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A.1. Absorption and bioavailability after dermal, inhalatory or oral exposure
Bioavailability is defined as the fraction of compound in a certain matrix that reaches the systemic
circulation. Three separate processes can be distinguished in bioavailability: (1) release of the
compound from its matrix (bioaccessibility), (2) absorption of the released fraction, and (3)
metabolism before reaching the systemic circulation (Versantvoort et al. 2005; Oomen et al. 2003).
Existing approaches have been summarised in Table A1.
Release of the compound from its matrix is required for transport across the dermal, lung or
intestinal epithelium and bioavailability of a compound to the body. Therefore, the release depends
mainly on the matrix, while absorption and metabolism depend more on the compound specific
properties and physiology. In order to predict the bioavailability of a cosmetic ingredient from a
cosmetic product, it is therefore important to determine the three different processes involved.
However, the bioaccessibility could be used as a measure for the maximal bioavailability.
A.1.1. Bioaccessibility models
Dermal exposure. The bioaccessibility in vitro model representing the release on skin is a test based
on human sweat with the pH of human skin. The preparation of artificial sweat is described in DIN
EN ISO 105-E04 (2008). This model could be useful to determine the skin bioaccessibility of a
cosmetic ingredient from a cosmetic product.
Inhalatory exposure. Artificial lung fluid could be used as in vitro bioaccessibility model for
inhalatory exposure. Beeston et al. (2010) described such a model to determine the lung absorption
after inhalatory exposure to nanoparticles. In vitro lung bioaccessibility models have also been used
to determine exposure after inhalation of soil, man-made fibres and various metals. Inhalatory
bioaccessibility model will not be available for pre-validation within the coming few years.
However, such a model could be useful to determine the lung bioaccessibility of an ingredient in
cosmetics.
Oral exposure. In vitro methodologies to study the human oral bioavailability of compounds from
consumer products, food (including herbs and spices) and soil have been set-up. Most of the in vitro
digestion models simulate in a simplified manner the digestion processes in the mouth, stomach and
small intestine, in order to enable investigation the bioaccessibility during transit in the
gastrointestinal tract. These models could be used to determine the bioaccessibility of a cosmetic
ingredient from a cosmetic product (Daly et al. 2010; Peters et al. 2010; Amiard et al. 2008;
Veda et al. 2007; Brandon et al. 2006; Versantvoort et al. 2005; Oomen et al. 2003 and many
others).
Bacterial metabolism in bioaccessibility. Bacteria (large intestine and skin) could be involved in the
metabolism of a cosmetic ingredient. In vitro metabolism studies with skin or intestinal bacteria
should be performed if bacterial biotransformation is expected (Platzek et al. 1999; van de Wiele et
al. 2005).
A.1.2. Absorption models
1
Dermal exposure. Different in silico QSAR models are available for the skin permeability
prediction of compounds (Lee et al. 2010; Hansen et al. 2008; Ottaviani et al. 2007). In vitro
models that might be useful to study the absorption of cosmetic ingredients from a cosmetic product
are artificial membrane PAMPA-skin, the stratum corneum and keratinocytes. Also the
spontaneously immortalised human keratinocyte cell line HaCaT has frequently been used as in
vitro model to study skin absorption. Furthermore, the cell line has been used in several
dermatopharmaceutical studies (Doebis et al. 2002; Boderke et al. 2000; Altenburger and Kissel
1999). However, it is noted that some of these in vitro models have active metabolising enzymes,
thus besides absorption, also metabolism may occur (Oesch et al. 2007; Swanson 2004; Baron and
Merk 2001).
Inhalatory exposure. The lung can be anatomically divided into several parts: trachea, bronchi,
bronchioles and alveoli. Absorption of inhaled compounds can be throughout the entire lung. In the
upper respiratory airways the absorption is low and is mostly happening in the lower part. With
respect to disposition in the airways, CFD (Computational fluid dynamics) could be a useful
approach (Kimbell and Subramaniam 2001). No QSAR models predicting lung absorption are
known in public literature. In vitro models to study the translocation of compound in the lung are
various types of pulmonary epithelial cell lines (e.g. Calu-3 cell line, A549 cell line) and primary
lung cells of either human or animal origin (e.g. primary rat type II pneumocytes) (Geys 2009; Geys
2006). The integrity of the cell monolayer was verified by measuring the transepithelial electrical
resistance (TEER) and passage of sodium fluorescein. Furthermore, a system of co-cultures of
relevant cells (pneumocytes (e.g. A549), macrophages (e.g. THP-1), mast cells (e.g. HMC-1) and
endothelial cells (e.g. EAHY926) can be used to study lung absorption (Alfaro-Moreno et al. 2008).
These in vitro methods have been used to study the translocation of ultra-fine particles, but could be
useful to study the absorption of a cosmetic ingredient after inhalatory exposure. However, the in
vitro techniques have active metabolising enzymes to varying extent, thus besides absorption
metabolism is investigated (Mansour et al. 2009; Foster et al. 1998).
Oral exposure. In silico QSAR-like models can predict specific parameters for an unknown
chemical based on structural and physicochemical similarities to various known chemicals.
Sophisticated commercial in silico software is available that can predict absorption via the
gastrointestinal tract. These in silico tools predict the overall oral absorption of a chemical based on
available physicochemical parameters. In vitro experiments (artificial membranes and monolayers
of specific cells) can predict the absorption over a single barrier. These in vitro studies are rather
standard and could be incorporated in a medium-throughput test strategy. However, cell monolayers
(e.g. Caco-2 cells) and artificial membranes lack (completely or to some degree) specific in vivo
metabolic and active transport systems. Therefore, these membrane studies can only rank a
chemical compared with known reference chemicals and can provide semi-quantitative data. As
with in silico models, the validity of these in vitro model predictions for cosmetics remains to be
established, because most, if not all, of these models were developed for pharmaceuticals. The
ongoing Liintop project provides relevant experimental data on the use of hepatic intestinal in vitro
methods to study absorption (http://www.liintop.cnr.it/index.php?PG=project&action=project).
A.1.3. Bioavailability models
Dermal exposure. OECD guideline 428 (2004)1 describes the in vitro method for measuring dermal
absorption of animal and human skin. Three different types of skin preparation may be used:
epidermal membranes, consisting of stratum corneum and epidermis, full thickness skin, consisting
1
The EPA guideline on Dermal exposure assessment:Principles and Applications (1992) provides a detailed
procedure for in silico calculation of dermal exposure.
2
of stratum corneum, epidermis and dermis (total thickness ca. 1 mm or more) and split thickness
skin, consisting of stratum corneum, epidermis and part of the dermis (total thickness ca.
