Drug Metabolism and Pharmacokinetics xxx (2016) 1e10

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Drug Metabolism and Pharmacokinetics

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pharmacokinetics

Regular article

Development of a permeability-limited model of the human brain and

cerebrospinal fluid (CSF) to integrate known physiological and
biological knowledge: Estimating time varying CSF drug
concentrations and their variability using in vitro data
Lu Gaohua a, *, Sibylle Neuhoff a, Trevor N. Johnson a, Amin Rostami-Hodjegan a, b,
Masoud Jamei a
a
Simcyp Limited (a Certara Company), Blades Enterprise Centre, John Street, Sheffield, S2 4SU, UK
b
Manchester Pharmacy School, Stopford Building, Oxford Road, Manchester, M13 9PT, UK

a r t i c l e i n f o a b s t r a c t

Article history: A 4-compartment permeability-limited brain (4Brain) model consisting of brain blood, brain mass,
Received 9 November 2015 cranial and spinal cerebrospinal fluid (CSF) compartments has been developed and incorporated into a
Received in revised form whole body physiologically-based pharmacokinetic (PBPK) model within the Simcyp Simulator. The
4 March 2016
model assumptions, structure, governing equations and system parameters are described. The model in
Accepted 27 March 2016
particular considers the anatomy and physiology of the brain and CSF, including CSF secretion, circulation
Available online xxx
and absorption, as well as the function of various efflux and uptake transporters existing on the blood
ebrain barrier (BBB) and blood-CSF barrier (BCSFB), together with the known parameter variability. The
Keywords:
Brain
model performance was verified using in vitro data and clinical observations for paracetamol and
Transporter phenytoin. The simulated paracetamol spinal CSF concentration is comparable with clinical lumbar CSF
Lumbar puncture data for both intravenous and oral doses. Phenytoin CSF concentrationetime profiles in epileptic patients
Bloodebrain barrier (BBB) were simulated after accounting for disease-induced over-expression of efflux transporters within the
Blood-cerebrospinal fluid barrier (BCSFB) BBB. Various ‘what-if’ scenarios, involving variation of specific drug and system parameters of the model,
Physiologically-based pharmacokinetic demonstrated that the 4Brain model is able to simulate the possible impact of transporter-mediated
(PBPK) model drug-drug interactions, the lumbar puncture process and the age-dependent change in the CSF turn-
In vitro-in vivo extrapolation (IVIVE)
over rate on the local PK within the brain.
Inter-individual variability
© 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. This is an open access article
Modelling and simulation
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction integration of existing information on various aspects of drug

Recently, the use of human physiologically-based pharmacoki-
netic (PBPK) models has had a resurgence within pharmaceutical
companies both in drug discovery and development [1e5]. In many
cases these applications were answering questions which might
have otherwise required extensive clinical studies or where the
conducts of such studies were not possible. Further, regulatory
agencies use these PBPK models for assessing applications made by
pharmaceutical companies both in the IND and NDA stages [6e8].
The value of PBPK models which provide a framework for
* Corresponding author.
E-mail address: gaohua.lu@certara.com (L. Gaohua).
characteristics with the knowledge of physiology and biology has
been highlighted previously [9]. Developing a detailed brain PBPK
model and applying it to simulate and predict brain exposure to
drug candidates is still a challenge, mainly due to lack of adequate
brain anatomical and physiological data and suitable human clin-
ical data to evaluate the model's performance.
Ball and co-workers developed a rat PBPK model dividing the
brain into 2 compartments, namely brain vasculature and brain
tissue [10]. While the cerebrospinal fluid (CSF) and the blood-
cerebrospinal fluid barrier (BCSFB) were not included, the model
was evaluated using rat brain microdialysis data for morphine and
oxycodone. The PBPK model structure and in vitro-in vivo
extrapolation (IVIVE) approach were extended to human but the
model has not yet been validated due to lack of human brain

http://dx.doi.org/10.1016/j.dmpk.2016.03.005
1347-4367/© 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
2 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10

