Human-Based In Vitro Systems for Predicting
In Vivo Hepatic Clearance of Drugs
Name: Ruthmila F. Paskel
Student number: 3858413
Course: MSc Thesis, MSc Toxicology and Environmental Health
Examiner: Nynke I. Kramer
Second Reviewer: Bas J. Blaauboer
Date: December 6, 2012
Table of contents
Abstract .......................................................................................................... 3
Introduction .................................................................................................... 4
In vitro systems.............................................................................................. 6
Human liver microsomes .............................................................................. 6
Hepatocytes ................................................................................................. 8
Fresh and cryopreserved primary human hepatocytes ............................. 8
Human hepatic cell lines ........................................................................... 9
Precision-cut human liver slices ................................................................. 10
Conclusion ................................................................................................... 12
References ................................................................................................... 14
2
Abstract
Unfavorable drug metabolism is the main reason for attrition of new chemical entities
and withdrawal of drugs from the market, costing pharmaceutical companies a great
deal of money and time. Therefore, obtaining information about the drug metabolism
of a new chemical entity via the hepatic clearance in the early stages of the drug
development process is crucial in guaranteeing its success. Human-based in vitro
systems, especially liver microsomes, hepatocytes and liver slices are increasingly
applied in the drug development process for this purpose. The aim is to determine
which of these commonly used systems is the most appropriate for predicting hepatic
clearance in humans. Although there is no “perfect” in vitro system, primary
hepatocytes and the HepaRG cell line are the most representative systems of the
liver at present. However, it has to be kept in mind that besides their potential to
accurately predict in vivo clearance other influencing factors that may interfere with
their ability to do so also have to be taken into consideration when deciding which is
the most appropriate system.
3
Introduction
The development of a new chemical entity (NCE) is a very expensive and
time-consuming process. It can cost pharmaceutical companies between 800 million
to 900 million USD and take well over 10 years before the NCE is put on the market,
if it ever reaches the market at all 4, 9. It is, therefore, of utmost importance to obtain
enough information about the pharmacokinetic properties of the NCE early in the
development process. Since unfavorable drug metabolism is one of the main
reasons for attrition of a NCE during the drug development process and the
withdrawal of a drug from the market, sufficient information about drug metabolism
early in the drug development process is crucial in guaranteeing the success of this
process 34.
Although there are several organs contributing to metabolism, the liver is the major
organ in biotransformation of drugs in mammals 52. Biotransformation or hepatic
metabolism is an enzymatic process in which a drug is chemically modified to
facilitate its elimination via the urine or feces. It can be divided into two phases,
namely phase I and phase II. During the former phase drugs become slightly watersoluble by exposing or incorporating functional groups mainly via the oxidation
pathway. The key enzymes involved in this pathway include enzymes belonging to
the cytochrome P450 superfamily, also known as CYPs. CYPs can be found
throughout the body, but is predominantly present in the liver. Moreover, the CYPs
superfamily can be divided into several subfamilies of which the CYP1A, CYP2C,
CYP2D, and CYP3A are responsible for the metabolism of nearly all drugs 4. At
present, CYP3A alone is responsible for an impressive 50% metabolism of all drugs
on the market 38. After phase I, phase II largely increases the water-solubility of drugs
by conjugating polar groups to the drug.
An important aspect of a NCE is its metabolic stability. The metabolic stability gives
information about the susceptibility of a NCE to metabolism in the body, often by the
liver 9. In other words, the higher the metabolic stability, the slower the chemical
compound is metabolized whereas the lower the metabolic stability, the more rapidly
it is metabolized. In practice it is difficult to directly measure the stability of a NCE,
therefore, this aspect is often measured as the kinetic parameter, clearance 9.