200-400 μm). The last skin preparation is most representative of the in vivo distance between skin
surface and dermal blood circulation. The skin preparation may or may not be metabolically active
and thus may lead to the determination of absorption alone or the bioavailability of the compound
after dermal application (Oesch et al. 2007). Two different types of exposure conditions may be
used: finite and infinite dose conditions. Under finite dose conditions, skin load (mg of
chemical/cm2) the concentration of dissolved chemicals will change with exposure time, while
under infinite conditions they will not, or at least not significantly over the total exposure time.
Absorption under finite dose conditions is often expressed as percentage of the applied dose. Its
value is specific for the specific exposure time, concentration and skin load employed in the study.
Due to this limitation, it is difficult as input parameter for PBPK-models. Absorption under infinite
dose conditions is often expressed as a permeation constant, Kp, in cm/h. It is calculated by dividing
the steady state flux of the chemical across the skin (mostly expressed in mg/cm2/h) by its
concentration in the donor fluid. Unless the chemical investigated or the vehicle in which it has
been dissolved alters skin permeability in a concentration dependent fashion, Kp is concentration
independent. It can serve as an input parameter for PBPK-models. By multiplying it with the
exposure concentration in mg/ml, steady state flux (in mg/cm2/h) can be calculated. The steady state
flux across the skin does not scale with body weight, as many other input parameters do. Chemicals
may show a significant lag time before reaching steady state flux. This lag time is usually also
reported, and can be taken into account when modelling. To conclude, Kp can only be calculated
for chemicals in solution or for liquid chemicals. When the chemical is applied as a solid, only
(maximum) steady state flux under infinite dose conditions can be calculated.
Inhalatory exposure. Currently there are in vitro methods described in public literature concerning
lung tissue used to study the bioavailability of compounds after inhalatory exposure (Geys et al
2007). There is a need to investigate whether and how lung preparations of human or animal origin
could be used to study the lung bioavailability of a cosmetic ingredient.
Oral exposure. The Ussing chamber utilises small sections of intestinal mucosa that are clamped
between two chambers containing buffer (Grass et al. 1988; Ussing et al. 1951). This technique
enables a fast and reproducible study of intestinal absorption of molecules across the inserted tissue
(Gotoh et al. 2005). An additional advantage of the Ussing chamber system is that next to
transepithelial drug transport also gut metabolism can be studied (van de Kerkhof et al. 2006).
Furthermore, in the everted sac method a segment of the intestine is inverted and placed over a
glass rod (Wilson et al. 1954). The mucosal epithelium with villi is turned inside out, while the
mucosal membrane forms the inner surface of the everted sac. This system provides information on
the mechanism of absorption (Kato et al. 2004).
With both in vitro models, the differences in absorption along the length of the gastrointestinal tract
can be studied and they can provide quantitative absorption information (absorption over time; ka
and Fabs). However, fresh tissue should be used (Bohets et al. 2001; Versantvoort et al. 2000).
3
Table A1. Replacement methods for Absorption and Bioavailability (A).
Test name
Endpoints
measured
QSAR dermal
exposure
Skin
permeability
Origin of test
Area(s) of
system (cell
applications;
line, ex vivo,
known
primary cells,
limitations
in silico, in vivo
In silico
Used mainly for
pharmaceuticals
Artificial skin
(e.g. PAMPAskin)
Absorption
In vitro
Developed for
pharmaceuticals
Stratum
corneum
Absorption
Ex vivo
Developed for
pharmaceuticals
Status
Comments
Suitability for
specific
subcategories of
cosmetic
ingredients
should be
evaluated
Suitability for
cosmetics
should be
evaluated
Not used for
cosmetics
No validation
>2013
necessary, methods
have to follow the
OECD principles for
the validation of
QSARs for
regulatory purposes
Might be useful for
>2013
cosmetics as a
screening tool
Stratum corneum is
mainly composed of
dead cells and is the
first skin barrier.
Provides
information on the
maximal absorption
Estimated
time to enter
prevalidation
process
<2013
4
Animal or
human skin
Absorption
Ex vivo
Developed for
pharmaceuticals
, the method is
in use for
cosmetics
Frequently used
for cosmetics.
Primary
keratinocytes
Bioavailability
(absorption +
metabolism)
In vitro
Developed for
pharmaceuticals
Suitability for
cosmetics
should be
evaluated
Immortalised
Bioavailability
keratinocyte cell (absorption +
line
metabolism)
In vitro
Developed for
pharmaceuticals
Suitability for
cosmetics
should be
evaluated
Pig skin resembles
<2013
more closely the
human skin than that
of laboratory
animals. Pig skin
from slaughter
animals can be used.
Also human skin
from cosmetic
surgery (e.g. facelift). An advantage
of this model is that
phototoxicity can be
studied
No barrier (stratum
>2013
corneum) present,
thus bioavailability
could be
overestimated
Absorption and
>2013
metabolism are
studied, providing
information on the
bioavailability. No
barrier (stratum
corneum) present,
thus bioavailability
could be
overestimated
5
Inhalatory
bioaccessibility
Maximal
inhalatory
absorption
In vitro
Currently not
frequently used,
used to
determine the
bioaccessibility
of metals, soil
and man-made
fibres
Developed for
the absorption
of ultra-fine
particles
Model under
development
Not developed for
cosmetics and still
under development,
but might be useful
for cosmetics
Primary lung
cells
Bioavailability
(absorption +
metabolism)
In vitro
Pulmonary
epithelial cell
lines
Bioavailability
(absorption +
metabolism)
Co-culture of
pulmonary cell
lines
Bioavailability
(absorption +
metabolism)
>2013
Suitability for
cosmetics
should be
evaluated
In vitro
Developed for
the absorption
of ultra-fine
particles
Suitability for
cosmetics
should be
evaluated
In vitro
Developed for
the absorption
of ultra-fine
particles
Suitability for
cosmetics
should be
evaluated
These models have
>2013
been shown to be
useful to study the
absorption of
ultra-fine particles.
Thus mainly
informative of
absorption in the
alveoli. Animals are
needed to obtain
primary lung cells
These models have
>2013
been shown to be
useful to study the
absorption of
ultra-fine particles.
Thus mainly
informative of
absorption in the
alveoli
Resembles more
>2013
closely the lung than
lung cell lines and
primary lung cells
6
Oral
bioaccessibility
Maximal oral
absorption
In vitro
QSAR oral
exposure
Oral absorption
prediction
In silico
Cell
monolayers,
including
transporter
transfected
(e.g.Caco-2,
MDCK)
Absorption
In vitro
Frequently used
to determine
bioaccessibility
of soil, clay and
food
components.
