Download Microdose: The New Drug Development Approach Tania Perestrelo

Microdose:
The New Drug Development Approach
Tania Perestrelo
Msc Toxicology & environmental health
University of Utrecht
Supervisor: Wouter Vaes & Babs Fabriek (TNO)
2nd Reviewer: Bas Blauwboer (IRAS)
1
Table of content
Summary
pg 3
Introduction
pg 4
 Traditional drug development process
The microdosing concept

AMS

PET
Predictability of pharmacokinetic
pg 8
pg 11
 CREAM
 EUMAPP
 PBPK modeling technique
Pediatric drug development
pg 19
 Traditional dosage correction
Differences in children vs adults
pg 21
 Absorption
 Distribution
 Metabolism
 Elimination
Dosage & effects of drugs in children
pg 27
 Paracetamol
Conclusion
pg 28
Discussion
pg 29
Reference list
pg 31
2
Summary
The development process of new drugs is very complex, expensive and time
consuming process. The development process consists of several steps, including
animal studies and human clinical trials. The process relies in general on the
understanding of the metabolism pathways and the pharmacokinetics of a drug.
During the development process approximately 40% of drug candidates that enters
the clinical trials fail to pass through the first human studies (phase 1). Reason for
most of the failures is due to inappropriate toxicological and/or PK properties,
therefore predetermining the PK value in humans before the drug is tested in the first
human clinical trials will lead to a reduced amount of drug failures. Minimizing the
amount of failures will also result in a reduction of the drugs developmental cost, from
which approximately 75% was based on drug failures in the early stages of the
process. Microdosing is a new promising approach that may help reduce both the
amount of animal studies needed and the drug development costs. The approach
consists of determining pharmacokinetic and pharmacodynamic properties of the
candidate drugs in humans in the early phase of the developmental process. In the
study a very small dose of the drug defined as less than 100 µg will be administered
to human subjects, following the collection of samples in different time intervals
and/or the real-time scan of the patients to observe how the compound is distributed
in the body. Drug candidates with better PK properties will be selected for further
study, whereas the other candidates will be banned. For the practice of microdose
study there are ultrasensitive analytical methods needed. The accelerator mass
spectrometry (AMS) and the positron emission tomography (PET) are needed to
determine the pharmacokinetics and pharmacodynamics of the candidate drugs in
humans. Projects such as the CREAM and EUMAPP confirm the predictability of the
microdose to determine PK values related to the therapeutic dose. Pediatric drug
development process is very complicated, because children are less likely to
participate in clinical trials due to ethical issues. Traditionally the dosage of drugs
administered to children is obtained from correction of the adult therapeutic dose with
bodyweight and body surface area. This indicates that there is the possibility that
children could be underdosed or overdosed in relation with the related effect exerted
by the drug. A proper dosage of drugs could be obtained by applying the microdose
study in children.
3
Introduction
The introduction of new drugs to the market compromises a very long, complex and
expensive development process 1. The baseline for the development process of drugs
consists of understanding the mode of action, toxicological effect, metabolism and
pharmacokinetics of a drug. Traditionally, all data needed to determine among others the
toxic effect is obtained from experiments performed on in-vivo, in-vitro and in-silico
models. For example the toxicological data such as the disposition of the drug and also the
effect of the drug at target organs is obtained from a well-conducted study, which is then
analyzed properly before the drug can further be tested on humans in a clinical trial.
Understanding the pharmacokinetics (PK) and pharmacodynamics (PD) of a drug is also of
great importance for the development process of drugs, for it can predict the possibility of
the drug to succeed in performing the desired mode of action. The PK and PD properties
determine the absorption, distribution, metabolism and excretion (ADME) characteristics of
a compound 1. The amount of drug available to perform a mode of action can be
determined or evaluated from the ADME characteristics of a drug. Therefore drugs with
better PK and PD properties are more likely to succeed in the development process and will
eventually be further tested in the clinical trials, whereas for the others drugs the
development process will end.
Besides the PK and PD properties also toxicological information such as the no-observed
adverse level (NOAEL), effect dose (EC), toxic dose (TD) and lethal dose (LD) is obtained
from the performed experiments/ These data are then used to determine the therapeutic
index and margin of safety of the drug. The therapeutic index (TI) is determined by the
ratio of the required dose to produce a toxic effect and the dose needed to obtain the
desired therapeutic response. The most commonly used dose for the index of effects is the
median dose, independent of its beneficial or toxic effects. The median dose is referred as
the dose required to induce an effect in 50% of the population or the dose that induce
50% of the maximum response. Therefore the TI is calculated by the following equation
TI= LD50 / ED 50. The median dose however does not consider the slopes of the doseresponse curve for the therapeutic and toxic effects, which indicate how fast a response
increases as result of increased dose. This questions the safety aspect of the drug in an
increased dosage. A way of resolving the problem is by determining the margin of safety
for the drug. The margin of safety is then calculated by dividing the dose of the drug
needed to obtain an undesired effect (LD1) by the dose needed for the desired effect
(ED99). Hereby LD1 is referred to the dose that produces a lethal toxic effect in 1% of the
population and ED99 is referred to the dose required to induce an effect in 99% of the
population 4.
The different data obtained from the experimental settings is then extrapolated for
prediction of the drug in the human population. Extrapolation consists of dividing the data
by uncertainty factors, which takes several variations into account such as the intra- and
interspecies variation. In other words the potential differences between the species and
within the (human) species itself is taken into consideration. Extrapolation occurs by the
mathematical modeling process called allometric scaling, with whom the values for the
human population are predicted.
After the extrapolation step the candidate drugs are tested in a clinical setting on humans.
The clinical trials can be divided in 3 phases: 1) phase I, 2) phase II and 3) phase III.
In the human clinical phase I researchers will test the drug on a small group of people (2080) to evaluate the safety of using the drug and also to determine a safe dosage range. In
this development phase also the possible side effects of the drug can be observed. If the
drug is considered safe it is further tested in the phase II clinical trial to evaluate its safety
and also determine its effectiveness in a larger group of people (100-300). After passing
these trials the drug is then tested on a larger group of people (1000-3000) in order to
confirm the effectiveness of the drug. In these phase the drug effect will be also compared
to the effects observed by other commonly used treatments. The possible side effects of
the drug will also be monitored. In general all data regarding the safety use of the drug
will be collected. After passing the third human clinical phase study the drug will be first
submitted for registration and after approval will be introduced in the market and become
4
available for the patients. Hereafter the drug can still be post-markedly tested in studies to
give additional information regarding its risk, benefit and optimal use.
Approximately 40% of drug candidates that enters the clinical trials during the
development process fail to pass through the first human studies (phase 1) 5. The reason
for most of these failures is due to toxicity problems and most likely inappropriate PK and
metabolism properties. It is therefore of great concern to estimate whether the
metabolism pathway and PK of a drug can differ between those in humans and the ones
predicted in the model studies, despite the well established extrapolation process which
includes the correction for several uncertainty factor. This raises the question: How well
can the selected model predict the human situation?
Reports can be found in the literature
database illustrating how the bioavailability
of various drugs can differ between species
(Table 1). This emphasizes the difficulty of
selecting the appropriate candidate that
should be used as model for human
predictions 1, 2. The study compared the
data of 25 newly developed drug candidates
obtained from animal studies to establish
the differences in oral bioavailability
between species. These drug candidates
were ready to enter the human phase 1
clinical trial. The bioavailability of the
compounds resulted to be highest in dogs
for 32% of compounds, whereas 16% of the
compounds had high bioavailability in rats
and none in monkeys. This indicates that
there is probably no better model for the
prediction of pharmacokinetics of a drug in
the human population than the human itself.
Figure 1 also illustrates that there is no
correlation between the bioavailability in humans and animals. The lack of correlation is
most probably due to differences in physiology, which can be described by differences in
absorption and metabolism, etc 6. Therefore as already mentioned an accurate prediction
of pharmacokinetics in human is not possible from animal data.
5
Predetermining the PK value in humans before the drug is tested in the first human clinical
trials will minimize the amount of drug failures during the development process, which also
results in a reduction of the drugs developmental cost. Approximately 75% of the
development costs are based on drug failures in the early stages of the process 1.
Therefore by reducing the costs more drugs can be tested for the same or lower costs,
which suggests that a variety of new medicines can be placed in the market for the
patients and maybe also at a lower price.
Here for a new experimental approach called microdosing has been introduced aiming to
solve the problems of drug failures. The microdosing approach consist of obtaining the
information needed to enhance the probability of the drug development success before
entering the clinical phase 1 human study (Figure 2) 1 . With this approach the precise
value of the drug in humans is determined by obtaining the human PK values in less time
than is needed while conducting a phase 1 human study. In other words it can establish
whether the development of a drug should continue or not, according to the obtained PK
values for the specific drug 1-3.
Traditionally potential drug candidates with high affinity towards a target protein are
identified in the early stages of the drug development process. The pharmacological
activity of the drug candidates is then confirmed by in-vitro and animal studies, which will
finally be tested in a clinical trial starting with the human phase 1 study. With the new
approach however the candidate drugs are tested in a microdose study (also known as the
human phase 0 study) prior to human phase 1 study of the clinical trial. Small doses of the
drugs candidates will be administered to the human subjects to determine the
pharmacokinetic and pharmacodynamic properties in the human data 1. Data such as
clearance, absorption, distribution, metabolism and half-life of a drug can then be obtained
in a much earlier stage compared to the traditional drug development process. This
indicates that the best drugs can be filtered out for further investigation.
Early human pk data can also be used to determine the best candidates or model for the
prediction of human values for further research, suggesting that the amount of animal
studies needed for a specific research can be reduced.
Figure 2:
A schematic view of the
traditional drug development
process and the new
microdosing approach 1 .