Clearance describes the depletion of an unchanged compound from a given volume
of plasma or blood per unit of time 29. For example, if clearance is 100mL/minutes,
100mL of plasma or blood is cleared of the compound each minute. Thus, given the
relation between metabolic stability and clearance, this is a suitable parameter for
obtaining information about the drug metabolism of a NCE. Most importantly,
clearance is very useful when considering the therapeutic dose range and regimen of
a NCE in the later stages of the drug development process 13. Various organs in the
body can contribute to clearance and the sum of all these clearances is referred to as
the total body clearance. Furthermore, clearance by the liver is referred to as the
hepatic clearance (CLh). Due to the fact that the liver is a major player in
biotransformation of drugs, as previously stated, the CLh is often used.
Traditionally, laboratory animals have been used in metabolic studies as models for
humans. The fact that these models are intact systems containing the complex
interactions among different compartments like in humans makes them attractive to
use in predicting the clearance of NCEs in humans. However, similarities in
physiology among species do not always imply similarities in drug metabolism.
4
For example, the human liver contains high levels of CYP3A in contrast to the liver of
laboratory animals, like rats 33. Moreover, large variations in the expression levels
and metabolic activity of CYP2D and CYP3A have been reported within the human
population 36, 38, 48. If even humans differ in their levels and activity of important
metabolic enzymes, animal models cannot accurately reflect the full span of
clearance values in humans.
Thus, human-derived materials would be much more suitable as candidates for
predicting drug clearance in humans. Not surprisingly, in vitro systems with human
tissue have been increasingly used in drug development in the past decade. With
the increasing application, different classes of in vitro systems have been developed.
The aim of this thesis is to determine what the most appropriate human-based in
vitro system is for predicting hepatic clearance of drugs in humans. The thesis
discusses how in vitro systems can be used to predict CLh in humans. It considers
the three most commonly used human-based in vitro systems for this purpose and
weighs their advantages and disadvantages.
5
In vitro systems
In vitro systems can be defined as subcellular fractions, cells or tissues cultured for
experimental purposes under artificial conditions mimicking the physiological milieu
in organisms. Some reasons why these systems are increasingly used in early
prediction of drug clearance are that they are relatively easy to maintain and their
use may be automated in a high throughput setting. Most importantly, in vitro
systems can be obtained from different human-derived organs or tissues what makes
them so suitable for predicting clearance in humans.
The prediction of CLh from in vitro clearance data is a stepwise process 32. First, the
in vitro system is incubated with the test substrate and the depletion of this substrate
in the in vitro assay medium is chromatographically measured at different time points.
From this parameter the rate constant for the depletion of unchanged compound, k,
can be calculated. The rate constant k can be then used to calculate the in vitro
intrinsic clearance (CLint). CLint is the maximum ability of the liver to clear a drug in
the absence of other physiological determinants such as hepatic blood flow and nonspecific binding of drugs to plasma proteins within the blood matrix 9. Nevertheless,
non-specific drug binding within the in vitro matrix can also be a crucial determinant
for accurately predicting the CLh. This matter will be further discussed in the next
subsections. The next step is scaling the calculated in vitro CLint to the whole liver.
Although there are several scaling factors (SF), the biological SF is the easiest and
most often used. For example, in the case of hepatocytes as the in vitro system, first,
the average number of hepatocytes per gram of liver is considered and then the
average grams of liver per kilogram of body weight. Now that the in vivo CLint has
been calculated the final step is the in vitro-in vivo extrapolation (IVIVE) by liver
models. The most used liver model is the well-stirred model, which states that the
free fraction of a compound is constant throughout the hepatic compartment and is
equal to the free fraction within the plasma or blood matrix 32. In other words, only
that fraction of the compound that has not bind to plasma proteins is able to reach
the enzymes and be cleared. Thus, by incorporating the aforementioned
physiological determinants via the well-stirred model, the CLh in humans can be
predicted from in vitro data. The three commonly used human-based in vitro systems
for this purpose are liver microsomes, hepatocytes and precision-cut liver slices.
Each system will be further discussed in the following subsections.