Infrequently
used for
consumer
products
Absorption
prediction is
based on
available
physicochemica
l parameters
Absorption over
a single layer
Some validation
for various soil
types
Not one single
method, different
models developed
which vary. Useful
for cosmetics to
determine maximal
bioavailability
>2013
Developed for
pharmaceuticals
and software is
commercially
available
Might be useful, but
remains to be
established
>2013
Developed for
pharmaceutical,
transfected cell
lines mainly
used to identify
transporters;
problem is that
for nonpharmaceutical,
low absorption
range is more
crucial
Standard in vitro test >2013
for the
pharmaceutical
industry. Provides
also information on
involved
transporters (e.g.
P-gp, OAT)
7
Artificial
intestinal
membranes
Absorption
In vitro
Ussing chamber
Bioavailability
(absorption +
metabolism)
Ex vivo
Everted sac
Bioavailability
(absorption +
metabolism)
Ex vivo
Absorption. DSPAMPA
combines a pH
gradient and
presence of
chemical
scavengers to
the acceptor to
mimic the
presence of
serum proteins
in blood
Absorption over
parts of the
intestine of
animal or
human origin.
Developed for
pharmaceuticals
Developed for
pharmaceuticals
as early
screening tool
Used for early
screening. Only
information on
passive diffusion. A
draw-back of this
method is the
underestimation of
the permeability of
highly lipophilic
drugs
>2013
Developed for
pharmaceutical,
but also used
for food
components
(e.g. vitamins)
<2013
Absorption over
parts of the
intestine of
animal or
human origin.
Developed for
pharmaceuticals
Developed for
pharmaceutical,
but also used
for food
components
(e.g. vitamins)
The bioavailability
depends on
intestinal section.
Human intestine is
available from
surgery and pig
intestine is available
from
slaughterhouses.
Information on
active transport
The bioavailability
depends on
intestinal section.
Human intestine is
available from
surgery and pig
intestine is available
from
slaughterhouse.
Information on
active transport
<2013
8
A.2. Distribution
After absorption, the distribution of a compound and its metabolites inside the body is governed by
three main factors: (1) the partition of the substance with plasma proteins and (2) between blood
and specific tissues; and (2) the permeability of the substance to cross specialized membranes, so
called barriers (e.g. blood-brain barrier/BBB, blood-placental barrier/BPB, blood-testis
barrier/BTB).
Existing approaches have been summarised in Table A2.
Concerning QSAR methods, a general review of the application of in silico approaches for
predicting ADME properties has been carried out by Cronin et al. (2009) and Madden (2010). Even
though a considerable number of substances has been included in the development of these models,
most of them considered pharmaceutical compounds – mainly “successful” drugs - and further
investigation would be required for the application of these approaches to cosmetic ingredients. In
addition, it would be also interesting to have data on drugs with poor pharmacokinetic profile to
improve QSAR predictions (Madden, 2010). Furthermore, QSAR methods should comply with the
OECD Guidelines for the validation of QSARs for regulatory purposes (OECD, 2004).
A.2.1. Estimation of plasma protein binding (PPB)
Only the free (unbound) fraction of a compound is available for diffusion and transport across cell
membranes. Therefore it is essential to determine the binding of a compound to plasma or serum
proteins. The easy availability of human plasma has made it possible to determine the unbound
fraction of compounds by performing in vitro incubations directly in human plasma.
In vitro approaches. There are three methods generally used for PPB determination: 1) equilibrium
dialysis (ED) (Waters et al., 2008); 2) ultrafiltration (UF) (Zhang and Musson, 2006); and 3)
ultracentifugation (Nakai et al., 2004). All methods can be automated for high-throughput, are easy
to perform and have good precision and reproducibility. The use of combined LC/MS/MS allows
high selectivity and sensitivity. Every method has its specific advantages and disadvantages.
Actually, ED is regarded as the “gold standard” approach (Waters et al., 2008). However, Skor et al.
(2005) argued that similar results may be obtained with UF when compared with ED, but in a
higher throughput fashion.
Commercial instruments are available and used routinely by pharmaceutical industries, there is no
reason why they should not been used for cosmetic ingredients. This test should be requested under
present cosmetics practices/regulations.
In silico approaches. Two recent reviews on the in silico approaches for the estimation of PPB have
been carried out by Wang and Hou (2009); and Mostrag-Szlichtyng and Worth (2010). A general
correlation, based on the octanol-water partition coefficient was proposed by de Bruyn and Gobas
(2007) after a compilation of literature data and using a broad variety of chemicals, i.e., pesticides,
polar organics, polychlorinated biphenyls, dioxins, furans etc. This work points out that with a
simple linear estimation, the errors would be in the range of ±2 log units. In addition, the authors
found that for compounds with log Pow <2, PPB did not depend on logPow, but a constant average
value could be assigned.
A2.2. Estimation of blood-tissue partitioning
The fate of a compound in the body is determined by partitioning into the human tissues. Therefore,
the knowledge of this partitioning is of fundamental importance for the understanding of a
compound's kinetic behaviour and toxic potential. The measurement of tissue storage and a
molecular understanding of tissue affinity have, historically, not been studied to the same extent as
plasma protein binding; however, the knowledge of these partitioning coefficients is essential for
9
the development of PBTK models. Fortunately, a quite large number of approaches have been
developed over the last years.
In vitro approaches. The available system is the vial-equilibration technique. A spiked sample of
organ tissue-buffer homogenate is equilibrated and subsequently, the free (unbound) concentration
of the test chemical is determined. The tissue-blood partition coefficient is calculated using results
from pure buffer, tissue-buffer and blood-buffer incubations. Tissues can be mixed to obtain
average values for e.g. richly perfused tissue groups. Olive oil or octanol are often used instead of
adipose tissue. The free (unbound) concentration is typically assessed by one of the following
techniques: equilibrium dialysis, ultracentrifugation, headspace analysis (for volatiles) or solid
phase (micro-) extraction followed by a classical analysis such as HLPC UV or MS. The purpose of
this technique is the prediction of the in vivo tissue blood partitioning and the prediction of an in
vivo volume of distribution (Gargas et al., 1989; Artola-Garicano et al., 2000).
In silico approaches. A critical review on QSAR estimation of blood-tissue partitioning can be
found in Payne and Kenny (2002) who concluded that no single method was able to work in all
cases, and that the correlation to apply depended on the species, the tissue and the chemicals
lipophilicity. However, in a series of papers Ballard and co-workers (2000, 2003a,b) have carried
out a comparison between in vitro and in vivo tissue distribution and have shown that the
methodology was viable and comparable results were obtained.