Traditionally the human data of the
drug metabolism pathway and
pharmacokinetics can only be
obtained when the drug is tested in
human phase 1 studies for
approximately 18 months, whether
with the new approach the data can
be obtained within 4 months.
Hereby it can estimate the precise
value of the drug before continuing
a very long and expensive
development process.
6
In general, microdosing is a promising approach that helps reduce both the amount of
animal studies needed and the drug development costs. By lowering the drug development
costs it also facilitates the introduction of new life-saving medicines into the market at
lower costs. This makes microdosing an interesting approach for several pharmaceutical
industries. In order to better understand the microdosing approach, this thesis will briefly
summarize the concept together with its advantages and disadvantages. As already
mentioned with the aid of microdosing both the pharmacokinetic and pharmacodynamic
properties of a drug can be determined. Therefore the data obtained from the microdosing
studies can be analyzed to determine if the obtained values are in accordance with the
known values for therapeutic dosage in the literature. If these values for the therapeutic
dose can be predicted by a microdose, than it could be questioned whether microdosing
can be applied for the prediction of PK values in children.
The PK values of adults and children are more likely to be different because of the maturity
differences of certain processes as for example the metabolism pathway. Doses for
children are mostly obtained by adjusting the normal dose of an adult subject for the
height and the weight of the young patient. Therefore applying microdose studies in
children may help to establish whether the calculated dosage is actually the right dosage
for the children after all. This will also be addressed in the thesis.
7
The microdosing concept
Microdosing is a new experimental approach that has been recently introduced as the
promising most successful method for the development of drugs. As already mentioned in
the introduction most drugs fail to pass through human phase 1 study during the drug
development process due to among others inappropriate pharmacokinetic (PK) properties.
With the aid of microdosing (or human phase 0) study these properties can be obtained
much earlier in the developmental process of the drug without having to go through the
long and expensive human phase 1 study. The proper value of the drug can then be
estimated before starting the whole phase 1 study and thus determining if the drug should
be further tested or not. Drugs that are most likely destined to fail due to inappropriate PK
properties will then be banned from further research.
Microdose as the name already implies is a very small dose, defined by both the European
agency for evaluation of medicinal products (EMEA) and also the Food and drug
administration agencies (FDA) as 1/100th of the predicted pharmacological dose derived
from in-vitro and animal models or 100 micrograms 5,1. From the definition of a microdose
from the agencies it can already be noticed that it is still not exactly clear what the real
dose should be for a microdose study. The agencies however also stipulates that the
smallest dose of the drug candidate, whether the 1/100th of the predicted dose or 100
micrograms should be taken into consideration for the human microdose study 1. This very
small dose is considered to be much safer than the pharmacologically active dose and is
therefore considered safe to start testing the candidate drugs in a human phase 0 clinical
study with such low level of drug concentration. Although the dose is considered safe for
testing in a human phase 0 study, it is important to elicit that no efficacy or safety data
can be obtained from such study due to the lower dose used. Therefore a conventional
phase 1 study should always be conducted to demonstrate the tolerability and safety of
subjects to the candidate drug 1.
Before any human clinical trials including a microdose study can be carried out the
candidate drug should have been tested on several studies in advance ensuring that
exposure of human subjects to the drug during the study will cause no harm to the
subjects (Table 2) 1. The safety evaluation of nonclinical studies as referred in the M3
guideline of the international conference on harmonization of technical requirements for
registration of pharmaceuticals for human use (ICH), should estimate the initial safe
starting dose and dose range for human trials and also identify parameters for clinical
monitoring of possible adverse effects 25. Therefore as expected one of the studies consists
of determining the safety margins, which is established by estimating the no-observed
effect level and the maximum tolerated dose in a single species. Drug candidates that pass
these tests with proper results are considered safe and can then be further investigated in
a human microdose study.
Subjects in a microdose study are exposed to very low dose concentration of a compound,
indicating that the presence of ultrasensitive analytical methods capable of measuring
concentration range of pictogram to femtogram are of extreme importance 1. The only
ultrasensitive analytical methods nowadays available that can be used in a microstudy are
the accelerator mass spectrometry (AMS) and the positron emission tomography (PET).
With the aid of the AMS in a microstudy the pharmacokinetic data of a compound can be
determined, whereas the PET can be used to analyze the pharmacodynamics.
For the AMS microstudy body fluids are analyzed to determine the concentrations of the
drug and metabolites, which is the used to estimate the PK properties of the compound.
These fluids are in general as for most analysis collected from the exposed subjects at
different time intervals after dosing. For the analysis of pharmacodynamics on the other
hand, real-time data is needed. Therefore subjects have to go through a complete PET
body scan after exposure, which will illustrate the disposition of the drug through the body
1, 6. Usage of these ultrasensitive analytical methods however are only possible if the drug
candidate that is administered to the subject is labeled. The drug can be labeled in two
ways: 1) attaching an isotope-ligand or 2) the drug itself can be radiolabeled. The most
frequently used technique is labeling of the drug with an isotope-ligand. This is a much
easier way of labeling a drug compared to the difficulty of synthesizing a radiolabelled drug
itself.
8
Table 2: Proposed toxicology tests to permit a human Phase 0 microdose study
AMS microdosing A small group of human volunteers are exposed to a single dose of
14C-labelled drug in a concentration range of 1-100 micrograms either intravenously or
orally through ingestion of the drug. The study design is a randomized control trial, in
which subjects are exposed in a parallel or cross-over manner to a compound followed by
a washout period 1. After exposure samples of blood and urine is collected from the
volunteers during a 60-hour period, which should be sufficient to retrieve plasma half-lives
of the administered drug. The concentration of the total drug or metabolite is obtained
from the blood samples, whereas the amount of drug excreted from the body through
urine is obtained from the urine samples. After sampling the compound is extracted from
the samples and chromatography separated in order to obtain sample fractions that are
analyzed by the AMS to detect and quantify both the parent compound as the metabolite.
The advantage of the AMS technique is that from a single run both information about the
parent drug and its metabolite can be obtained. With this information the capability of
humans to metabolize the compound can be determined, which can be used to rank
compounds in an early stage of drug development. Ranking of the compound according to
the metabolizing capacity of humans should reveal compounds with promising features
depending on the desired effect of the designed drug. For example if the drug was
designed to reduce pain by blocking a receptor, than the compound should not be easily or
rapidly metabolized for it will only result in minor effect. These way drug candidates with
better properties can be selected to continue further in greater clinical trials during the
drug development process.
PET microdosing Subjects participating in the microdose study will be administered a
single dose of radiolabeled drug, followed by a non-invasive scan by the positron emission
tomography (PET). This scan as already mentioned illustrates the disposition of the
compound in the body, determining therefore the pharmacodynamic properties of the
compound. In general, results obtain from the PET-scan informs about the ability of the
drug to reach the target tissues 1. Distribution of drugs in the body is of great interest for
it determines the target organs that will be affected. Therefore also information about
possible adverse effects due to the effect of the drug on non-desired target organs can be
obtained. For example the nervous system such as the brain, distribution of a drug in the
brain is of great interest for the determination of possible side effects such as sleepiness.
In general PET imaging can be used to determine the distribution of the drug in the body,
illustrating the amount of drug that reached a specific target. The amount of drug in the
image can therefore not be distinguished as bound or unbound drug in the tissue, hence
the total effect of the drug on the target cannot be predicted. Therefore the images can
only predict whether the drug will reach a specific target organ or not. This is also of great
importance for ranking drug candidates with the promising features the drug was
designed. For example if the drug was designed to target the brain in the case of psycho-
9
medications it should be shown in the PET images of the head, otherwise it does not reach
the target and is therefore destined to fail in the drug development process.
As already mentioned above the results obtained from a microdose study can be used to
rank drug candidates according to the desired pharmacokinetic and pharmacodynamic
features. Ranking of the compounds should facilitate the selection of the drug candidates
that are most likely to succeed in inducing the desired effect it was designed for. Selection
of candidate drugs before starting a phase 1 clinical study should reduce the costs and
time needed for the development of drugs, because only candidates with the proper
features will be further tested whereas for the other compounds the developmental
journey ends.
10
Predictability of pharmacokinetic
The dose in a microdose study is not a therapeutic dose, suggesting already that there
might be differences in the estimated PK values from both doses. At therapeutic dose the
metabolic enzymes and drug transporters may become saturated which can influence the
estimation of the PK value. This is certainly not the case when using a microdose, which
raises the concern if a microdose can properly predict the values from a therapeutic dose 2.
Therefore it is of great importance to determine if the pharmacokinetics obtained from a
microdose can successfully predict those from the therapeutic dose. Several studies
attempted to try justifying the microdosing approach, including the well-known studies the
European Union Microdose AMS Partnership Programme (EUMAPP) project and the
Consortium for Resourcing and Evaluating AMS Microdosing (CREAM) study.
EUMAPP A project funded by the European union as an international multi-centre
research study that involves the collaboration between industry and academia. Candidate
drug selection for the study was based on the known prediction problems the drugs have
when using the traditional models (e.g. in-vitro and animal species) to determine the
pharmacokinetics 5. 7 drugs were selected, consisting of 6 generic drugs and one failed
development drug named S-19812. Table 3 summarizes the selected drugs and their main
problems impeding any pharmacokinetic prediction 5. The synthesized drugs were
isotopically labeled with 14C and a microdose of 100µg, 7.4 kBq (200nCi) was administered
either by oral or through intravenous route (through an infusion over 30min) to the study
group, which consisted of 6 healthy male volunteers. Depending of the drug tested a
different study design was used differing in the routes of exposure. According to the study
design the dose could be 1) orally administered, 2) intravenously administered or 3)
administered by a combination of both routes. All drugs were exposed both orally and
intravenously in different period of time, with the exception of the drug phenorbital that
was only exposed orally. All drugs, with the exception of phenorbital and paracetamol was
tested in a 3-way cross-over study, which included besides oral and intravenous exposure
also of the combined exposure consisting of an oral exposure with the unlabelled drug and
intravenous exposure with the 14C labelled drug in the same period of time. In the
combined exposure study design, the intravenous exposure with the labeled drug acts as a
tracer. The dosing pattern used for the EUMAPP study is summarized in table 4. Because of
the different route of exposure studied it is possible to compare the similarity of the PK
data obtained from the microdose (period 1) with that obtained from the therapeutic dose
(period 3). Also because of the combined exposure from different routes of exposure, the
bioavailability of the drug can also be determined in a single clinical study. Samples were
taken from all subjects and also analyzed.