morphine and oxycodone clinical PK data [10]. A brain PBPK
model with 4 CSF sub-compartments was developed by West-
erhout and co-workers based on rat brain microdialysis data for
paracetamol [11]. The model was then fitted to the human lumbar
CSF paracetamol data by adjusting model parameters (clearances
between plasma and brain compartments). However, to use such
models in prospective studies, in particular at the drug discovery
stage, their performance should be proven using independent
observed data that were not used to build the model. Moreover,
the predictions should include the measures of population pa-
rameters (physiology, biology and anatomy) including their vari-
ability so that a confidence interval can be built around any given
prediction.
The aim of this study is to provide a detailed description of a 4-
compartmental permeability-limited brain (4Brain) model, which
integrates the known physiology and biology of the human cere-
brospinal system relevant to drug disposition. This 4Brain model is
nested within the human whole body PBPK model of the Simcyp
Simulator [9,12e15]. To evaluate the performance of the model,
paracetamol and phenytoin were selected as two model com-
pounds due to availability of observed concentrations in both brain/
CSF and plasma. These two compounds signify distinct mechanisms
of brain penetration. Paracetamol penetrates into brain purely via
passive permeability [16] whilst active transport is known to
contribute to brain distribution of phenytoin, a substrate of the
human Permeability-limiting glycoprotein transporter (P-gp) [17].
(Supplementary 1). Based on the anatomy and physiology of the
human brain, the 4Brain model (Fig. 1) was developed assuming:
✓ The whole brain is represented by 4 compartments, namely
brain blood, brain mass, cranial CSF and spinal CSF.
✓ The BBB is a barrier between brain blood and brain mass and
BCSFB between brain blood and cranial CSF. Passive, i.e. non-
saturable, non-inducible and non-inhibitable, penetration
across these barriers is defined using the permeability-surface
area product.
✓ Transport across BBB and BCSFB depends on the transporter
location and its direction and is described by overall efflux/up-
take transporter clearance.
✓ The barrier between brain mass and cranial CSF (brain-CSF
barrier) is of high passive permeability and without any
transporter.
✓ No barrier exists between the cranial CSF and the spinal CSF
while the CSF shuttle flows link the two together.
✓ Metabolic clearance due to brain enzymes is only considered
within brain mass.
✓ All compartments are well-stirred with constant volumes.
✓ CSF is circulated within various intracranial compartments and
reabsorbed into the brain blood compartment, thus fluid bal-
ance is maintained in the model.
✓ Unbound and unionized drug can passively pass through all
barriers while transporters act upon unbound drug (both
unionized and ionized species).

2. Materials and methods

2.1. Model assumptions
The current knowledge available on the ultrastructure of human
brain and its surrounding CSF system has been reviewed
2.2. Model structure and fluid balance
The equations describing the fluid balance within the 4Brain
model are given in details in Supplementary 2, while the human
whole body PBPK model has been described in previous report [12].

Fig. 1. The 4-compartmental brain model within the human whole body PBPK model. The blood and CSF flows are shown while the fluid flow and drug mass conservation are
enforced. The 4Brain model consists of brain blood, brain mass, cranial CSF and spinal CSF compartments. QSin and QSout denote the CSF shuttle flow rate from the cranial to the
spinal part and vice versa, respectively. QCsink and QSsink denote the CSF flow out of the cranial and spinal CSF compartments, respectively. QBCSFB represents the CSF secretion by the
choroid plexus and Qbulk represents the bulk flow from the brain mass to the cranial CSF. QBBB represents the water transfer from the BBB because of the hydrostatic pressure
between the brain blood and brain mass compartments. Qmet represents the water generation due to the glucose metabolism within the neurons, which is considered as water
permeation from brain blood to brain mass via BBB to maintain fluid balance within the model. CLBin, CLBout, CLCin and CLCout are the overall clearance of uptake and efflux
transporters expressing at BBB and BCSFB. PSB, PSC and PSE are passive permeability-surface area products for the three barriers, BBB, BCSFB and brain-CSF barrier, respectively.
CLmet (L/h) is the metabolic clearance due to brain enzymes. Qbrain is cerebral blood flow through which the 4Brain model is connected to the human whole body PBPK model.