Recently, different methodologies have been developed, starting from QSAR, correlations with
physicochemical properties, up to mechanistic approaches. Using a mixed approach, Poulin and
Theil (2002, 2009) have developed a method to predict tissue partition coefficients based on in vitro
data on drug lipophilicity and plasma protein binding as input parameters in a mechanism-based
approach. Around 80% of the drugs tested (63 and 123) fall within a twofold error range. The main
problem for the generalization of QSAR correlations has been the poor results obtained for charged
molecules under physiological conditions and with charged phospholipids. However, in a new
mechanistic approach proposed by Schmitt (2008) applied to 59 drug compounds, a deviation less
than 3-fold was found for 73% of the 474 experimental partition coefficients. The brain was the
organ responsible for most of the errors, but errors were in the conservative side, i.e. higher
predicted dose than the measured one. Thus, from a risk assessment perspective, errors are on the
safe side.
A new surrogate method, biopartitioning micellar chromatography (BMC), that uses micellar
mobile phases of Brij35, that structurally resembles the ordered array of the membranous
hydrocarbon chains, has demonstrated to be useful in mimicking the drug partitioning process into
biological systems (Martín-Biosca et al., 2009). This method combines experimental determination
and in silico calculation and offers a new approach to obtain partition coefficients that does not
require human or animal tissues.
A.2.3. Estimation of substance permeability through Blood-Brain Barrier (BBB)
The BBB is a regulatory interface that separates the Central Nervous System (CNS) from systemic
blood circulation and may limit or impair the delivery of certain compounds, which makes the brain
different from other tissues. There are several mechanisms of transport through the BBB
(Mehdipour and Hamidi, 2009): passive diffusion, carrier-mediated transport, receptor-mediated
transport, absorptive-mediated transport and active efflux transport.
In vitro approaches. Prieto et al. (2004) have reviewed the in vitro models of the BBB and their
application to toxicology, whereas the results of a study carried out by Garber et al. (2005) to test
the in vitro-in vivo correlation of BBB permeability and several transport mechanisms, using
primary bovine and human brain endothelial cells co-cultured with astrocytes, two different
immortalized brain endothelial cells and cells not derived from the BBB (ECV/C6, MDCK and
Caco-2 cell lines) were unsatisfactory. It was argued by Lai and Kuo (2005) that the absence of
10
pericytes – a CNS cell type - was the main reason for the lack of a clear correlation, but also the
absence of close contact between endothelial cells and astrocytes (Cohen-Kashi et al., 2009) and the
absence of blood flow that occurs in vivo (Santaguida et al., 2006) were also contributing.
New in vitro BBB models have now being developed that integrate pericytes with endothelial cells
and astrocytes (Nakagawa et al., 2009) and that allow close contact between endothelial and
astrocytes cells (Cohen-Kashi et al., 2009). In both cases, preliminary results confirmed that this
new BBB models showed higher barrier integrity and better correlation with in vivo permeability
(Pe) and transendothelial electrical resistance (TEER) data. An in vitro BBB model for high
throughput screening has been developed by Culot et al. (2008) and used integrated with neuronal
cell lines (SH-SY5Y) in neurotoxic assessment of 16 compounds (Hallier-Vanuxeem et al., 2009).
Furthermore, MDR-MDCK transfected cell lines have been proposed as one of the standard models
to study BBB permeability (Wang et al., 2005, Garberg et al. 2005)
In silico approaches. Two recent literature reviews on in silico models for BBB penetration based
mainly on the prediction of log BB (log Cbrain/Cblood) has been carried out by Mehdipour and Hamidi
(2009) and Mostrag-Szlichtyng and Worth (2010). The first generation of QSAR models tried to
predict BBB permeability as a function of several parameters such as lipid solubility, hydrogen
bonding, molecular weight and surface area, using generally linear regressions. In the second
generation more advanced statistical techniques have been implemented (Mostrag-Szlichtyng and
Worth, 2010) and more descriptors have been used: size type (topological indices, molecular weight,
surface area, etc.) and polarity descriptors (partial charges, hydrogen bonding, etc.). They concluded
that several models exist which are able to provide logBB predictions around 0.35-0.45 log units.
However, the data sets used are relatively small and the majority of compounds are drugs. In
addition, most of the methods are focused on the prediction of log BB which are an imperfect
measure for BBB penetration (Martin, 2004). Furthermore, the vast majority of these approaches do
not consider the type of transport mechanism taking place. Recently, some molecular models have
been developed to consider the BBB transporters (Allen et al., 2006). However, there is still the
need of the 3D structures of these membrane proteins from human cells.
A.2.4. Estimation of substance permeability through Blood-Placenta Barrier (BPB)
The BPB serves to transport nutrients and waste, and other compounds such as hormones. However,
the placenta does not provide a true barrier protection to the fetus from exposure to compounds
present in the mother’s systemic circulation, although it might reduce the transport of certain
molecules. The transfer across the placenta can occur by four kinds of mechanisms (Myren et al,
2007): passive diffusion, carrier mediated or facilitated diffusion following a concentration
gradient, carrier mediated active transport against a gradient and pinocytosis through the formation
of a vesicle.
In vitro approaches. Experimental methods to study human transplacental exposure to toxic
compounds have been reviewed by Vähäkangas and Myllynen (2006). Primary trophoblasts are
difficult to cultivate and do not form monolayers, whereas BeWo cells (an immortalized
trophoblastic cell line) seem to be the most promising cell lines for transport studies. BeWo has
been used to study the placental transport properties by Poulsen et al. (2009) obtaining the same
ordering in terms of transfer but much lower rate of transfer when compared with ex vivo human
perfused placental experiments.
Ex vivo: human perfused placenta cotyledon. This method offers information about transplacental
transfer, placental metabolism, storage, acute toxicity and the role of transporters. In addition, it
provides an estimation of foetal exposure. The method has recently been reviewed by Pacifici
(2006) who showed that all examined antibiotics cross the human placenta including those with a
molecular weight greater than 1000 kDa. The ex vivo human perfused placenta cotyledon approach
has a minimum number of ethical problems since placentas are normally discarded and incinerated
11
after birth. The only limitations are that the cells remain viable for up to 48 h and that the system
may not reflect the placenta during the first months of foetus development (Myren et al., 2007). In
vitro models are still under development to obtain data similar to those in ex vivo data. Placental
perfusion method is already available. However, the development of a new tiered approach to
determine when this test is necessary should be performed for cosmetic ingredients.
In Silico approaches. A recent review of QSARs developed for this aspect has been carried out by
Hewitt et al. (2007). They found that molecular size, hydrogen bonding and hydrophobicity were
the most useful descriptors for passive diffusion. However, the other transport mechanisms and the
fact that metabolism can occur in placenta were not addressed. Similar results were found by
Giaginis et al. (2009) using 88 structurally diverse drugs.