CREAM A project with the primary goal of assessing how good a microdose can predict
the pharmacokinetics of a therapeutic dose. The candidate drug selection was based on the
known problems the drugs have impeding a proper estimation of the PK data. 5 drugs
were chosen for the study, consisting of 4 drugs that can found in the market and 1 failed
development drug named ZK253 (Table 3) 8. A cross-over study design was chosen for the
project. The drugs were isotopically labeled with a radioactive carbon (14C) and were
administered orally or intravenously to the study group, which consisted of 6 healthy
volunteers for each compound. The dosing regimen used in the project is summarized in
table 4. All drugs were exposed both orally and/or intravenously in different period of time,
with a wash out period of 14 days between each dose administration time. Besides the oral
and intravenous exposure of the compounds ZK253, midazolam and erythromycin, the
volunteers have also been exposed to the drugs through both routes of exposure
simultaneously. These volunteers received a 14C-labeled intravenous microdose and also
an unlabeled oral therapeutic dose at a certain time.
Samples were taken from the subjects and further analyzed.
11
Table 3: List of drugs tested in EUMAPP and CREAM study and the possible problems
impeding an accurate prediction of the PK data.
Study
Drug
Fexofenadine
EUMAPP
Paracetamol
(acetaminophen)
Phenorbarbital
Sumatriptan
Propafenone
Clarithromycin
S-19812
CREAM
Warfarin
Midazolam
Erythromycin
Diazepam
ZK253
Problem impeding PK predictions
A probe P-gp and OATP substrate. The Limited absorption is partially
controlled by P-gp and it is excreted in urine principally as the parent drug.
Primarily metabolized by sulphate and glucuronide conjugation. The
metabolites are difficult to quantify from in vitro-data.
100% orally bioavailable, with very low clearance and moderate volume of
distribution justifying its long half-life time of approximately 100 hr.
Low oral bioavailability (ca 15%) and metabolism-dependent elimination.
Clearance and first-pass loss in humans are difficult to predict from in-vitro
data.
Saturable first-pass metabolism resulting in a dose-dependent PK at
therapeutic doses.
P-gp substrate undergoes metabolism and exhibit limited oral bioavailability
(ca 50%).
Trial drug forms an active form S-32361. The ratio of these two are
significantly different between data obtained from human phase-1 study
and those from animal and in-vitro studies.
100% orally bioavailable with low clearance. Difficulty in determining
clearance in human in-vitro systems.
Low bioavailability due to extensive first-pass CYP-P450 metabolism.
Known substrate for CYP3A. PK in influences by transporters e.g. Pglycoprotein.
Low clearance and moderate tissue distribution. Primarily metabolized by
CYP enzymes.
Oral absorption is uncertain due to conflicting animal data.
The pharmacokinetics of a drug obtained from a microdose study can in general be
compared to those reported in the literature. Pharmacokinetic data for most of
therapeutics used can be found in books. By comparing both data an overall conclusion
regarding the reliability of the microdosing approach can be taken. Besides these 2 major
projects, there are also several other studies that were carried out to determine the
predictability of PK data of therapeutic dose by a microdose for a specific compound.
However the same persons Lappin and Garner that are promoting the microdosing
approach writes most of the microdosing studies. Therefore only the compounds tested in
the EUMAPP and CREAM projects will be further referred to in this paper. The
Pharmacokinetic data of the compounds tested in the projects are summarized in table 5,
including data of these compounds published in books.
The general rule that is applied to predictions obtained by allometric scaling of animal data
to human data is also applied to the data obtained from microdose study or literature,
which implies that the difference between the data obtained from literature and/or
prediction can only vary within factor 2. In other words prediction of the PK from a
microdose study is only accepted if the difference between the pk obtained from the study
and the literature is less or equal to factor 2. The results of both projects predicted similar
pk data as described in the books. Most of the results differed by a factor 2 from the
literature data, indicating that there is a good concordance between the pharmacokinetics
of a microdose and that of a therapeutic dose. Hereby both projects ensure the
predictability of the pk of a pharmacological dose by a microdose, which justifies the
approach. This clearly shows that microdosing can be used for the selection of drugs in the
earlier phase of drug development.
12
Table 4: Clinical dosing regimen used in EUMAPP and CREAM
CREAM
EUMAPP
Study
Drug
Clarithromycin
Dose period 1
14
C oral (100µg)
Paracetamol
14
C oral (100µg)
14
C intravenous (100 µg)
Fexofenadine
14
C oral (100µg)
14
C intravenous (100 µg)
Phenobarbital
14
C oral (100µg)
Sumatriptan
14
C oral (100µg)
14
C intravenous (100 µg)
Propafenone
14
C oral (100µg)
14
C intravenous (100 µg)
S-19812
14
C oral (100µg)
14
C intravenous (100 µg)
Warfarin
14
C oral (100 µg)
oral (5 mg)
Erythromycin
14
C oral (100 µg)
Diazepam
14
oral (2.50 mg) +
14
C intravenous (100 µg)
14
C intravenous (10 mg)
Midazolam
14
C oral (100 µg)
14
C intravenous (100 µg)
ZK253
14
C oral (100 µg)
14
C intravenous (100 µg)
C intravenous
(100 µg)
Dose period 2
14
C intravenous (100 µg)
-
Dose period 3
14
C tracer dose +
oral therapeutic dose (250mg)
C tracer dose +
oral therapeutic dose (120mg)
14
C tracer dose +
oral therapeutic dose (50mg)
14
C tracer dose +
oral therapeutic dose (150mg)
14
C tracer dose +
oral therapeutic dose (100mg)
14
oral (7.5 mg) +
14
C intravenous (100 µg)
oral (50 mg) +
14
C intravenous (100 µg)
13
Table 5: Pharmacokinetic properties of therapeutic drugs tested in the EUMAPP and CREAM
project
Drug
Study
Route
Dose
Clarithromycin
EUMAPP
Diazepam
Literature 1
Literature 2
CREAM
Oral
Oral
IV
Oral
Oral
IV
IV
250 mg
100 µg
100 µg
250 mg
500 mg
100 µg
10 mg
Literature 1
Literature 2
Oral
Oral
10 mg
2 mg
Erythromycin
CREAM
Fexofenadine
Literature 1
Literature 2
EUMAPP
IV
Oral
Oral
Oral
Oral
Oral
IV
Oral
Oral
Oral
IV
100 µg
250 mg
250 mg
250 mg
120 mg
100 µg
100 µg
60 mg
120 mg
5 mg
5 mg
Oral
IV
Oral
Oral
Oral
IV
Oral
Oral
7.5 mg
100 µg
10 mg
2 mg
100 µg
100 µg
100 µg
1400
mg
325 mg
100 µg
90 mg
50 mg
150 mg
100 µg
100 µg
50 mg
100 µg
100 µg
200 mg
3 mg
100 mg
25 mg
100 µg
5 mg
6.1 mg
5 mg
Midazolam
Literature 1
Literature 2
Schwagmeie
r (1998)
CREAM
Paracetamol
Literature 1
Literature 2
EUMAPP
Tozuka 2010
Literature 1
Propafenone
Literature 2
EUMAPP
Literature 1
Literature 2
EUMAPP
Sumatriptan
EUMAPP
Warfarin
Lacey
(1995)
Literature 1
Literature 2
CREAM
Phenobarbital
Literature 1
Literature 2
Oral
Oral
Oral
Oral
Oral
Oral
IV
Oral
Oral
IV
Oral
IV
Oral
Oral
Oral
Oral
Oral
Oral
T1/2
(h)
3.4
4
4.1
3.3
4.5
45.
1
35.
7
43
32.
9
2.5
2.5
1.6
1.4
12
16
8
14
5.1
143
181
.5
3.3
2.6
1.9
1.2
5.8
4.6
2.4
2.0
Cmax
(ng/ml)
958
188
1200
4.7
322
V
(L/kg)
1.9
2.6
3.3
1.3
1.8
CL
(L/h/kg)
0.3
0.4
0.5
0.02
0.02
Fa
(%)
39
22
55
50
-
317
-
1.1
2.0
0.02
0.04
100
100
3.4
717
900
318
0.3
286
55.9
-
1.1
0.8
0.6
1.7
4.6
-
0.3
0.2
0.3
0.2
0.6
1.0
5.2
14
35
40
30
41
74.5
-
34
3.0
78
1.1
1.6
0.02
1.1
1.1
3.3
1.8
1.0
0.3
0.4
0.8
0.3
0.3
0.3
22.1
44
88
88
2.5
108
99
90
2.6
3.8
5.4
1.4
1.9
6.5
2.9
1.6
1.0
2.5
274
48
37
46
2.6
98
0.02
26
0.1
95
77
54
5.0
493
500
-
1.1
0.5
0.7
3.9
6.1
2.4
2.0
2.4
1.0
0.3
0.1
0.1
0.3
0.003
0.06
0.7
0.6
0.2
1.0
1.3
0.7
0.002
0.004
0.003
0.002
85
100
90
13
5.8
7.6
20
14
14
15
93
90
a) F defines the absolute bioavailability and is shown in percentage (%)
Literature 1 8.
Literature 2 9.