Human liver microsomes
Human liver microsomes (HLM) are vesicles of endoplasmic reticulum (ER) prepared
by centrifugation of liver homogenates from either fresh or frozen human liver tissue
30
. Coming from the ER, these HLM contain most of the CYP enzymes, which, as
stated earlier, are the most important metabolizing enzymes for most drugs.
Nevertheless, it has been reported that microsome preparation from frozen liver
tissue causes a decrease in the CYP catalytic activity and content 47, 55. Therefore,
microsome preparation from fresh human liver tissue is preferred. In contrast to
some other in vitro systems, microsomes can be stored for a long period of time at 80C without any significant loss of neither CYP catalytic activity nor its content 62.
Moreover, Pearce et al. showed that freezing at -80C followed by thawing at room
temperature for up to 10 times did not cause significant loss of the CYP activity in
6
HLM 47. Due to this important aspect, HLM are commercially available thereby
circumventing the limited availability that some in vitro systems face. Although high
preparation-to-preparation variation in the CYP activity of HLM from different donors
has been reported, pooling the HLM provide an easy and successful solution to this
problem 51. In this way the pooled microsomal preparation reflects an average man
and the predicted CLh may be representative for a given population. In addition to
the CYP enzymes, HLM also contain UGTs, which are one of the phase II enzymes
involved in conjugation of drugs. Microsomes are not intact systems and, therefore,
they do not contain cofactors that are essential for metabolizing enzymes. By either
supplementing the CYPs or the UGTs with cofactors, each metabolism pathway can
be thoroughly studied separately 34. This makes liver microsomes among the most
popular in vitro systems used in pharmacological studies.
On the other hand, this system is only a simplistic model for a far more complex
situation. During preparation of the HLM the outer plasma membrane is removed and
along with it cytosolic phase II enzymes. Not surprisingly, these systems inaccurately
predict the in vivo CLint of drugs predominantly metabolized by phase II enzymes 2, 55.
Another crucial component that is being removed along with the plasma membrane
are transporters. Nowadays, the importance of transport-mediated uptake as a
determinant of the plasma clearance of drugs in humans is being more and more
acknowledged 28, 54. For slow diffusing drugs, in particular, this can be a rate-limiting
step in drug clearance 7. In addition, some drugs can influence their own clearance or
that of other co-administered drugs by accelerating the rate at which they are
metabolized by CYPs. Many CYP-inducing drugs exert their effects at a
transcriptional level and because HLM do not contain cell nuclei, this is not taken into
consideration when using these systems for predicting in vivo clearance 55. One
other major limitation of this system is its limited incubation time. After only 2 hours of
incubation the enzyme activities start decreasing making liver microsomes an
inappropriate model for slow metabolizing drugs 9. Most importantly, it has been
shown that the incubation matrix can have significant influence on the catalytic
activity of CYP3A4 in microsomal preparations 24.
In the literature different results regarding the ability of HLM to predict in vivo
clearance are reported. One of the main causes of these differences is the nonspecific drug binding to the phospholipid bilayer membrane of HLM 45. Austin et al.
showed using rat liver microsomes that the extent of non-specific microsomal binding
of a given compound correlates with its lipophilicity 5. Most importantly, they showed
that it correlates with the physicochemical properties of a given lipophilic compound.
McLure et al. further support these findings by showing that acidic and neutral drugs
do not bind appreciably to the microsomal membrane, but basic drugs bind to a
greater extent 41. The reason behind this is thought to be due to favorable
electrostatic interactions between the positively charged basic compounds and the
negatively charged phosphate groups on the phospholipid bilayer 5. Due to this nonspecific binding the bound fraction of a given drug is no longer available for
conversion by metabolic enzymes resulting in potential underestimation of the in vivo
clearance of basic lipophilic drugs. It is, therefore, of importance to also take the nonspecific binding into consideration when predicting in vivo clearance for such drugs.
On the other hand, Obach et al. showed that despite the unappreciable binding of
acidic compounds the in vivo clearance of these drugs were accurately predicted
when the non-specific binding was also taken into consideration 43, 44. Suggesting
that this factor is an important determinant for accurately predicting clearance in
humans from microsomal data.