A.2.5. Estimation of substance permeability through Blood-Testis Barrier (BBB)
In the testis, the BTB is a physical and physiological barrier which assures functions in hormonal
regulation and spermatogenesis (Fawcett et al 1970). BTB is a testis-specific structure composed of
side-by-side arranged tight junctions, the basal ectoplasmic specialization, the basal tubulobulbar
complexes, the desmosome-like junctions and gap junctions (Siu and Cheng 2008; Xia et al 2005).
In vitro approaches. Although there has been a lot of research activity on the exposure of testis to
xenobiotics (perhaps due to widely known work on sperm quality), in vitro test systems are still far
from being predictive. Actually, few models are presented as in vitro BTB model for toxics studies.
Indeed primary Sertoli cells culture are used to study the effects of phorbol ester or cadmium on
tightness junctions (Janecki et al 1991; 1992). Culture of Sertoli cells lines on impermeable support
has also improved the knowledge about the effects of toxicant on junctional membrane proteins of
seminiferous epithelium (Fiorini et al 2004, 2008). Another study was performed combining
bicameral chamber and 3D culture with only Sertoli cells and presented another dynamic model of
BTB for studying their integrity after bisphenol A exposure (Li et al 2009). To date, no in vitro
reprotoxicity test on the male reproduction function allowed the demonstration of the toxic effects
both on BTB and spermatogenesis. Only one type of testicular cells (Sertoli or Leydig cells) was
tested limitating the interpretation of the observed toxic effects to in vivo extrapolation.
Development of an in vitro engineering blood-testis barrier (eBTB) model appears to be the best
effective alternative for major step of reprotoxicity test in male: toxicants BTB clearing and further
adverse effect on germ cells.
Many systems have been tested as organ culture, culture or co-culture of Sertoli cells and germ cells
on impermeable support or permeable support as bicameral chamber with or without gel matrix (i.e
collagen, matrigel, etc.) (for review see Staub 2008). However these models seem adequate for
study dynamics in vitro but is not the exact reflect of in vivo BTB which is composed of a 3D
structure with three compartments (interstitial, basal and apical) delimitated by many cell junctions,
all contributing to barrier function and germ cells differentiation. A model with a 3D culture in
bicameral chamber, the apical and basal compartment has been set up with three types of testicular
cells isolated from 18-day-old rat testis (peritubular cells, Sertoli cells and germ cells) (Legendre et
al. 2010).
In silico approaches.Due to the scarcity of the data sets there are few QSARs for blood-testis
transfer (Cronin and Hewitt, 2007) and these are far from being predictive.
12
Table A2. Replacement methods for Distribution (D).
Test name
Endpoints
measured
Distribution
Bound/unbound
fraction
of
compounds in
plasma, specific
tissues
and
organs
and
permeabilities
to
cross
specialised
membranes
Estimation of Unbound
plasma protein fraction
binding
blood
Origin of test
system
(cell
line, ex vivo,
primary cells,
in silico, in vivo
In silico
Human plasma
in
Area(s)
of Status
applications;
known
limitations
Comments
Estimated time
to
enter
prevalidation
process
Data available
mainly
for
“successful”
drugs but of
general
applicability to
cosmetic
ingredients
Estimation
of
partitioning of
compounds
between blood
and plasma and
specific tissues
more developed
that estimation
of permeabilities
No
validation
necessary,
methods have to
follow
the
OECD principles
for the validation
of QSARs for
regulatory
purposes
Many
tools
currently
available.
Further
improvement
necessary with
focus on data
for
cosmetics
ingredients.
Results depend
on the validity
domain defined
for the method
Mainly
Routinely used
pharmaceuticals by
but applications pharmaceutical
reported
for industry. High
other chemical throughput
classes
systems.
Optimised; not
formally
validated, but
extensive
inhouse
evaluation
studies.
Three methods Formal
available with validation
commercial
necessary
instruments:
1) Equilibrium
dialysis;
2)
Ultrafiltration;
3)
ultracentrifugati
on
not
13
Protocols
established,
hundreds
of
compounds
tested by a large
number
of
laboratories
Estimation of Partition
Ex-vivo
blood
tissue coefficient
partitioning
between blood
and tissues
Permeability
Blood
barrier
–brain In vitro, mixed
cultures:
pericytes with
endothelial cells
and astrocytes
Method
produces same
results than invivo
Research
to
obtain
higher
barrier integrity
and
better
correlation with
in
vivo
permeability
(Pe)
and
transendothelial
electrical
resistance
A considerable
number
of
reported
applications
during PBTK
model
validation
If
ex-vivo <2013
animal tissues
not
accepted
then
>2013
since alternative
approaches are
still
under
development
Still research is
>2013
needed
to
develop a more
realistic in vitro
barrier
14
Permeability
Blood
– Ex-vivo human
placental barrier perfused
placenta
cotyledon
SOP
well
established and
used by several
laboratories.
Minimum
<2013
number
of
ethical problems
since placentas
are
discarded
after birth.
Permeability
Blood
barrier
Still research is
needed
to
develop a more
realistic in vitro
barrier
>2013
The
only
limitations are
that the cells
remain viable
for up to 48 h
and that the
system may not
reflect
the
placenta during
the first months
of
foetus
development
–testis In vitro, 3D In vitro systems
mixed
do not reflect in
cultures:peritub vivo BTB
ular,
Sertoli
and germ cells
15
A.3. Metabolism (Biotransformation)
Metabolism or biotransformation is the principal elimination route of organic chemicals;
roughly 70 to 80 % of pharmaceuticals are partially or practically completely eliminated by
metabolism (Zanger et al 2008). Due to a multitude of xenobiotic-metabolizing enzymes
possibly acting on a chemical with different metabolic pathways, the first screen should
preferably be as comprehensive as possible. Because liver is the principal site of xenobiotic
metabolism, the enzyme component in in vitro systems should preferably be liver-derived
(Coecke et al 2006; Pelkonen et al 2005; 2008) and of human origin, to avoid species
differences (see e.g. Turpeinen et al 2008). There is a generally accepted consensus that
metabolically competent human hepatocytes or hepatocyte-like cell lines are the best enzyme
source to perform the first primary screening of metabolism (Gomez-Lechon et al 2003, 2008;
Houston and Galetin 2008; Riley and Kenna 2004). The two most important end points
measured are 1)intrinsic clearance which can be extrapolated into hepatic metabolic clearance
and 2) the identification of metabolites (stable, inactive, active or reactive metabolites of
concern). At the same time it is possible to have indication about the enzymes participating in
the metabolism which allows a number of predictions about physiological and pathological
factors affecting the kinetics of a compound of interest.