14
The microdosing approach is very promising for the selection of candidate drugs with the
desirable human ADME properties in the early phase of drug development. Hence only
compounds that are capable of inducing a desired response or effect is tested in the
human phase 1 clinical trial. With the microdose study the pk properties of a compound
can be predicted, however it should be noticed that this predicted value might differ from
the therapeutic value because the compound can for example bind to an enzyme or
transporter. In this case saturation of the enzyme or transporter plays an important role
and will affect the bioavailability of the compound. The bioavailability of a compound is of
great importance for it is the free amount of concentration that is available to induce an
effect at the target organ or tissue. The doses applied in a microdose study is very low
compared to the therapeutic dose, indicating that saturation of enzymes or transporters
will not take place and thus in such a case a good indication of the pk paramaters cannot
be given.
In these cases the data obtained from a microdose study can still be used to predict proper
PK values with the aid of other studies such as for example the physiologically based
pharmacokinetic (PB-PK) computer model.
Figure 3 : Schematic view of PBPK model analysis simulating PK profiles of a drug
The physiologically based pharmacokinetic (PBPK) computer model is a mathematical
modeling technique, in which all type of dosage forms and delivery methods can be used
to predict the time-course behavior of compound in biological matrices (for example blood,
urine, exhaled air, tissues) of humans or animal species 6,26. The model is based on a
multi-compartment model, which corresponds to the different tissues of the body that are
connected by the circulating blood system figure 3. Each compartment is defined by the
tissue volume and tissue blood flow rate that is specific for the species of interest.
Therefore for the proper use of the technique essential parameters such as for example the
pharmacokinetics, bodyweight and organ weight should be available for input in the model.
A database with such physiological parameters can be found for humans and/or animals on
the web or can also be found in the literature. The physiological parameters can be
obtained from in-vitro, in-silico or in-vivo studies. Representative PBPK models include the
in vitro determination for the estimation of ADME (iDEATM) model and the
compartmentalized absorption and transit (CAT) model. The technique relies in general on
the input of species specific physiological parameters and compound specific information
(as for example metabolic clearance (CL)), to predict the plasma concentration profile of
the compound in the species of interest after intravenous (i.v.) or oral administration. As
already mentioned all information needed such as the physiological parameters can be
gathered from the literature. The model attempts to describe mathematically all relevant
physiological processes in order to predict any interaction of the compound with the
system. The model can predict the amount of drug distributed to target or non-target
15
organs, which is important in determining the physiological response or side effects of the
drug. The performance capability of PBPK modeling in predicting oral absorption in humans
has been tested by the iDEATM simulation system. The fraction of the dose absorbed to the
portal vein (FDp) for some compounds have been predicted by the model and compared to
the FDp values obtained from human clinical trials (Figure 4) 6. Plotting of the values
against each showed a correlation between the predicted values and the human values,
validating the performance of such a model.
BPK modeling is a promising tool for the prediction of the behavior of the drug in the body
after exposure. There are many reports including the report of the iDEATM simulation
system that demonstrates the prediction of PK profiles of drugs in humans by PBPK model.
However validation of such simulations for some compounds is difficult when the in-vivo
evidence is lacking 2. Therefore introducing the microdosing approach will help fill the gap
of the in-vivo data. Combining the microdosing approach and the PBPK modeling technique
will therefore lead to a better understanding of the behaviour of a candidate drug during
the development process Figure 5. The in-vivo evidence for the verification of the PBPK
model simulation can then be obtained from the microdosing study. On the other hand
PBPK modeling technique can be also used to analyze the problems that microdosing can
encounter when predicting PK values. For example saturation due to binding of the
compound to proteins is less likely occur with a microdose, indicating that the PK values
cannot be predicted accurately. Hereby a more accurate prediction of PK profiles of a drug
or test compound at therapeutic dose in humans can still be obtained from PBPK model
analysis based on the microdose clinical study in combination with in-vitro study with
human materials 2.
Figure 5: Method that includes both the microdosing approach and the PBPK-model
analysis for a more accurate prediction of PK profiles of a new drug candidate in human.
16
The PK profiles of a compound can be studied by evaluating the ADME properties of the
compound. For the evaluation of these properties there are several methods available,
however only a few will be shortly discussed. Evaluating these properties can help to
predict the outcome of the microdose study.
The absorption properties of a compound can be obtained from in vitro study with human
colon adenocarcinoma (Caco-2) cells 27. These cells have similar characteristics as the
intestinal enterocytes, such as formation of microvilli, tight junctions, P-glycoprotein (P-gp)
and other transporter expression 28. Several studies have found good correlation between
Caco-2 permeability and the oral drug absorption in humans. The method consists of
measuring the amount of compound that is transported from the medium to the cells. The
data is then expressed as the apparent permeability coefficient (Papp) with the unit cm∙sec1, using the following equation: P
app = (area × initial concentration × time)⁄ amount transported
The difference between the transports of the compound can also be studied by using
specific inhibitors. For example the role of P-gp in the uptake of the compound can be
evaluated by using Verapamil (inhibitor of P-gp). The presence of the inhibitor would
inhibit the efflux from the cells and therefore would enhance the Papp of the compound.
The outcome of the result can determine if it is worth to be further studied in a microdose
study or not. For example if the uptake of the compound is mostly influenced by the P-gp,
indicating that the compound is poorly absorbed through the intestine, it has no
significance of performing a microdose study especially not through oral exposure.
Binding of compounds to proteins or tissues can influence their distribution across the
body. Plasma protein binding can be studied with the methods ultrafiltration and
equilibrium dialysis, from which the equilibrium dialysis is the most used method 28. This
method is most used because it is not affected by nonspecific binding. However it should
be pointed out that it is unsuitable for compounds that are known to be unstable in plasma
due to the long time (approximately 20 hours) needed for the system to reach equilibrium.
The percentage of plasma protein binding can then be derived from the measured amount
of compound remaining in the plasma (Cpl) and the amount of compound found in the
buffer (Cbu), with the equation percentage plasma protein binding = 100 × (Cpl − Cbu)⁄ Cpl
According to the plasma protein binding percentage a compound can be categorized as
highly, moderately or poorly protein bound. Only compounds that show more than 95%
binding to proteins is considered highly protein bound. However protein binding
interactions have little clinical implications unless the drug has a narrow therapeutic index
and is also highly protein bound 28. Nowadays most of the drugs approved have high
therapeutic indices.
The bioavailability of a compound can also be influences by metabolism of the compound.
After oral exposure the drug is absorbed in the intestine and transported through the
circulation system to the liver, where it is subjected to the hepatic metabolism followed by
the elimination of the compound through the bile or urine via the kidneys. The metabolism
process can be divided in two phases: 1) phase 1 (oxidation, reduction or hydrolysis step)
that makes the compounds more hydrophilic, and 2) phase 2 (conjugation step) that
makes the compounds inactive. Enzymes responsible for the metabolism process are
present in the hepatocytes, therefore incubating these cells with the compound followed by
measurement of the amount of compound present in the buffer (Cafter incubation) should
determine the metabolic stability of the compound 27. In vitro systems that can be used for
this study can consist of hepatocytes or microsomes. Microsomes are usually used to
determine P450-mediated metabolism (phase 1), however hepatocytes being intact cells
are believed to be similar model of the liver and therefore should deliver better drug
clearance measurements 29. The metabolic stability is generally expressed as the
percentage of parent compound that disappeared and can be calculated with the equation
percentage compound disappeared = 100 × (1− (Cafter incubation⁄ Cbefore incubation))
The ability of the liver to metabolize and therefore help eliminate the compound can be
determined by estimating the intrinsic hepatic clearance. The apparent intrinsic clearance,
the following equation is used
Clint,app = (0.693⁄ in vitro t1/2)( incubation volume⁄ mg microsomal protein)(45 mg microsomal protein/
g of liver)(*20 g of liver/ kg bodyweight), where * refers to the values depending on the selected
species. Human values are given in the equation.
The apparent intrinsic clearance is then corrected for the free fraction of the incubated
compound to determine the intrinsic clearance 29. The amount of parent compound
remaining also known as the free fraction can be calculated with the equation
Fu,mic = (concentration at receiving side×1.5)⁄(concentration at donor side×3)
17
The in vitro apparent intrinsic clearance (CLint) of the compounds by the liver can be
determined from the enzyme kinetic data by calculating the ratio of Vmax⁄ Km, known as the
maximal rate (Vmax) at which metabolism occurs and the michaelis constant (Km) which
indicates the concentration substrate needed to fill half the active sites of the enzyme thus
yielding half the maximal velocity (Vmax). By observing the formation of a specific
metabolite at constant enzyme concentration exposed by different concentration of
compound both the Vmax and the Km properties can be estimated (fig 6). At saturation rate
the amount of compound metabolized per unit time can be calculated by [S]×Vmax⁄ Km+[S]
After determination of the kinetic properties in vitro, the data can be extrapolated to
determine the in vivo situation.
As can be observed in figure 6 the plotted data does not show a linear curve, however this
can be linearized by using the lineweaver burk method. Hereby 1/reaction velocity is
plotted against the 1/ substrate concentration, resulting in a straight line where the slope
is defined as Km/ Vmax also known as the CL.
Figure 6: Michaelis menten kinetics 30.
Plot of the reaction velocity (V0) as
function of the substrate concentration [S].
Figure 7: conversion of michaelis menten
kinetics plot using the lineweaver burk
method 31.
As can be imagined from the plot at therapeutic dose the maximal reaction velocity is
mostly reached indicating saturation of the system, which is not the case for a microdose.
Therefore it can be assumed that in a microdose study the compound would be mostly
metabolized, indicating that no clinical effect would be obtained from the parent
compound. This also points out how safe a microdose study is.