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10 3

2.3. Governing equations The bulk flow from brain to cranial CSF is estimated to be about
1/4 of total CSF production [20,24]. The CSF flow from the spinal to
Drug disposition in various brain compartments are described the cranial part is reported as 7 mL/h [25].
based on the mass conservation law, as detailed in Supplementary
3. 2.4.1.3. pH. The pHs of the interstitial fluid, intracellular fluid, CSF
and the plasma are 7.44, 7.05, 7.31 and 7.41, respectively [23].

2.4. Model parameters
2.4.1. System-related parameters
Various system-related parameters were collected from the
literature for adult healthy Caucasian subjects and after meta-
analysis the mean and inter-individual variability values were
incorporated into the model.
2.4.1.4. Brain transporter. Major apical efflux transporters at the
BBB are the Adenosine-triphosphate (ATP) dependent transporters:
P-gp, MRPs and BCRP. Their functionality along with any other
possible efflux and/or uptake transporters at BBB and/or BCSFB is
considered in the 4Brain model.
2.5. Model verifications

2.4.1.1. Volume. In adults, the intracranial blood volume is about
75 mL [18]. The cranial CSF compartment is around 130 mL and
spinal CSF about 30 mL [19]. There is huge inter-individual vari-
ability in CSF volumes. For example, the lumbar volume in younger
healthy individuals varied between 28 and 81 mL, with a mean of
50 mL [20]. Data from Magnetic Resonance Imaging (MRI) in 64
healthy volunteers aged 18e64 years indicate wide variations in
the total cranial CSF volume (57e286 mL), as well as gender dif-
ference (males 146 mL vs. females 115 mL) [21].
In order to assess the 4Brain model performance, the model was
used to simulate paracetamol and phenytoin concentrations in
brain/CSF. The model building follows the workflow as shown in
Supplementary 4 Figure 4.1. The main focus of the workflow is to
deal with the drug-related aspects while the system parameters are
already included in the 4Brain model. For these two example
compounds, either the first order absorption model or the
Advanced Dissolution, Absorption and Metabolism (ADAM) model,
the whole body PBPK model and the 4Brain model in the Simcyp
Population Based Simulator (Version 14 Release 1) were used.

2.4.1.2. Flow rate. The rate of CSF formation in humans is
0.3e0.4 mL/min, or 430e580 mL/day [22]. It takes about 5e7 h to
replace the total CSF volume [23]. The secreted CSF is absorbed
completely into the systemic circulation through two pathways, the
cranial CSF absorption and the spinal CSF absorption, the latter is
about 38% of total CSF production in human [20].
2.5.1. Drug-related parameters
In vitro and PK parameters for the two test compounds, para-
cetamol and phenytoin, were collated from the literature (Tables 1
and 2). Together with the physicochemical parameters, absorption,
distribution and elimination data for the human whole body PBPK
model, the drug-related parameters for the 4Brain model include:

Table 1
Drug-related parameters used for paracetamol simulations.

Parameter Description Value Unit Reference Note

Physicochemical
MW Molecule weight 151.165 g/mol [46]
logP Octanol: buffer partition coefficient 0.46 [46]
pKa Acid dissociation constant 9.38 [46] Monoprotic Acid
a
BP Blood-to-plasma partition ratio 1.09
fu Fraction unbound in plasma 0.855 [47]
Absorption
S Intrinsic solubility 11.4 mg/mL [46]
fugut Unbound fraction of drug in gut enterocytes 1 Simcyp default assumption
PCaco-2 Caco-2 permeability 31.6
106 cm/s [48] (6.5:7.4), calibrated using verapamil data
from the same study.
Peff,man Human jejunum permeability 2.27
104 cm/s Simcyp prediction based on Caco-2 data
Distribution
Vss Distribution volume at steady-state 0.84 L/kg [49] Prediction based on Kps and tissue
volumes, same as clinical observation
(using Simcyp Kp prediction Method 2).
Elimination
CLiv Intravenous clearance 22.8 L/h [50]
CLr Renal clearance 0.78 L/h [50]
CLint-HLM Human liver microsomal protein 8.44 mL/min/mg Simcyp retrograde calculation
in vitro intrinsic clearance protein based on CLiv and CLr.
Brain transport
b
PSB Passive permeability-surface area product on the BBB 1.875 L/h [51]
PSC Passive permeability-surface area product on the BCSFB 0.9375 L/h [51] Half of PSB was assumed.
PSE Passive permeability-surface area product on the 300 L/h Assumed so it is not a barrier.
brain-CSF barrier
fubm Fraction unbound in brain mass 0.747 [47,52] The same as Simcyp prediction.
fucsf Fraction unbound in CSF 1 Assumed based on low protein
concentration in CSF.
Brain metabolism
CLmet Brain metabolic clearance 0 L/h Assumed.
a
Calculated from BP ¼ 1Ht þ EP*Ht where Ht ¼ 0.45, EP ¼ 1.2 [50].
b
Based on PS ¼ 25 mL/h/kg body weight, assuming 75 kg body weight.