Existing approaches have been summarised in Table A3.
A.3.1. Biotransformation, In silico approaches
Scientific relevance and purpose. The available systems are of three types: 1) expert systems
based on structure-metabolism rules; 2) SAR and QSAR modeling; and 3) systems based on
pharmacophore (ligand) or target protein modeling (e.g. CYP2C9 or CYP3A4 molecular
models). There are a large number of commercial software available for predicting
biotransformation, in various phases of development They provide useful information
although none of them allows reliable predictions on the different aspects that should be
covered, that is identification of possible metabolites, involved metabolising enzymes, and
quantitative kinetics. A recent trend has been to use meta-models (i.e. a combination of
models) for partial processes: discussions of various approaches can be found in recent
reviews (Testa et al 2004; de Graaf et al 2005; Kulkarni et al 2005; Crivori & Poggesi 2006;
Lewis & Ito 2008; Muster et al 2008; Mostrag-Szlichtyng and Worth 2010).
Fields of application and limitations. Both knowledge-based algorithms and molecular
modelling approaches are currently useful in giving a rapid but only qualitative overview of
the potential metabolic fate of a compound. Useful predictions are usually restricted to the
class of chemicals which has been used as a training set, but recently promising advances
have been achieved (de Groot 2006; Hansch et al 2004; Lewis et al 2008). Standardization of
different systems has mainly been in-house of biotechnological enterprises within the
pharmaceutical sector.
Extrapolation and prediction from in vitro to in vivo human biotransformation from limited in
vitro human systems with the help of in silico approaches still represents a major challenge
(Pelkonen et al 2009; EPAA 2010). It is worth of stressing that in silico models are usually
based on existing human data, so their predictions are directly applicable to humans.
Current status and future efforts needed. Although in silico approaches are developing rapidly,
they are still inadequate for the production of results which are accepted by regulators and
new approaches are needed to predict the major metabolic routes when there are a number of
potential metabolic pathways
16
Reliable and good-quality databases (not limited to pharmaceuticals) are of the utmost
importance for the development of reliable software for application to a wider assortment of
chemicals including cosmetic ingredients and they are still in great need.
A.3.2. Biotransformation: Metabolic clearance/stability
Scientific relevance and purpose. The metabolic stability test is a relatively simple, fast-toperform, but specialised analytical equipment-based MS study, to find out whether a
compound is metabolically stable or labile. It is based on the disappearance of the parent
compound over time (with the appropriate analytical technique) when incubated with a
metabolically competent tissue preparation (e.g. a human liver preparation, preferably a
homogenate added with appropriate cofactors, or human hepatocytes), The rate of parent
compound disappearance gives a measure of its metabolic stability and allows for the
calculation of intrinsic clearance and extrapolation to hepatic (metabolic) clearance (see e.g.
Grime and Riley 2006; Hallifax and Houston 2009; Pelkonen and Turpeinen 2007; Pelkonen
et al 2005, 2008; Plant 2004).
With the advent of modern MS techniques, the test is suitable also for studying both the
detailed qualitative and quantitative metabolic profiles of a compound (Anderson et al 2009;
Dalvie et al 2009; Pelkonen et al 2009; Tolonen et al 2009). The use of recombinant enzymes,
transfected cells and metabolically competent human (liver-derived) cell lines or subcellular
fractions are being very actively employed in pharmaceutical industry and academia.
Fields of applications and known limitations. The use of liver based experimental systems
should give a fairly reliable view of hepatic intrinsic clearance. However, to be able to predict
in vivo clearance, a number of assumptions concerning the substance under study must be
made, so an extrapolation model is needed (see e.g. Pelkonen and Turpeinen 2007; RostamiHodjegan and Tucker 2007; Webborn et al 2007). To cover also extrahepatic
biotransformation the above described method for metabolic stability can be combined with
the use of other tissues.
For cosmetic substances, dermal uptake is the most prominent intake pathway and
consequently methodologies for skin metabolism would be of considerable significance.
Likewise, inhalation (spray applications) is also an important uptake route and metabolism
should be taken into consideration in in vitro pulmonary tests. Some efforts would be needed
to standardize metabolic stability in skin and pulmonary tissues.
Current status and future efforts needed. Although standardization has mainly been made inhouse by pharmaceutical companies and academic groups, reports have been published (e.g.
Houston and Galetin 2003; Soars et al 2009; Webborn et al 2007). No formal validation
studies are known. However, the stability screening test should be relatively ready for
validation after the availability of common procedures and related SOPs. ECVAM has
supported a study in which 55 ECVAM/ICCVAM validation substances have been studied in
in vitro human and rat liver preparations, to survey metabolic stability and metabolite
formation (Pelkonen et al 2009) as well as analytical performance (Rousu et al 2010)
As a part of the in vitro testing strategy, the test could replace animal biotransformation
studies to a certain extent. It is worth noting that preparations from different species,
including man, used under comparable conditions, can provide valuable information on
species differences.
17
A.3.3. Biotransformation: Bioactivation assays
Scientific relevance and purpose. The formation of reactive metabolites by biotransformation
seems to be the cause of deleterious effects for a large number of compounds (Park et al 2006;
Williams 2006). Even though mechanistic details of relationships between toxicities and
reactive metabolites are still somewhat unclear, there is ample indirect, albeit circumstantial,
evidence for their associations (Baillie 2006; 2008; Tang and Lu 2010). Nevertheless, it has
been extensively demonstrated that reactive metabolites form covalent adducts with
nucleophiles on cell proteins or DNA, or initiate free radical chain reactions within cells
which then may lead to drug-induced toxicity, cell damage or other adverse reactions.
There are direct and indirect methods to test the potential formation of reactive metabolites.
Most direct assays use trapping agents (i.e. glutathione or its derivatives, semicarbazide,
methoxylamine or potassium cyanide), that are able to trap both soft and hard electrophiles:
conjugates are then analytically measured. Recently, a simple and efficient LC/TOFMS-based
screening method for reactive drug metabolites was presented, utilizing several stable isotope
labeled trapping agents (Rousu et al 2009).
The Ames test is a prime example of an indirect method for the bioactivation assay making
use of metabolically competent enzyme system (which could be human-derived, if needed)
and properly engineered bacteria to detect reactive, DNA-bound metabolites. It has been in
use since 1970s and extensively validated. Another indirect method to detect the role of
reactive intermediates in toxicity is based on employing metabolically competent cells in
culture (primary hepatocytes, genetically engineered cell lines, etc.) and non-metabolising
cells (cell lines are often used). By comparing the concentration–toxicity curves (or IC50
data) of the compound in both models, it is possible to know whether a bioactivation of the
xenobiotic is required to elicit the toxic effects. An additional possibility consists in the
addition of inhibitors of metabolising enzymes to the hepatocyte culture medium: the
reduction of toxicity indicate that it is associated to the bioactivation of test compound.