18
Pediatric drug development
As already mentioned in the introduction, the development process for drugs consists of
in-vitro and in-vivo studies to obtain toxicity data of the studied compound. The obtained
data from experiments with laboratory animals is then extrapolated to the human
population to predict the effect of the compound in the human subject. During
extrapolation the variation between species and within species are taken into
considerations, including the differences between adults and children. Therefore correcting
for the variations during extrapolation is expected to correct for the differences between
children and adults, indicating that the compound should be considered safe for both
adults and children. This however can always be questioned, because most of the
toxicological data obtained are from studies with adult animals. Information from studies
with juvenile animals would be more relevant to children. On the other hand this could also
lead to great uncertainties for the risk assessment in children due to the different
maturation rates of several systems such as the enzyme activity, which may affect the
pharmacokinetics of a compound between the species. Maturation of the enzyme system
for example is known to start in the first 2-3 months in humans, whereas in rats it occurs
in the first 2-3 weeks of life 10.
Generally dosage of the compounds is mostly determined for the adult human. For children
the dosage is therefore corrected for bodyweight and/or body surface area in order to be
more indicative. The most preferred method for prescribing therapeutic dosage of drugs in
children is by correcting for body surface area instead of correcting for bodyweight.
Correction for the body surface is expected to give better adjustment parameters, because
it describes the total body water and extracellular water better 10. Therefore dosing based
on correction for the surface area may avoid underdosing of water-soluble drugs. Another
advantage is that both the measures of height and bodyweight are required to estimate
the size. This indicates that most variables are considered in the correction of adult dosage
to a proper dosage in children.
Correction of the adult dosage to a more proper dosage in children however does not
indicate that the dosage is good for children of different ages. Children of different ages
can show great differences in for example the absorption of drugs, indicating that the
corrected proper dosage is more indicative in children that have equal PK properties as the
adults. Variability of pharmacokinetics can be observed in both children and adults.
However PK variability can also be observed within the group of children, due to the
grouping of different ages (e.g. 1 week or 1 year of age). Variability in pharmacokinetics of
children is shown in figure 6. Neonates and infants are shown to have great variability,
indicating that the corrected dosage can lead to an overdosing or an underdosing with the
related adverse effect in children of this age. The variability differences is of great
importance for the risk assessment in children 11.
Table 6: Children classification
according to age.
Term
Neonates
Infants
Age
(month)
0–1
Figure 7: Child/adult half life ratios across
drugs and age groups 11.
Plotting the half life ratio of different compounds for
both children and adults according to their age in days
gives an indication of the variations according to age.
1 -12
Toddlers
12 – 36
Children
36 – 120
Adolescent
120 - 228
19
Either overdosing or underdosing of a drug in children indicates that the efficiency of a
drug is unknown, resulting in different toxicity effects in children of different ages. This can
be mostly explained by pharmacokinetic and pharmacodynamic differences in children. As
an example the drug acetaminophen or better known as paracetamol is taken. Adults are
more sensitive to an overdose of paracetamol than children. In adults paracetamol is
normally eliminated from the body through glucuronidation, however the glucuronidation
level observed in children are low indicating that children have other elimination routes
compared to adults. Higher rate of glutathione turnover and also more active sulfate
conjugation is observed in children, which compensates for the low level of glucuronidation
resulting in a faster elimination of the active compound 12. This example illustrate that
there are important differences between children and adults that need to be consider in
order to obtain a therapeutic dosage for children or for the development of new drugs,
besides only the adult data obtained from experimental studies. Differences in growth or
maturation of specific systems that may have an impact on the absorption, distribution,
metabolism and elimination of a compound may explain the observed variability in
pharmacokinetics.
20
Differences in children vs adults
The growth and development process of different organs of either animals or humans
occur at different rates, indicating that differences in pharmacokinetics and toxicokinetics
between the various age groups is most likely to occur. Therefore the differences should be
taken into consideration for the development of generic drugs. The main physiological
differences observed in human adults and children have been well studied during the years
and are summarized in table 7. These physiological differences, such as the immature gut
development and immature enzyme activity, can lead to different PK properties of a drug
according to age of the treated patient.
Researchers have studied the effect of age on the pharmacokinetics in humans both invivo and in-vitro, and concluded that the most dramatic physiological changes that may
affect the pharmacokinetics of a drug occur in the first 6-12 months 10. Research for the
physiological changes in animals from different ages on the other hand is minimal. The
pharmacokinetics is mostly affected by changes in the absorption, distribution, metabolism
and elimination process of compounds, better known as the ADME.
Table 7: Summary of developmental changes affecting pharmacokinetic
Physiological system
Gastrointestinal tract
Body compartments
Plasma protein binding
Metabolizing enzyme
capacity
Renal excretion
Observation in
children
Immature gut, less
surface for the
absorption of
compounds.
Low P-glycoprotein
expression.
Decreased fat,
decreased muscle mass,
increased total body
water.
Limited serum protein
binding capacity.
Immature enzyme
function
Decreased glomerular
filtration rate and active
tubular function
Pharmacokinetic implication
Slower uptake of compounds. Without
affecting the bioavailability. For some
compounds such as metals, an increased
uptake is observed.
Low P-glycoprotein results in less excretion
and thus more absorption of compounds.
Less distribution and retention of lipid soluble
chemicals, whereas the water soluble
chemicals have larger volume of distribution
(Vd).
More free toxicants in the blood circulation.
Slower metabolic clearance, less metabolic
activation or elimination of activated
metabolites.
Reduced plasma clearance and increased
elimination half-life of compounds
Absorption The primarily route of exposure for most medications is through oral
ingestion and therefore the absorption of drugs by the gastrointestinal tract is of great
importance. The major function of the small intestine is to facilitate the absorption of
nutrients and compounds. Children have relative smaller surface area than adults,
indicating that there are less receptors and transport proteins present per square unit
intestine and thus will result in slower absorption of compounds. A study with 580
hospitalized children receiving drugs through a feeding tube, showed that the rate of
absorption is related to age 10. The absorption was much slower in neonates and young
infants than in older children.
The absorption of nutrients and ions in the intestine occurs through active transport
processes present in the intestine. The expression of these transport processes is in line
with the needs of a growing child. Therefore absorption of compounds that involves these
transport processes is most likely to be better absorbed in the children. A classic example
to illustrate this is the absorption of lead. The default oral absorption factor used by
USEPA’s biokinetic model for lead uptake in children is defined as 50% for lead in
diet/drinking water and 30% for lead in soil/ house dust. However the absorption of lead in
the human adult gastrointestinal tract only accounts for 10%. The great difference in lead
absorption between adult and children is partially attributed to the greater pinocytic
activity of the intestinal epithelium in young children before closure or maturation of the
21
gastrointestinal tract 11. However there are also evidences that suggest that the presence
of milk in the gastrointestinal tract may promote the absorption of metals in neonates.
One of the many transporters that plays an important role in the absorption of xenobiotics,
are the transporters belonging to the ATP binding cassette (ABC) superfamily such as the
P-glycoprotein. The P-glycoprotein has been detected in the apical surface of epithelial
cells of excretory organs, suggesting its protective role against xenotoxins. The Pglycoprotein can protect the host by enhancing the excretion of these compounds or
preventing their uptake from the gut. The presence of the transporter is already detected
in the early human fetal life (11-14 weeks), however this is far not the adult levels. The
expression of P-glycoprotein in mice was reported to be low at birth and increased
significantly with the maturation of the intestine.
Distribution After absorption the compound is mainly distributed in the body according
to its chemical properties, which indicates that the body composition of a subject plays an
important role in the distribution of chemicals within the body. Newborns and infants have
different body composition of water and lipid compared to older children and adults. At
birth there is more body water (expressed as percentage of total bodyweight) than in
adults, e.g. 78% in neonates and 55% in adults. The total body water of children reaches
the adult level by 12-years of age 10. Besides the high body water and thus low lipid body
content, the adipose tissue consists also partially of water. The adipose tissue of infants
consists of 57% water and 35% lipids, whereas the values in adults are 26% and 71%
respectively. Therefore in general the lipid soluble compounds are less likely to distribute
through the body at birth, whereas the water soluble compounds on the other hand will
have a large volume of distribution (Vd). Body lipids of children will increase after birth in
its first 9 months of life, and then will decrease again until the ages 4-7 years. Hereafter it
will increase again reaching adult levels. This illustrates that the distribution of lipid soluble
substances and therefore the half-life of the lipid compounds can change during the
development process and growth of children.
Besides the body composition there are also other factors that can influence the
distribution of compounds. The binding capacity of plasma proteins for example can affect
the distribution of compounds. Binding of the compounds to plasma proteins has two
consequences, which are: 1) a slow elimination process of the compound and 2) that the
compound is not free to exert its effect. Only free compounds can exert effect to target
cells, indicating that binding of the compounds to proteins limits the amount of free
compounds. The measured amount of total plasma protein concentrations in neonates is
approximately 59 g/L, whereas in adults it is approximately 72 g/L. At first the difference
in concentration does not seem so dramatic, but due to different binding capacities of
proteins it can have a great impact. The protein binding capacities of neonates is low
regarding both albumin and alpha-1-glycoprotein, indicating that relative high levels of
circulating free active drug can be found in children.
Metabolism Clearance of a drug usually starts with metabolism. The primary organ
responsible for the elimination of compounds through metabolism is the liver. For the
metabolism process however maturation of enzymes involved in the process is needed.
Maturation of the enzyme system starts in humans in the first 2-3 months, whereas in rats
it occurs in the first 2-3 weeks of life. Observations in infants showed that some drugs tend
to have longer half-life (t1/2) and lower clearance, indicating the metabolism capacity of
children. The enzymes activity responsible for metabolism will increase after birth until it
reach adult levels by 1-3 years of age 10. Metabolism in general is divided in two steps: 1)
phase 1, and 2) phase 2.
Phase 1 involves mostly oxidation, hydrolysis and reduction of a compound, whereas in
phase 2 the compound is conjugated with for example glutathione making it more water
soluble and thereby increases its elimination from the body.