7
Hepatocytes
Fresh and cryopreserved primary human hepatocytes
Fresh primary human hepatocytes (pHH) are liver cells isolated from liver tissue of
humans, which are directly cultured afterwards. Consequently, their greatest
advantage as in vitro systems for the prediction of in vivo drug clearance in humans
is their intact plasma membrane. In contrast to microsomes, primary hepatocytes
have the entire array of metabolizing enzymes of phase I and phase II, cofactors
essential for these enzymes, and transporters that are responsible for drug uptake.
Altogether, these components result in a representative scenario of the in vivo
situation making pHH a “gold standard” for drug clearance studies.
Unfortunately, fresh pHH in culture are subject to dedifferentiation. This process is
triggered by the isolation method, which destroys tight and gap junctions between the
cells 15. Under such trauma the quiescent primary hepatocytes enter the cell cycle
and start proliferating what is also seen after partial removal of the liver in vivo 15. By
starting proliferating the hepatocytes are no longer differentiated cells. Therefore,
transcription factors involved in the expression of CYPs are altered resulting in
decreased CYP mRNA levels and increased protein degradation 15, 50. Consequently,
CYP catalytic activity and content is rapidly reduced in cultured pHH. However, it has
been reported that the mRNA levels of some CYP enzymes is recovered to some
extent after 72-96 hours of culture 18, 20. Moreover, the loss in CYP content has
shown to be CYP specific 10. In addition to the dedifferentiation issue of pHH in
culture, this in vitro system faces yet another issue, namely its short life span. They
can only be kept for a maximum of 1 week in conventional monolayer cultures 10.
Culturing of pHH sandwiched between layers of collagen, for example, can prolong
their life span and to some extent preserve their catalytic activity 12. Drug clearance
studies using sandwiched rat hepatocytes showed that these systems successfully
predicted in vivo CLh for slow and fast metabolizing compounds 56. High preparationto-preparation variation in CYP activity is also found in pHH from different donors 17.
This interindividual variation is seen both in the total CYP activity as well as individual
CYP activity. As with HLM, pooling pHH from different donors is an easy and
successful solution to overcome interindividual variation and obtain a relatively
average human biotransformation profile. The only problem is that their limited
availability makes it difficult to pool pHH from sufficient donors. Healthy fresh liver
samples are often obtained from transplantation and surgical waste tissue 18. The
limited availability of good quality samples continues to hinder the widespread use of
pHH as in vitro systems for drug clearance.
One way of dealing with the scarce availability of pHH is by optimizing preservation
methods for these in vitro systems. Indeed, cryopreservation has led to the
successful storage of pHH. By storing living material at the temperature of liquid
nitrogen (-196C), all biological processes come to a standstill 12. With the help of this
technique convenient use of isolated hepatocytes is possible as both phase I and
phase II drug metabolizing activities are retained in cryopreserved pHH 37.
Furthermore, transport activity and viability are also retained at similar levels as seen
in fresh pHH 1, 11. Due to cryopreservation, pHH can be pooled thereby circumventing
donor-to-donor variation, which can have major impact on the prediction of in vivo
drug clearance. However, it was shown that the metabolic activity in cryopreserved
pHH in culture decreased in the same way as in fresh hepatocytes in culture 37.
Moreover, it has been reported that after thawing their ability to attach onto the
8
culture plate was impaired 35. Although cryopreservation made it possible to store
fresh primary hepatocytes for convenient use and circumventing limited availability,
the viability of these in vitro systems is still low.
Several drug clearance studies showed that cryopreserved hepatocytes accurately
predict in vivo clearance 8, 31, 40. On the other hand, Naritomi et al. showed that the
predicted in vivo CLint of these systems for drugs with a wide range of in vivo hepatic
clearance differed between 12.4 and 199-fold from the observed CLint 42.