Fields of application and limitations. Subcellular screening tests are simple, robust and can be
employed in high throughput screening conditions. The most important limitation is that the
most convincing evidence about an association between reactive metabolites and toxicity
come from limited sets of compounds, which are mostly drugs and hepatotoxicants. Due to
the species differences in metabolism, there is a great need for the application of these
methods to human-based in vitro models that offer better predictions of potential hazard to
humans.
Current status and future efforts needed. A number of in vitro systems are available for
studying the production of reactive metabolites or biotransformation-mediated effects.
However, apart from the Ames test, no in vitro methods for bioactivation have been properly
validated.
A.3.4. Biotransformation: Induction assays
Scientific relevance and purpose. Since induction has a complex underlying mechanism, it is
a good indicator for high quality metabolic competent systems that can be used for long-term
purposes (Coecke et al 1999; Pelkonen et al 2008): that is why developments are ongoing to
assess CYP induction in bioreactor-based systems. Obviously, the most relevant intake routes
18
for cosmetics (dermal, inhalation) should be considered when in vitro test systems are
developed.
Fields of application and limitations. A large number of test systems ranging from nuclear
receptor binding assays to induction-competent cell lines is currently available All these test
systems are widely used in pharmaceutical industry to help early drug development and are
designed to detect induction of CYP enzymes relevant for the pharmaceutical area. This can
represent a potential limitation since for cosmetics, other CYP forms might play an additional
or more prominent role (e.g. CYP 2E1, 1A1). Thus, further progress is needed to cover this
potential gap.
Current status and future efforts needed. For two human hepatic metabolic competent test
systems ECVAM is carrying out a multi-study validation trial for cytochrome P450 induction
providing a reliable human metabolic competent standard model using the human
cryopreserved HepaRG® (CryoHepaRG®) cell line and human cryohepatocytes (Cryohep).
Two detailed standard operating procedures (SOPs) have been established for the CYP
induction test methods for both cells (Kanebratt and Andersson, 2008; Antherieu et al 2010;
Richert et al 2010; Lubberstedt et al 2010; Stephenne et al 2010).
However, again probe substrate are pharmaceuticals, and it would be useful to investigate its
use with a wider assortment of chemicals of interest, e.g. typical cosmetic ingredients with
known toxicokinetic properties.
A.3.5. Biotransformation: Inhibition assays
Scientific relevance and purpose. Due to the broad substrate specificity of metabolizing
enzymes, there is always a possibility that compounds would interfere with each other’s
biotransformation. Inhibition of biotransformation leads to higher concentrations and delayed
clearance and may cause adverse effects. At the site of entry (i.e. gi-tract, skin, lung)
inhibition of the first-pass metabolism would increase the blood concentration of the parent
compound.
Fields of application and limitations. There are currently available a large number of test
systems ranging from recombinant expressed enzymes (principally CYP and UGT enzymes,
but increasingly also other xenobiotic-metabolizing enzymes) to primary cells (hepatocytes)
and permanent cell lines (Li 2008; Farkas et al 2008). All these test systems are widely used
in pharmaceutical industry and can be judged to be validated at least for pharmaceuticals. This
can represent a potential limitation since for cosmetics, other CYP forms might be relevant. In
addition some cosmetics contain complex plant-derived mixtures and it is not elucidated to
what extent current inhibition assays would be applicable. Thus, further progress should cover
chemical and compositional peculiarities characteristic for cosmetics field.
Current status and future efforts needed. There is an extensive literature available about the
current situation (see e.g. Pelkonen et al 1998, 2008) and both FDA and EMA have published
revised draft guidelines for testing interactions, including inhibition, of drugs under
development. Because inhibition tests have been used principally for drugs and within
pharmaceutical companies, it would be useful to apply them with a wider assortment of
chemicals of interest, e.g. typical cosmetic ingredients with known toxicokinetic properties.
19
Table A3. Replacement methods for Metabolism (M).
Test name
Endpoints
measured
Biotransformation
/
in
silico
approaches
Metabolite profile,
principal
metabolites,
metabolizing
enzymes
Metabolic stability
/
metabolic
clearance
/
metabolite
formation
Disappearance of
parent compound
and/or
appearance
of
(major)
metabolites
Metabolite profile
Origin of test
system
(cell
line, ex vivo,
primary cells, in
silico, in vivo
Expert systems
QSAR
Pharmacophore
or
molecular
protein modelling
Human
(cryopreserved)
hepatocytes,
permanent
hepatocytederived cell lines,
Subcellular
organelles
Recombinant
enzymes
Area(s)
of
applications;
known
limitations
Status
Comments
Estimated time to
enter
prevalidation
process
Developed mostly
for
pharmaceuticals
(drug
development
tools)
Quantitative
predictions
not
reliable
Mainly
pharmaceuticals
but applications
reported for other
chemical classes
Used during early
drug development
for screening and
“negative”
selection
Predictive
capability heavily
dependent
on
selected parameters
and
model
compounds
No
validation
necessary,
methods have to
follow the OECD
principles for the
validation
of
QSARs for
regulatory purposes
Optimised;
not
formally
validated,
but
extensive
inhouse evaluation
studies, Protocols
adequately
established,
hundreds
of
compounds
tested; a large
number
of
laboratories
With
suitable
analytical
techniques,
covers
also
elucidation
of
primary
metabolite profile
ready
prevalidation
to
20
Metabolic
activation
Adducts
with
trapping agents
Suicide inhibition
Production
of
oxidative stress
Metabolismdependent
toxicity
As above
Mainly
pharmaceuticals
but applications
reported for other
chemical classes
Some
in-house
evaluation;
mostly
in
development
phase;
no
consensus about
pros and cons of
different methods
Correlations
between
activation
and
toxic
outcomes
not
well
established
the Ames test is
validated
Other approaches
>2013
Induction
metabolism
of
CYP
induction
(function and/or
expression
of
mRNA)
Cultured human
hepatocytes;
permanent
cell
lines
(e.g.