The Phase 1 metabolism step consists mainly of the cytochrome P450 enzyme system. The
CYP P450 enzymes are shown to remain stable from the first trimester of gestation till 1
year of age, and accounts for 30- 50% of the adult level. The enzyme levels in neonates
and infants ranged from 0.08 to 0.13 nmol/mg protein, whereas in adults levels of 0.3 ±
0.037 nmol/mg protein is found 10. Several CYP’s, such as CYP1A2 and CYP2E1 are present
at low level at birth and increases hereafter rapidly in the first year of life. The low levels of
CYP1A2 at birth is also supported by in-vitro studies 11. Theophylinne is compound that is
22
primarily metabolized by CYP1A2. The metabolic clearance of theophylline is low in
neonates and infants with a limited metabolizing capacity and therefore it is mostly
excreted as the parent compound itself. The level of the CYP1A2 enzyme is shown to
approach adult level by 2-6 months of age. Expression of the enzyme CYP2E1 is also
shown to be low at birth, however in contrast to CYP1A2 it increases its level rapidly during
the first 24hr after birth achieving adult levels in children at 1-10 years of age. Another
enzyme of the CYP P450 family is the CYP3A4 enzyme. This enzyme is the most abundant
enzyme expressed in adults and is known to be responsible for the metabolism of allot of
chemicals. In neonates however the enzyme is found in very low levels. The mRNA levels
of the enzymes in the foetal liver microsomes accounted for 10% of adult levels. After
birth the levels of the enzyme increases rapidly reaching 50% of adult levels at 6-12
months of age 10. Surprisingly there are also studies that show that CYP3A4 activity in
children may exceed the adult levels at the second months until the first 2-3 years of life.
Another enzyme belonging to the CYP3A subfamily is the CYP3A7. In contrast to the other
enzymes, the enzyme activity level of CYP3A7 is higher in newborns relative to adults.
Although both CYP3A7 and CYP3A4 have similar substrate specificities, they are shown to
have different functional activity. Midazolam is metabolized by CYP3A4, suggesting that
the compound may also interact with the CYP3A7 enzyme due to the similar substrate
specificity. The compound is shown to be slightly metabolized by CYP3A7 in neonates,
resulting in a reduced clearance of the compound. This also support the low CYP3A4
activity present at birth 10. Despite the high levels of CYP3A7 present in newborns, it is still
not clear what the metabolizing capacity of the enzyme is.
Enzymes responsible for the phase 2 metabolizing step are among others the uridine
glucuronosyl transferases (UGT), glutathione S-transferases (GST) and sulfotransferases.
UGT is in general responsible for the glucuronidation of most hydrophobic compounds
making them more hydrophilic and therefore enhances it elimination from the body.
Studies have shown that neonates and infants have limited glucuronidation capacity
compared to adults. This could be illustrated by studying the elimination of the compound
acetaminophen, which is mainly eliminated through glucuronidation. Children should be
expected to have a reduced excretion, because of their reduced glucuronidation activity.
Despite the reduced enzyme activity, paracetamol is yet eliminated as a sulfate conjugate
in children. Until 9 years of age the sulfation capacity in children is increased, which
compensates for the limited glucuronidation activity enhancing the elimination process of
compounds such as for example paracetamol 11. This indicates that compounds may be
cleared in early life stages by alternative pathways.
The enzyme glutathione S-transferases (GST) play an important role in the detoxification
of electrophillic alkylating agents 10. The ability of glutathione conjugation is already
present in utero and at birth thanks to the dominant presence of the GST-pi (foetal)
isoform 11. Other isoforms such as GST-mu and GST-alpha are also present but in much
lower levels compared to adults. The presence of high level of GST-pi in the early life
stages does not indicate that its ability to detoxify all compounds is efficiently. The
substrate specificity of the different isoforms is not well known, which suggests that it is
unknown if the substrates that are specific for GST-mu may be detoxified by the GST-pi or
not. A study showed that human fetal liver cytosol was 33% less active compared to
adults. The conjugation of glutathione with benzo(a)pyrene-4,5-oxide was lower compared
to adults 11.
Another enzyme responsible for the phase 2 metabolizing process is glutathione Stransferases (GST). The ability of GST to detoxify compounds by conjugation with
glutathione depends of the amount of glutathione (GSH) available. GSH can be depleted,
which means less GSH available and therefore subjects are more susceptible to the toxic
effects of compound. Plasma GSH levels in infants increases from 1µM during the first
week of life to approximately 3µM at 1 year of age, which is similar to the adult levels of 34µM 11.
Besides the enzymes present in the liver, there is another factor that plays an important
role in the metabolism process of compounds in the intestinal flora. The bacterial flora of
the gut lumen varies according to the different ages of life, and is also capable of
metabolizing compounds. The enterobacteria and enterococci are the dominant bacteria
present at birth, however after 7 days of birth the bifidobacteria increases becoming at 13
days of birth the dominant bacteria. The change in microflora is accomplished by the
ingestion of fresh milk, which will promote the growth of the bifidobacteria. The food intake
23
of neonate’s changes according to their development and growth enhancing further
changes of the intestinal flora by decreasing the presence of lactobacilli and increasing the
putrefactive bacteria. The change in bacterial flora is important for the hydrolosis of
compounds that are conjugated and secreted in the bile. Absorption of unconjugated
compounds by the intestinal epithelium is facilitated by changes in the microflora 10.
Besides the bacteria there are also another enzyme namely ß-Glucuronidase, responsible
for the metabolizing capacity in the gut. ß-Glucuronidase is an enzyme responsible for the
unconjugation of glucuronidated compounds. This enzyme is known to be absent in the
human adult gut, whereas in children it can be detected. This indicates that the neonatal
gut is capable of converting glucuronides to their unconjugated state which can then be
enterohepatically reabsorbed. In general it can be concluded that the ability of children to
metabolize certain compounds in the gut depends on the maturity of the bacterial enzyme
system present in the gut lumen.
Elimination Excretion of compounds occurs through the bile into the faeces or through
the kidneys into the urine. Renal excretion is for most compounds the predominant step in
the elimination process. The excretion capacity of the kidneys is reflected by the clearance
rate and half-life of compounds. Neonates are shown to have high bodyweight-normalized
clearance compared to adults, indicating their fast elimination rate. The elimination rate
depends on the amount of compound that is filtered per unit time defined as the
glomerular filtration rate. This can be influenced by the renal plasma flow and binding of
compound to proteins.
The kidneys of neonates are known to receive only 5-6% of the cardiac output compared
with 15-25% for adults, resulting in a renal plasma flow average of 12ml/min at birth and
140ml/min in adults which is achieved by 1 year of age 10. A reduced renal plasma flow
also suggests a reduced glomerular filtration rate (GFR). In neonates the GFR is found
between 8 and 20ml/min that then immediately increases achieving the adult levels at 1.56 months of age. Besides this factor the proximal tubules of the kidney in infants are
small, indicating that there are less functional tubular cells present which leads to a
reduced transporter (secretory) system. The tubular function present in neonates is
established to achieve the adult level at 30 weeks of life after correction for the body
surface area. By using the tubular transport maximum for para-amino hippurate (TmPAH)
as an indicator for tubular function, it could be observed that in the first year of life a 10fold increase in TmPAH occurs. Another important transporter is the organic anion
transporter (OAT) family, which removes anionic substances from the blood via a transport
mechanism present in the basolateral membrane of renal epithelial cells. OAT-gene
expression in rats has been shown to increase through birth, following a decrease after
birth to reach adult levels 10. Many drugs such as penicillin are excreted by the transport
system present in the proximal tubules. This indicates that the deficiency in the system
may cause longer half-lives for the compounds.
24
Dosage and effects of drugs in children
Since 1997 there have been new rules related to the pediatric drug development due to
the variability in both the efficiency and toxicity potency of drugs between adults and
children. In 2007 the pediatric research equity act (PREA) has been reauthorized, which
describes the practice of pediatric clinical studies of certain drugs to obtain pediatric
labeling for new indications, new dosage forms, new route of administration, new dosage
regimens, and/or new active ingredients 12. Indicating that candidate drugs that passed
the clinical trials during the drug development process and are therefore approved for the
market, can be tested in a pediatric setting to obtain information relative to its use in
children. Hence for the development of pediatric drugs more studies are needed, including
time and the participation of more patients, which eventually will result in increased
developmental costs.
The human investigational ethical assurance has kind of impeded the participation of
children in clinical trials with elements as “respect for persons” and “human autonomy
leading to concepts of voluntary informed consent” 12. This makes pediatric clinical trials
challenging and therefore a proper balance between scientific, ethical and practical
concerns is required. One of the most challenging drug trials are those with analgesic such
as paracetamol, because during the trial subjects are most likely to experience pain for an
extended period of time 13.
The most common pain relievers used in the human population are among others
paracetamol, diclofenac and sumatriptan. The mechanism of action and effects of these
compounds are in general well known in adults, especially for paracetamol. However the
efficacy of the drug in children depending on their age is mostly unknown and requires
more studies. As already mentioned in section Differences in children vs adults, maturation
of the enzymes involved in the metabolism process reaches adult levels at approximately 3
years old of age. Indicating that extrapolation of adult data to predict efficacy of drug in
children is justified, due to equal metabolic clearance of the compound between children
and adults. For children younger than 3 years of age the efficiency and toxicity data of
compounds are needed for the proper selection and dosage of the medicated drug. Overall
conclusion is that there are more studies needed to elicit the pharmacokinetic and
pharmacodynamic properties of a drug in children.
Determining these properties will result in the selection of the proper drug for treatment
and the proper dosage according to the age of the subject.
Prescriptions of drugs to children are preferably based on the available information from
studies with young subjects. However in the absence of such information the prescription
of medicines relies on the efficiency and toxicity data obtained from experiments with adult
subjects. In general this indicates that a proper dosage for the young generation is not
known.
