledon
• Advantages: Determining
– Placental transfer of substances
– Effects of endogenous and
exogenous substances to placental
transport
– Release of endogenous
substances into maternal and fetal
perfusions
• Disadvantages:
– Metabolically static system instead
of dynamic state
– Term placenta
– Results affected by procedures of
sample preparing
Methods to Study Placental Drug
Transport – in vivo Animal Studies
• i.v. infusion of the drug to the mother and the fetus
• Determine maternal and fetal plasma concentrations at steady state
• Calculate ratio of CLmf/CLfm
=1: passive or facilitative
>1: active maternal-fetal transport
<1: active fetal-maternal transport
Cssf and Cssm: steady-state drug conc.
CLmf and CLfm:drug clearances
CLfo and CLmo: irreversible drug clearances(
metabolisms etc)
Methods to Study Placental Drug Transport
– in vivo Human Studies
Most commonly:
Measure the fetal umbilical cord : maternal plasma concentration at the time of delivery.
Must be interpreted very carefully
Usually obtained at a single time point
Under non-steady state conditions
Unadkat JD, et al (2004) Curr. Drug Metab. 5, 125-131.
Directionality of Drug Transport
Direction of transport determined by :
•Substrate concentration gradient and/or energy driving force
•Location of drug transporter
Feto - maternal
direction
Materno - fetal
direction
Placental Biotransformation
Enzymes Category Enzymes
Phase I Cytochrome P450
(CYP)
First Trimester: CYP1A1, 1A2, 2C, 2D6,
2E1, 2F1, 3A4, 3A5, 3A7, 4B1, 19 and 11B
Full term: CYP1A1,2E1,2F2,3A3/4,3A5,
3A7, 19 and 11B
Others Quinone reductases(QR),
Nitric oxide synthase (NOS)
Phase II Glutathione s-
transferases (GST)
GSTP1-1, GSTA4
Others Epoxide hydrolase (EH),
Sulphotransferases (STs),
N-acetyltransferase (NAT)
Pasanen M.(1999) Adv. Drug. Deliv. Rev.,38,81-97.
Placental Drug Transporters
Unadkat JD, Dahlin A and Vijay S, (2004) Curr Drug Metab. 5, 125-131.
Efflux transporters: P-
glycoprotein (P-gp) • Best characterized drug
efflux transporter in the placenta
• Transmembrane glycoprotein encoded by the human multidrug resistance gene MDR-1
• Polymorphism of MDR gene affects placental P- gp expression levels.
Polymorphism in the human MDR-1 gene.
Mutations result
in decreased
levels of P-gp.
Tanabe M, et al (2001) J. Pharmacol.Exp.Ther.297,137-1143.
Efflux transporters – P-gp
• Expressed on the maternal side of the placental trophoblasts (BBMV)
• Controversy of P-gp expression levels in the placenta during pregnancy
P-gp is expressed and active throughout pregnancy (most researchers)
v.s. P-gp activity decreased in the last stage of pregnancy
• Preventing the entry of potentially toxic compounds into the fetal circulation
Ushigome F, et al (2000) Eur.J.Pharmacol.,408,1-10.
Importance of P-gp: First
Evidence • In 1998, first evidence to
suggest that P-gp can significantly reduce fetal exposure to toxic xenobiotics.
• CF-1 mice (naturally occurring mdr-1a knock-out) outbred mouse stock have no placental Mdr1a P-gp.
• This absence is associated with increased sensitivity to avermectin, a teratogenic pesticide.
• Increased incidence of cleft plate correlate with the conc. of avermectin in fetal tissue.
Lankas GR, et al (1998) Reprod. Toxicol.12, 457-463.
Importance of P-gp: Further Evidence
• Absence of P-gp expression in mdr1a/b-deficient placenta is associated with increased exposure of the fetus
– Digoxin (2.2-2.4 fold),
– Saquianavir (5-7 fold)
– Paclitaxel (16 fold).
• Co-administration of PSC833, a P-gp inhibitor, increase penetration of these drugs to fetus.
Smit JW, et al (1999) J.Clin.Invest. 104,1441-1447. Marzolini C,et al (2002) AIDS,16:889-893.
Mdr1a+/+1b+/+
Mdr1a+/-1b-/-
Mdr1a-/- 1b-/-
• ~25% of the children of these mothers become infected with HIV, and most of these (~80%) appear to occur during late pregnancy and (particular) birth.
• Co-administration of effective P-gp inhibitors during late gestation: – Increase infant loading with drugs
– Limit adverse effects on development stages
Importance of P-gp: Further
Evidence Drugs Plasma Conc(ng/ml) Ratio (C/M)
Cord Mother
Ritonavir < 250 1113 0-0.2
Saquinavir < 100 350 0-0.3
Lopinavir < 250 3105 0-0.1
Nelfinavir < 250 1110 0-0.3
• Transplacental passage of protease inhibitors in pregnant HIV-1 infected women: – The cord-to-
maternal blood ratio extremely low
Marzolini C,et al (2002) AIDS,16:889-893.