HepaRG)
Mainly
pharmaceuticals;
a number of other
compounds
studied;
in
principle
any
chemicals
CryoHepaRG and
cryohepatocytes
are
under
validation
at
ECVAM
Suitable also for
the
study
of
inhibitory
interactions with
probe substrates
<2013
Inhibition
metabolism
of
CYP
inhibition
(function)
Human
liver
microsomes and
other
preparations;
Recombinant
enzymes;
cultured human
hepatocytes
or
permanent
cell
lines
Mainly
pharmaceuticals;
a number of other
compounds
studied;
in
principle
any
chemicals
Optimised;
not
formally
validated,
but
extensive
inhouse evaluation
studies, Protocols
adequately
established,
hundreds
of
compounds
tested; a large
number
of
laboratories
High-troughput
screening
and
cocktail methods
established
practically ready
to prevalidation
21
A.4 Excretion
Predicting major excretion pathways of compounds is important in relation to their kinetic
behaviour and the relationship to pharmacological/toxicological effects. The kidneys and the
hepatobiliary system have the capacity to excrete either as the parent compound or as
metabolites and are important routes for elimination of xenobiotics and their metabolites.
Excretory processes seem to be the least developed area in the context of in vitro toxicokinetic
methods (see 5.6.). Consequently, some background on the current knowledge of excretory
processes is given and thereafter only a few examples of approaches are described, which may
point to definite advances for the field.
Background on renal excretion
Excretion by the kidney encompasses three different mechanisms: firstly, glomerular filtration
of the unbound fraction, secondly, secretion by transport mechanisms at the tubular site and
thirdly, tubular reabsorption. Whereas glomerular filtration and tubular secretion are
mechanisms by which the xenobiotic substance is excreted from the body, tubular
reabsorption is a process by which the substance is taken back from the intratubular fluid into
the renal blood flow and hence, into the body. The amount excreted by the urine is resulting
from all three processes. Tubular secretion of a variety of organic anionic drugs and polar
metabolites is mediated by a family of multispecific organic anion transporters (OAT genes)
that are part of the SLC22 family of solute carriers. Different OATs localize to the apical
(OAT2, OAT4, and RST/URAT) or basolateral (OAT1/NKT and OAT3) membranes of the
renal proximal tubule; the net transport of organic anions from blood to urine is believed to
require both apical and basolateral OATs. The extent of functional redundancy among OATs
remains uncertain, but closely related OAT genes are tightly linked in the genome. Hence, a
better understanding of human variation in organic anionic xenobiotic excretion may be
obtained by studying OAT genes in combination rather than individually (Xu et al. 2005) and
co-expressing them in specific cell lines. There are also transporters for cationic substances.
The process of reabsorption can be also mediated by transporters. For endogenous substances
like sugar, Tmax and Vmax have been characterised by in vivo studies in humans. Lipophilic,
not-charged substances are diffusing back from the primary urine during the transport through
the tubular system of the kidney. This process is very efficient so that for many drugs
excretion by urine is practically zero.
Even if there are examples how the involved transporters can be identified, it is difficult to use
the findings to feed into a physiological model of renal excretion which includes tubular
secretion and tubular reabsorption. A general review on transporters is given by Klaassen and
Lu (2008).
Background on biliary excretion
In humans, biliary excretion does not play an important role for most of the substances. To
undergo biliary excretion, a compound present in the portal venous or hepatic portal blood
must first pass through the sinusoidal membrane or be taken up by transporters and enter the
hepatocytes. Inside the hepatocytes the compound can be metabolised by phase 1 and phase 2
biotransformation enzymes, bind non-specifically, efflux back into the blood across the
sinusoidal membrane or to be transported across the canalicular membrane to be excreted into
the bile. As a result biliary excretion represents at least one active process involving
transporters embedded in the canicular membrane of hepatocytes. These transporters belong
to the ATP binding cassette (ABC) superfamily of transporters. The ABC-transporter
22
intercepts compounds at the level of the plasma membrane and effluxes them before they are
able to reach their intracellular target structures (Chiba et al., 2006). The major transporters
involved in the biliary excretion of xenobiotics include ABCB11 (P-glycoprotein, MDR1, Pgp), ABCC2 (multi-drug resistance associated protein 2 (MRP2)), and ABCG2 (breast cancer
resistance protein (CRP). ABCB4 (MDR3) and ABCB11 (bile salt export pump (BSEP)) are
two other ABC transporters present in the canalicular membrane, although they play a minor
role in biliary excretion. It is assumed that the molecular weight also plays a role (> 600 ) and
that the transporters specifically transport charged molecules. The recent review of Klaassen
and Aleksunes (2010) provides a general survey of hepatic transporters.
Examples of current approaches and future efforts needed.
There have been a few attempts for developing expert systems or computational approaches to
predict renal excretion from some basic molecular and physicochemical properties. Based on
the intravenous dose for 160 drugs, Manga et al. (2003) developed a decision tree model to
predict unchanged drug excreted in the urine as percentage of the i.v. dose. The data were
categorized into classes and the classes were assigned correctly in 90% of the cases using a
test set of 40 compounds. The dependence of the of properties such as the distribution
coefficient at pH 6.5, the H-bond donors, counts of OH and COOH groups, etc. shows that
ionization
has
a
prominent
role in
the
modeling
of
this
process.
Kusama and co-workers (2010) demonstrated on 141 approved drugs that only 4 physicochemical parameters (charge, molecular weight, lipophilicity, and protein unbound fraction in
plasma) are required to predict major excretion pathways. For those compounds major
clearance pathways were determined to be metabolism by CYP3A4, CYP2C9 and CYP2D6,
or renal excretion in unchanged form.
Efforts have been undertaken to use collagen-sandwich cultures of hepatocytes as an in vitro
test system. Using this system bilirubin conjugation and canalicular versus sinusoidal
disposition of bilirubin glucuronides has been studied. Biliary excretion index was estimated
by measuring disposition of bilirubin glucuronides into standard and Ca(2+), Mg(2+)-free
medium. From the results the authors concluded that sandwich-cultured primary hepatocytes
might provide a useful in vitro method to differentiate between sinusoidal and canalicular
disposition of bilirubin glucuronides (Lengyel et al., 2005).
Based on published data in silico modeling attempts are being made to evaluate the molecular
weight dependence of biliary excretion and to develop quantitative structurepharmacokineticc relationships to predict biliary excretion (Yang et al., 2009).
Due to the fact that progress in the field is very recent, no real global efforts have been
systematically undertaken to standardize the above mentioned approaches. Standardization
has mainly been in-house. Some pharmaceutical companies as well as academic groups have
published reports on their experiences. No formal validation studies are known.
An understanding of mechanisms that determine these processes is required for the prediction
of renal and biliary excretion. Physiologically based in-vitro/in-silico/in-vivo methods could
potentially be useful for predicting renal and biliary clearance. Whereas for biliary excretion
some advances have been made with in vitro models (i.e. sandwich-cultured hepatocytes), no
reports could be indentified in the literature on in vitro models of renal excretion nor were
reports available on in silico methods.
23
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