Paracetamol is a general safe and well tolerated analgesic and antipyretic drug that is
frequently prescribed in children. In general, the dosage of paracetamol in children is
based on a single dose of 10 mg per kg bodyweight which can be repeated every 4-6
hours. Despite the general dosage children are frequently being overdosed (young
children) or underdosed (older children) together with the associated toxicity risk or risk
for under treatment 14. In the emergency room pediatric patients are commonly exposed
to paracetamol for the reduction of pain. The prescription is mostly based on the
recommendations in books, which also includes the current best practice based solely on
experience rather than on scientific basis 15. Many of the recommendations involve the use
of unlicensed or off label medicines. Unlicensed medicines are used as an alternative for
the lack of licensed medicines. Off label is defined as medicines used without available
pediatric information or contraindication in children or that the drug is used different as is
indicated in the literature 15, 17. A study in a Spanish emergency room showed that 70% of
the drugs administered to pediatric patients were in an off-label manner 17.
A total daily dose of 90 mg/kg per day for paracetamol either orally or rectally for 48hr is
recommended in order to provide an adequate analgesic level of the drug and thus relieve
the patient of postoperative pain. 30% of all off label prescription is related to the
prescription of paracetamol, from which 20% is prescribed to children resulting in high or
25
low dosage exposure 14. Prescription of paracetamol to children is mostly based on the
concerns of potential hepatotoxiciy (observed at doses of 150mg/kg per day and above)
rather than the pk properties of the drug. In the literature there are PK properties for
paracetamol in children reported, however it is only valid for ≥5 years old children. Little
information is available for neonates and infants.
A clinical study involving children of 5-15 years of age having a tonsillectomy, determined
the pk properties of paracetamol. The children were orally exposed to 22.5 mg/kg
paracetamol after they had the surgery in order to determine the efficacy of the drug in
the reduction of pain. The obtained pk properties were T1/2 = 15H, Cmax = 12.7 µg/ml,
V=1.06 L/kg and CL = 0.68 L/H/kg, which is in concordance to the properties found in the
literature for adults (T1/2 = 2H, Cmax = 0.02 ng/ml, V=1.0 L/kg and CL = 0.3 L/H/kg) with
the exception of T1/2 and Cmax 18. Another pediatric study investigated the pharmacokinetic
changes in children of different ages. The study showed that the total body of clearance for
paracetamol increases whereas the volume of distribution decreases after birth. Clearance
of a newborn is estimated to be 62%, whereas the volume of distribution is 174% of older
children. Clearance of paracetamol is estimated to be 0.15 L/H/kg in neonates and 0.37
L/H/kg in infants. The study also estimated that for reaching target concentration higher
than 10 mg/L exposures of 45 mg/kg per day is needed at birth, whereas for 5 and 8 years
old children the exposure needed are 90 mg/kg and 75 mg/kg per day respectively 19.
Target concentration of 10 mg/L is defined as the steady-state concentration needs to
reduce 50% of the maximum possible temperature observed in febrile children and is also
the effect site concentration associated with pain scores less than 6/10 in children after
undergoing for example a tonsillectomy.
The pharmacokinetics of a drug is very important to determine the proper therapeutic
dosage for a drug to elicit its effect. The effects of a drug can be further explained by
studying the mechanism of action of the drug and the pharmacodynamic properties. The
pharmacodynamics of a compound should elicit how the compound is distributed around
the body of the subject and therefore determine in which compartment it mostly exerts its
effect. The mechanism of action of paracetamol for instance is well studied. However due
to the results it is still debated and elusive, indicating that the precise mechanism of action
is still not known. Among the different mechanism of action it is thought that paracetamol
is metabolized into an active metabolite after it crosses the blood-brain barier and thus
enters the central nervous system (CNS). Concentrations of 1.3–18 mg/L have been
detected in the cerebrospinal fluid of children undergoing surgery after a single
intravenous injection of 15 mg/kg paracetamol, whereas the plasma concentrations ranged
between 2.4 and 33 mg/L 20. The active metabolite will then promote endogenous
cannabinoid activity that results in a prostaglandin-mediated cyclo-oxygenase enzyme 5
(COX-5) effect 21. The analgesic capacity of paracetamol is associated with the availability
of cannabinoid receptors in the nervous system. Blockage of the cannabinoid CB1 receptor
prevents the analgesic effect of paracetamol 22. Further mechanism for the analgesic
capacity of paracetamol includes the suppression of the signal transduction from the dorsal
superficial layers to the spinal cord. Metabolites of paracetamol e.g. NAPQI, act on the
TRPA1-receptor in the spinal cord inhibiting the signal transduction pathway 16.
Besides the ability of paracetamol to reduce fever and pain in the treated patient, it can
also induce adverse effects. A study showed the use of paracetamol in the first year of life
is associated with a dose-dependent increased risk of asthma symptoms 23. Medium and
high use of the paracetamol resulted in an odss ratio (OR) of 1.61 [95% confidence
interval: 1.36-1.56], whereas no use resulted in OR 3.23 [95%-CI: 2.91-3.60]. The
calculated population-attributable risks rang between 22% and 38%, making paracetamol
a risk factor for the development of asthma in children.
26
Pediatric microdosing
Children are mostly treated by drugs that, although its usage is approved in adults, it is
not yet approved for use in this group of the population. The unapproved usage of these
drugs in children is mainly due to incomplete labeling guidelines. This can lead to
therapeutic incidences, such as for example the incidence with the drug thalidomide. In the
1960s several children were born with stunted arms or legs due to exposure of the mother
to thalidomide for the treatment of morning sickness during the pregnancy 12. This disaster
was the leading cause that triggered the regulatory affairs to facilitate the examination of
drugs in this sensitive group of the population. The involvement of children in clinical trials
has increased by approximately 1.5 fold between the years 2000 and 2006 12.
Pharmaceutical industries are paying to obtain pediatric data, however the barrier of
obtaining the consent of parents to use a drug in an off label manner on their children
(especially in studies using a placebo) still persists. Although a clinical study is a wellcontrolled study, not knowing what will happen to their children after exposing them to
such as drug certainly disturbs them.
A microdose on the other hand is known to be harmless because of the low dose used in
the study. However this does not imply that parents will give their consent easier. It is still
their child and depending on their interest in science will give permission or not.
Performing a pediatric microdose study will give information about for example the
pharmacokinetics in children, which then can be used to determine if further studies for
that compound are worth or not. If data from the microdose study demonstrates that the
compound is easily cleared from the body or maybe not absorbed then it is not worth
starting a clinical phase 1 study without changing the exposure properties of the
compound. Therefore a microdose study can determine if the compound that is approved
for usage in adults can be used for treating children.
Traditionally the dosage of drugs for children is obtained by correcting the dosage of the
drug in adults by the bodyweight and/ or body surface area such as mentioned in the
chapter pediatric drug development. However this can lead to an underexposure or
overexposure of children to the drug, depending on the physical properties of the children
for example children with obesitas. On the other hand correction for bodyweight and/or
body surface area does not consider the pharmacokinetic differences between adults and
children. As summarized in the chapter differences in childern vs adults, children have for
example a reduced amount of P-glycoprotein compared to adults, which indicates that
excretion of compound through this pathway will be reduced in children meaning that the
amount of drug in the blood circulation will be elevated compared to the level observed in
adults. The absolute bioavailability for the compound can be determined from the
microdose study, which is eventually the dose that exerts the effect eventually. From a
microdose study also the Cmax and Tmax for the compound can be determined, referred
as maximum concentration and time elapsed to reach maximum concentration. This
information can then be included for example a PBPK model to determine the effect or fate
of the compound in children. By running simulations of children of different ages exposed
to the dose normalized for the weight obtained from adult data, the exposure of the cells
or organ compartments can be predicted. Determining therefore if age-specific dose
changes are necessary to achieve a relatively constant drug exposure in adults and
children 32.
27
Conclusion
Microdosing is a promising approach that helps reduce the failures of drugs during the
developmental process and therefore also reduces the high developmental costs. More
drug candidates could be tested for the same developmental cost, indicating that a great
variety of drugs may be placed in the market. Therefore more drug variations are available
for patients in case of adverse effects due to ingredient present in the formulation of the
medicine. This is particularly the case with allergic reactions to certain compounds. Besides
for the development of drugs in adults, microdosing could also be used to predict PK
properties and proper dosage of drugs in children. It is extreme difficult to start a clinical
trial with children because of the ethical implications. However with such a low dose that is
considered to be safe, it should be possible to start a clinical trial with children. This would
help in the development of proper drugs for children. Drugs with better PK properties in
children should be more efficient for treatment. Determination of the PK properties of a
compound in children can also lead to proper estimation of the therapeutic dosage. These
properties can then be included in the PBPK modeling technique for simulation of the
weight-normalized dosage obtained from adult data to determine the exposure of the
different organ compartments. From results it can be estimated whether correction for
age-specific dose changes should be performed in order to obtain the same constant drug
exposure as observed in adults.
28
Discussion
Before a microdose study can be carried out, the candidate drug should pass a variety of
test to assure the safety of the subjects that will be exposed to the compound. One of the
tests describes the establishment of the NOAEL and MTD in one animal species. This
partially contradicts as mentioned in the introduction the aim of the microdosing approach
in reducing the amount of animals needed for testing of the compounds in an experimental
setting. Despite the extreme concern of safety, it should be noticed that the regulatory
agencies defined a microdose to be a 100th of the predicted pharmacological dose or
100µg. Therefore it could be questioned whether the amount of drug needed for the test
can be calculated by applying the standard toxicological safety factor of 100, after
considering the weight of the subjects 1, 15. This would result in less animal studies needed
before a microdose study can take place.
The microdosing approach looks very promising for the development of new drugs for the
children. Because the NOEL and MTD is determined before a microdose study can occur, it
can be considered to be safe to be tested in children. Allot of drugs that are being
administered to children has never been tested on children. The dosage of such drugs is
mostly based on correction of the therapeutic adult dose for the bodyweight and
bodysurface area. In this case a microdose study could help determine the proper dose for
children. The pharmacokinetics of drugs in human is shown to vary according to the age of
the subject. Great variability is shown within the group of children, namely in infants.