Furthermore, it was stated that underestimation of in vivo clearance by hepatocytes
is dependent on the clearance rate of a compound 63. Indeed, Foster et al. showed
that hepatocytes have the tendency to underestimate in vivo clearance of high
clearance drugs 16. Hallifax et al. further support this by showing that underestimation
by hepatocytes increased with increasing CLint 22. Both research groups suggest that
this tendency may be caused by limited factors such as cofactor exhaustion or cell
permeability of drugs, especially of slow diffusing compounds. As with HLM, lipophilic
compounds can also bind non-specifically to the plasma membrane of hepatocytes.
Moreover, Austin et al. showed that the extent of binding to hepatocytes also
correlates with the physicochemical properties of a given lipophilic compound 6.
Therefore, non-specific binding can affect the ability of this in vitro system to predict
in vivo clearance if not taken into account.
Human hepatic cell lines
As pHH, human hepatic cell lines are liver-derived cells. However, they are not
isolated from “normal” liver tissue, but often from primary tumors of the liver
parenchyma 10. In other words, human hepatic cell lines are hepatoma cells.
Therefore, these cells have long life spans and proliferate almost unlimitedly. Due to
this, human hepatic cell lines are readily available. Furthermore, they are easily
standardized among laboratories resulting in higher reproducibility for experiments
and they have a relatively stable gene expression in culture over time 10, 14. Not
surprisingly, human hepatic cell lines are sometimes considered as alternatives for
pHH in drug clearance studies. There are various types of hepatic cell lines. However,
HepG2 cell line is the most used and well-characterized one. In this subsection
HepG2 is only used as an example to illustrate typical limitations of these in vitro
systems in general and will not be used to discuss their ability to predict in vivo
clearance. The latter will be discussed for another more advanced hepatic cell line
below. When compared to pHH, HepG2 show similar expression of most phase II
enzymes, but they lack or poorly express some phase I enzymes 60, 61. Consequently,
the catalytic activities of these enzymes are also low. Westerink et al. showed that
mRNA levels of most CYPs are between 100 and 1000-fold lower than those of
primary hepatocytes 59. Moreover, it was reviewed that key transcription factors are
poorly expressed in these cell lines thereby causing the low mRNA levels and
catalytic activities of most of the CYPs 18. In addition, Guo et al. stated that cell
culture environments such as the oxygen concentration in the medium matrix or its
composition can alter the expression of drug metabolizing enzyme in HepG2 21.
Incubation with low oxygen concentrations resulted in down-regulation of drugmetabolizing genes and using different types of medium up-regulated the expression
levels of some CYPs. Furthermore, it was reported that CYP-induction is very poor in
these cell lines 14.
The newly established human hepatic cell line, HepaRG, is not subject to these
typical limitations of hepatic cell lines. This cell line was derived from a female patient
9
suffering from hepatocarcinoma and hepatitis C infection 19. A whole genome gene
expression profile comparison between HepaRG and HepG2 revealed that the
former transcribe genes at similar levels to in vivo 23. Moreover, transcription factors
in these systems are stable over a period of six weeks 27. Other studies showed that
expressed mRNA levels of CYPs and phase II enzymes and their activity in cultured
HepaRG are similar to those in pHH 20, 25, 63. Most importantly, the catalytic activity of
these enzymes in culture also remains stable for a maximum of 6 weeks and can be
induced at comparable levels to pHH 20, 57. Besides expressing metabolic enzymes
they also express other specific markers of differentiated hepatocytes like drug
transporters 14. However, as with HepG2, liver specific functions of HepaRG are
altered by the cell culture environment 25. HepaRG are progenitor-like cells that have
the ability to from hepatocyte-like colonies in culture. When cultured at low density
and after reaching confluence, some of the undifferentiated proliferating cells
differentiate into hepatocyte-like cells while others differentiate into biliary epitheliallike cells 3, 14, 19, 25. Altogether, these characteristics make this cell line a powerful tool
to study drug clearance in humans. Nevertheless, one should keep in mind that with
these similarities with pHH also come similar limitations. One such limitation is the
aforementioned clearance-dependent underestimation of in vivo clearance. Zanelli et
al. showed that extrapolation from in vitro CLint of HepaRG caused similar
underestimation of the in vivo situation for high clearance drugs as pHH and
suggested that this could worsen with increasing clearance 63. Moreover, their ability
to predict in vivo clearance is also influenced by non-specific binding of lipophilic
drugs.