Efflux transporters – Multidrug
Resistance-associated Proteins (MRPs)
Types Placental
Location
Bewo cell Substrates
MRP1 Trophoblast,
but mainly in
fetal capillary
Basolateral
and apical
membrane
Present Organic anions,
glutathione and
glucuronate
conjugates
MRP2 Trophoblast Apical
membrane
Unknown Similar to MRP1 & 3
MRP3 Trophoblast Basolateral
and apical
membrane
Unknown Similar to MRP1 & 2
MRP4-5 Trophoblast Unknown Present Nucleotide analogs
MRP6-8 Unknown Unknown Unknown
Young AM et al (2003) Adv. Drug. Deliv. Rev. 55, 125-132.
Efflux Transporters – MRPs
• Overall effects of the MRPs on placental xenobiotic removal is unknown
• Might be similar to P-gp
MRP1 MRP3MRP2
St-Pierre MV, et al (2000) Am.J.Physiol.Regul.Integr.Comp.Physiol.,279, R1495-1503.
Control MRP5MRP4
Efflux Transporters – Breast
Cancer Resistance Protein (BCRP)
• An ABC half-transporter encoded by ABCG2
• Forms a homodimer to function as an efflux pump
• Considerable overlap of substrate activity with P-gp
• High expression in placenta, localized in syncytiotrophoblasts
Litman T et al (2002) Cancer Res. 60:6-16.
Efflux Transporters –BCRP
• BCRP function as a maternal-fetal barrier to
passage of xenobiotic across the placenta
[14C]Topotecan Vehicle
treated
GF120918
treated
Ratio P*
Fetus, ng/g 15.6 ± 4.6 49.6 ± 5.5 3.18 <.001
Plasma, ng/mL 35.7 ± 6.2 57.9 ± 11.2 1.62 .029
Fetus/plasma 0.43 ± 0.07 0.87 ± 0.09 2.02 .001
At gestation day 15.5, GF120918 (50 mg/kg) was administered orally to pregnant dams; 2 hours
later, [14C]topotecan (0.2 mg/kg) was administered intravenously; 30 minutes later, the fetal
distribution and maternal plasma concentration of [14C]topotecan were determined. (n = 3 or 4
dams, n = 30 fetuses for vehicle, and n = 41 fetuses for GF120918).
GF120918 and [14C]topotecan in mdrla/1b(-/-) mice
Jonker JW, et al (2000) J.Natl. Cancer. Inst. 92, 1651-1656.
Other Placental Transporters -
Organic Cation Transporter 3 (OCT3)
• Human: Extraneuronal Monoamine Transporter (EMT)
• Rat: Organic Cation Transporter
• Mouse: Orct
– Na+ and Cl- independent monoamine transporter.
– Responsible for importing dopamine and norepinephrine into the fetal compartment.
• Orct3 transports neurotoxin MPP+
between the placenta and the fetus:
– In knockout mouse, 3 fold reduction of MPP+ accumulation in embryos.
Placental MPP+ transport. Wild-type (black bars)
and Orct3 mutant (white bars). MPP+, methyl-4-
phenylpyridinium acetate.
Zwart R, et al (2001) Mol. Cell. Biol. 21, 4188-4196.
Other Placental Transporters - Human Equilibrative Nucleoside Transporters 1 and
2 (hENT1 and hENT2)
Isoform Placental
localization
Substrate Affinity NBMPR*
inhibition
Placental
function
hENT-1 Brush border
membrane of
syncytio-
trophoblasts
Nucleoside
&
nucleoside
analogs;
Anticancer
& antiviral
drugs
Higher for
cytidine,
guanosine,
thymidine
adenosine
Potent
inhibitor
Move
nucleosides
&nucleoside
analogs across
the placenta from
mother to fetus or
vice versa,
depending on
concentration
gradient
hENT-2 Unknown Higher for
inosine
Moderate
inhibitor
Knipp GT.(1999) Adv. Drug. Deliv. Rev.38, 41-58 Sastry BV. (1999) Adv. Drug. Deliv. Rev.38, 17-39.
*NBMPR: nitrobenzylthionsine
Example: ribavirin, a possible substrate of hENT1/2, when administrated to
perinatal rats, results in fetal malformation
Other Placental Transporters- Amino Acid Transporters
Knipp GT1999) Adv. Drug. Deliv. Rev.38, 41-58
Transported drugs: gabapentin (an antiepileptic drug), arginine analogs (inhibitors
of nitric oxide synthases), and thyroid hormone mimics
Summary • Placenta functions as a selective barrier to passage
of drugs/xenobiotics into the fetal compartment.
• Placental transporters are important in modulating
the exposure of the fetus to drugs and, therefore,
the efficacy and toxicity of such drugs towards the
fetus.
• These transporters might be targeted to modulate
drug distribution across the placental barrier.
– Targeted delivery
References • Unadkat JD, Dahlin A, Vijay S. Placental drug
transporters. Curr Drug Metab. 2004 Feb;5(1):125-31.
• Young AM, Allen CE, Audus KL. Efflux transporters of the
human placenta. Adv Drug Deliv Rev. 2003 Jan
21;55(1):125-32.
• Sastry BV. Techniques to study human placental
transport. Adv Drug Deliv Rev. 1999 Jun 14;38(1):17-39.
• Knipp GT, Audus KL, Soares MJ. Nutrient transport
across the placenta. Adv Drug Deliv Rev. 1999 Jun
14;38(1):41-58.