Therefore dosage of drugs corrected from adult therapeutic dose could lead to underdosing
or overdosing in combination with its related effect in children. Microdosing in combination
with PBPK modeling technique could determine the behavior of the drugs after the children
are exposed, gaining more information about the dose-effect relationship based on
scientific evidence rather than only the results observed from years of experience.
Correcting of the therapeutic adult dose for dosing in children could lead to problems in the
case of obese children. The actual bodyweight of obese children may be in the range of the
adult bodyweight, indicating that these children may receive doses outside the allowed
dosing regimen for children of the same age. This is likely to occur in drugs that are
already licensed as for example with paracetamol. The amount of paracetamol used to
relieve pain in children after having an operation procedure is extremely high, compared to
the dose prescribed in the label of the drug. As already mentioned children are less
sensitive to a paracetamol overdose compared to adult, due to their rapid ability to
eliminate the compound.
90% of premature babies in a healthcare setting are exposed to drugs that are either
unlicensed or used in an off label manner. Unlicensed drugs are used as an alternative
when no commercially produced licensed drug is available 15.
This is extreme dangerous due to the lack of information about the dose-effect relationship
and adverse effects in babies. Children have always been considered as the most delicate
and vulnerable group for toxic effects. However they are more likely to be exposed to an
overdose of drugs in an acute or chronic manner depending on the treatment and thus are
also exposed to its toxic effect. For a proper risk assessment it should be determined if the
internal exposure of children is within the drug’s therapeutic window, or if it is more likely
to exceed the NOAEL of the compound 10.
The toxicological data obtained from animal studies used to determine the dosing regimen
in humans is obtained from studies conducted on adult animals. As in the case of humans
the physiological aspects can also differ in animals of different ages. Indicating that the
observed values in adults is not necessary the same for the younger animals. Research for
the physiological changes in animals from different ages is minimal, whereas in humans it
is well studied. Traditionally during the development process of drugs, all compounds are
first tested in animals before it can be tested in a clinical setting on humans. Therefore
dosing of animals in the first days of life would give more information about the
sensitivities in children due to their immature ADME-relevant proteins.
From the studies also the toxicological data such as the effective dose concentration
(EC50), NOAEL and margin of safety can be determined. When extrapolating these data
obtained from animal studies to predict the values in the human population it is important
to note the slope of the dose-response curve. The slope can be linear or curvilinear and
29
determines the toxicity of the compound. The NOAEL obtained from the studies is then
used to calculate the acceptable daily intake of the drug and also to calculate the reference
dose (RfD) for chronic oral exposure. This is achieved by dividing the NOAEL by
uncertainty factors (UF) and modifying factors (MF) from the selected study for a specific
critical endpoint. RfD = NOAEL / (UF x MF) 4, 24. A problem associated with using the
NOAEL to determine the RfD is that the dose must be a dose that has been tested during
the experimental phase and resulted in no observed effect. This indicates that the shape of
the dose-response curve in the lower part of the curve is ignored and unknown.
The benchmark dose (BMD) approach can in that case help resolve the problem. The BMD
approach models the dose-response curve by the software, from which the 95%confidence interval of a dose associated to a benchmark response (BMR) level is
calculated. The BMR is usually 1-10%, and therefore it indicates the dose associated with a
low level of risk, such as 1-10% 4, 24. The 95%-confidence interval of the BMD can be
divided by the lower range (BMDL) and the upper range (BMDU) of the interval. By looking
at the ratio of BMD associated to different BMR, such as for example 5% and 10%, the
steepness of slope of the dose-response curve could be established. This could be applied
to determine the toxic impact of a compound in a population group.
30
Reference list
1. Lappin G, Garner RC. Innovation: Big physics, small doses: the use of AMS and PET in
human microdosing of development drugs. Nature reviews drug discovery. 2003;2:p2338p.
2. Sugiyama Y, Yamashita S. Impact of microdosing clinical study - Why necessary and
how useful? Adv Drug Deliv Rev. 2011;63:494-502.
3. R.Colin G. Less is more: the human microdosing concept. Drug Discov Today.
2005;10:449-451.
4. Curtis D. Klaasen, John B. Watkins III. Casarett & Doull's Essentials of Toxicology.
the McGraw-Hill companies, Inc.; 2003.
5. Outcomes from EUMAPP- A study comparing in vitro, in silico, microdoe and
pharmacological dose pharmacokinetics. . 2007.
6. Grass GM, Sinko PJ. Physiologically-based pharmacokinetic simulation modelling. Adv
Drug Deliv Rev. 2002;54:433-451.
7. Wilding IR, Bell JA. Improved early clinical development through human microdosing
studies. Drug Discov Today. 2005;10:890-894.
8. Laurence L. Brunton, John S. Lazo, Keith L. Parker. Goodman & Gilman's: The
Pharmacological Basis of Therapeutics. 11th ed. ; 2006:1794.
9. Wolfgang A. Ritschel, Gregory L. Kearn. Handbook of Basic Pharmacokinetics
Including Clinical Applications. ; 2009:184-423.
10. de Zwart LL, Haenen HEMG, Versantvoort CHM, Wolterink G, van Engelen JGM, Sips
AJAM. Role of biokinetics in risk assessment of drugs and chemicals in children. Regulatory
Toxicology and Pharmacology. 2004;39:282-309.
31
11. Ginsberg G, Hattis D, Sonawane B. Incorporating pharmacokinetic differences between
children and adults in assessing children's risks to environmental toxicants. Toxicol Appl
Pharmacol. 2004;198:164-183.
12. Andrew E. Mulberg, Steven A. Silber, John N. van den Anker. Pediatric Drug
Development: Concepts and Applications. USA: Hoboken, New Jersey : Wiley-Blackwell;
2009.
13. Berde CB, Walco GA, Krane EJ, et al. Pediatric Analgesic Clinical Trial Designs,
Measures, and Extrapolation: Report of an FDA Scientific Workshop. Pediatrics.
2012;129:354-364.
14. Kazouini A, Mohammed BS, Simpson CR, Helms PJ, McLay JS. Paracetamol prescribing
in primary care: too little and too much? Br J Clin Pharmacol. 2011;72:500-504.
15. Conroy S, Peden V. Unlicensed and off label analgesic sue in paediatric pain
management. Paediatr Anaesth. 2001;11:431.
16. Andersson DA, Gentry C, Alenmyr L, Killander D, Lewis SE, Andersson A, Bucher B,
Galzi J-L, Sterner O, Bevan S, Högestätt ED, Zygmunt PM. TRPA1 mediates spinal
antinociception induced by acetaminophen and the cannabinoid Δ9tetrahydrocannabiorcol. Nature Communications 2: 551. 2011.
17. Morales-Capri C, Estan L, Rubio E, Lurbe E, Morales-Olivas FJ. Drug utilization and
off-label drug use among Spanish emergency room paediatric patients. Eur. J. Clin.
Pharmacol. 2010:315-316, 317, 318, 319, 320.
18. Romsing J, Ostergaard D, Senderovitz T, Drozdziewicz D, Sonne J, Ravn G.
Pharmacokinetics of oral diclofenac and acetaminophen in children after surgery. Paediatr
Anaesth. 2001;11:205.
19. Anderson BJ, Woollard GA, Holford NHG. A model for size and age changes in the
pharmacokinetics of paracetamol in neonates, infants and children. Br J Clin Pharmacol.
2000;50:125.
32
20. Kumpulainen E, Kokki H, Halonen T, Heikkinen M, Savolainen J, Laisalmi M.
Paracetamol (Acetaminophen) Penetrates Readily Into the Cerebrospinal Fluid of Children
After Intravenous Administration. Pediatrics. April 2007;119:766-771.
21. Elaine M. W. Systemic analgesics in children. Anaesthesia & Intensive Care Medicine.
2010;11:217-223.
22. Ottani A, Leone S, Sandrini M, Ferrari A, Bertolini A. The analgesic activity of
paracetamol is prevented by the blockade of cannabinoid CB1 receptors. Eur J Pharmacol.
2006;531:280-281.
23. Beasley R, Clayton T, Crane J, et al. Association between paracetamol use in infancy
and childhood, and risk of asthma, rhinoconjunctivitis, and eczema in children aged 6–7
years: analysis from Phase Three of the ISAAC programme. The Lancet. 2008;372:10391048.
24. Ernest Hodgson. A Textbook: Modern Toxicology. 4th ed. John Wiley & Sons, Inc;
2010.
25.
www.ICH.org M3 Safety Guidelines.
26. Kannan Krishnan, Melvin E. Andersen. Quantitative Modeling in Toxicology. WileyBlackwell 2010.
27. Albert P. Li. Screening for human ADME/ Tox drug properties in drug discovery. Drug
Discovery Today. 2001 ; 6 (7) : 357-366.
28. Mitchell N. Cayen. Early Drug Development : Strategies and Routes to First-inHuman Trials. Wiley & Sons, Inc. 2010.
29. Chuang Lu, Ping Li, et al. Comparison of intrinsic clearance in liver microsomes and
hepatocytes from rats and humans: evaluation of free fraction and uptake in hepatocytes.
Drug metabolism and disposition. 2006; 34 (9): 1600-1605.
33
30.
http://www.ncbi.nlm.nih.gov/books/NBK22430/
31.
http://www.graphpad.com/prism/tutorials/lineweaver-burk/linwvrburk.htm
32. Feras Khalil and Stephanie Laer. Physiologically based pharmacokinetic modeling :
methodology, applications, and limitations with a focus on its role in pediatric drug
development. Journal of biomedicine and biotechnology. 2011 (2011), article ID 907461,.
13 pages.
34