Precision-cut human liver slices
The introduction of slicer devices enabled the cutting of thin liver slices with precise
thickness ranging from 100 to 250 µm 12. Due to such small thickness considerable
amounts of slices can be prepared from only a small piece of liver tissue. In
comparison to the more disruptive isolation technique of hepatocytes, the preparation
technique for liver slices maintains most of the architectural integrity of the liver cells
thereby also maintaining the intercellular communication. In contrast to isolated
primary hepatocytes, precision-cut liver slices also contain supporting cells such as
Kupffer cells 30. This multicellular aspect is very important in maintaining the cell
viability and activity. As with primary hepatocytes, these liver slices still have
transporters after slicing 12. Most importantly, they contain active phase I and phase II
enzymes 53. The complexity of these in vitro systems reflects that of the liver. Not
surprisingly, they serve as mini-organ models in drug clearance studies.
Unfortunately, there is a rapid loss of CYP expression and activity in culture 30, 49.
This may be the cause of a loss in inductive stimuli in vitro compared to in vivo 12.
Consequently, CYP mRNA levels are decreased what results in the loss of their
expression and activity. The rate at which these cell functions decline was shown to
be CYP-specific 49. This differential loss appears to be the result of an imbalance
between production and degradation of some CYP proteins 12. Further proof for the
possible cause of loss of CYP expression and activity in cultured liver slices comes
from the fact that phase II enzymes are relatively stable in culture as these are less
dependent on stimuli in vivo 12, 58. However, there also exists evidence that this loss
might be due to dedifferentiation of liver cells in the slice edges presumably as a
result of the cutting procedure 12. As with hepatocytes, precision-cut liver slices have
also very short life span. If incubated with specialized incubation methods such as
the dynamic organ culture (DOC) system, where slices are intermittently exposed to
10
medium and oxygen, these in vitro systems can be viable for up to 5 days 12, 46.
However, the viability is still short. Another limitation is the scarcity of good healthy
liver tissue for the preparation of the slices. What makes this even worse is the fact
that there are no adequate protocols for preservation at present. Although Martignoni
et al. successfully cryopreserved human liver slices, their viability was decreased 39.
Not surprisingly, these in vitro systems are not commercially available yet. Several
papers stated that liver slices poorly predict in vivo clearance, especially that of high
clearance drugs 2, 10, 26, 55. By the rapid uptake of these drugs by the outer cell layer,
the diffusion to the inner cell layer is limited 12. Consequently, only a fraction of the
slice is intrinsically metabolically active thereby poorly predicting in vivo clearance.
De Graaf et al. stated that reducing the thickness of the slices helps solve this
problem 12. Despite of this many researches are still skeptical of the ability of this
system to predict in vivo clearance.
11
Conclusion
The drug development process costs pharmaceutical companies a great deal of
money and time. With so many drugs failing to reach or stay on the market because
of unfavorable drug metabolism, sufficient knowledge of the latter in an early stage of
drug development is of utmost importance. In practice this is often achieved by
determining the CLh of a NCE. This information gives insight into the susceptibility of
a NCE to metabolism by the liver thereby providing the opportunity to disapprove
NCEs with unfavorable hepatic metabolism in the earliest stages of drug
development. On the other hand, clearance is also useful in determining the
therapeutic dose range and regimen of a NCE in later stages. Laboratory animals
have been often used as models for humans in drug clearance studies. However, at
present species differences in drug metabolism is a well-known phenomenon. Most
importantly, even among humans some important metabolic enzymes differ. It is for
these reasons that human-derived materials are more preferred for predicting in vivo
drug clearance. Therefore, the aim of this thesis was to determine what the most
appropriate human-based in vitro system is for predicting hepatic clearance of drugs
in humans. In order to do so, the advantages and disadvantage of the three most
commonly used in vitro systems, namely HLM, human hepatocytes and precision-cut
human liver slices were discussed.
At present there is no “perfect” in vitro system to predict CLh in humans. Each
system has its own set of advantages and limitations. Therefore, the usefulness of
each in vitro system highly depends on the purpose of the study. HLM are plain
simple subcellular fractions containing only some CYPs enzymes and UGTs, but
they lack other determinants such as transporters, cytosolic phase II enzymes and
the ability to be induced. Altogether, these aspects make it impossible for HLM to be
representative models. While HLM may not be an appropriate in vitro system for
predicting CLh in humans, this system can be very useful for studying various and
separate metabolic pathways in drug metabolism studies. On the other hand,
precision-cut human liver slices are multicellular intact systems maintaining a
considerable architectural structure and intercellular communication. This complexity
gives them a major advantage in serving as mini-organ models. To date, there are no
adequate preservation techniques that allow their commercial availability. Most
importantly, the poor penetration of compounds into the inner cell layers poses a
threat to the accurate prediction of in vivo clearance from these systems. However, if
preservation techniques and slice thickness were to be optimized, this in vitro system
could be a powerful tool in drug clearance studies. As stated earlier, the liver is not
the only metabolically active organ. Thus, slices prepared from various metabolically
active extra-hepatic tissues allow direct inter-organ comparison of metabolism of a
NCE 12. To date, pHH and HepaRG are, therefore, the most promising in vitro
systems for predicting in vivo clearance. As intact systems possessing an intact
plasma membrane, the most important metabolic enzymes of phase I and phase II,
transporters, cofactors and the ability to be induced, they give a very well reflection of
the in vivo situation. Even though rapid loss over time of CYP activity and content in
cultured pHH due to dedifferentiation have been shown, sandwich culture between
collagen layers maintains to some extent the CYP activity. Furthermore, due to the
successful cryopreservation technique of fresh pHH, these in vitro systems are
commercially available and pooling is possible. In contrast to pHH, HepaRG, has
longer life span and a stable gene expression for up to 6 weeks in culture.
12
Progenitor-like cells like HepaRG can form hepatocyte-like colonies in culture
thereby reflecting the multicellularity of the liver.
Not only the capability of the in vitro systems have to be taken into account when
deciding which is the most appropriate system for predicting in vivo clearance, but
also influencing factors such as the culture matrix, culture method or preservation
techniques that may interfere with their performance. Most importantly, it also highly
depends on the class of compound to be studied and the prediction methodology
used to predict in vivo clearance from the in vitro CLint data obtained from these
systems. In the case of hepatocytes, more extensive researches have to be
conducted on the cause of the tendency this system has to underestimate in vivo
clearance of high clearance dugs. Finding the cause is crucial in choosing the most
appropriate extrapolation method for accurately predicting in vivo clearance for these
classes of drugs. Since the extent of non-specific binding is correlated with the
physicochemical properties of a given lipophilic drugs, it would be perhaps better to
use different models to correct for the unbound fraction for acidic and neutral
compounds than for basic compounds in the in vitro assay medium. Austin et al.
proposed using a model for acidic and neutral compound that only takes their
lipophilicity into consideration, as these compounds do not bind to the bilayer
membrane to a great extent 5, 6. For basic compounds he proposed a model that
takes the ionized fraction into consideration, as this is the cause of non-specific
binding to the phospholipid groups in the plasma membrane 5, 6. Finally, there is a
need for studies that only investigate the physicochemical properties of a group of
only a given class of compound in relation to the power of the in vitro systems
discussed in this thesis to accurately predict in vivo clearance. By doing so, it would
be much more easier to compare the different studies for a given class of compound
and to choose the most appropriate in vitro system.
13
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