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
4 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10

Table 2
Drug-related parameters used for phenytoin simulations.

Parameter Meaning Value Unit Reference Note

Physicochemical
MW Molecule weight 252.28 g/mol [53]
logP Octanol: buffer partition coefficient 2.47 [53]
pKa Acid dissociation constant 8.15 Monoprotic Acid
BP Blood-to-plasma partition ratio 0.61 [53]
a
fu Fraction unbound in plasma 0.3 [54,55]
Absorption (Not used in the simulations)
Distribution
Vss Distribution volume at steady-state 0.64e1.98 L/kg Prediction based on tissue
volumes and tissue:plasma partition
coefficients (using Simcyp Kp prediction
Method 2), adjusted with Kp scalar for
each of 6 subjects.
Elimination
Vmax-2C9 Maximum rate of metabolite formation 0.24 pmol/min/pmol [53,56]

Km-2C9 MichaeliseMenten constant 4.1 mM [53,56]

Vmax-2C19 Maximum rate of metabolite formation 1.53 pmol/min/pmol [56]
Km-2C19 MichaeliseMenten constant 36.8 mM [56]
CLint-HLM In vitro intrinsic clearance of microsomal protein 0.97 mL/min/mg protein [56]
CLr Renal clearance 0.015 L/h [56]
Brain transport
b
PSB Passive permeability-surface area product on the BBB 46.44 L/h
PSC Passive permeability-surface area product on the BCSFB 23.22 L/h Half of PSB was assumed.
PSE Passive permeability-surface area product on 300 L/h Assumed so it is not a barrier.
the brain-CSF barrier
fubm Fraction unbound in brain mass 0.0967 [57] Rat data
fucsf Fraction unbound in CSF 0.993 [57] Rat data
CLBout Clearance of active efflux transporter on the BBB 1.8e216 L/h ASA and PE on clinical data.
Brain metabolism
CLmet Brain metabolic clearance 0 L/h Assumed.
a
There was a high inter-individual variability in the free phenytoin concentrations reported in epileptic patients, for example, 6.7e33.3% [55] and 6.8e35.3% [54]. For this
exercise, fu is set to 0.3. In Wilder's study [32], neither the free fraction in plasma (fu), brain mass (fubm) and CSF (fucsf) of the 6 epileptic subjects, nor the serum albumin levels,
were reported. The rat data are used in these simulations mainly due to the high correlation between the brain tissue binding in different species (fubm is 0.095 and 0.097 in
humans and rats respectively) [57,58].
b
Calculated from PS ¼ 0.0086 mL/s/g brain weight, assuming brain weight 1500 g (Personal communication with Dr. Scott G. Summerfield).

✓ passive permeability-surface area product on BBB, BCSFB, as
well as the brain-CSF barrier;
✓ transporter-mediated clearances on BBB and BCSFB; and
✓ metabolic clearance due to brain enzymes.
2.5.2. Paracetamol case study
Paracetamol is known to penetrate into the brain only via pas-
sive permeability; hence no transporter is involved in its penetra-
tion into the brain [16]. Three independent clinical adult studies
have reported the lumbar CSF data together with the systemic
plasma data [16,26,27]. These studies were based on a standard
dose regimen of 1000-mg paracetamol intravenous infusion over
15 min, with a combination of younger and elderly subjects (18e84
years), males (n ¼ 39) and females (n ¼ 22), patients (n ¼ 55) and
healthy volunteers (n ¼ 6). In particular, 6 male healthy volunteers
(18e45 years) were enrolled in Singla's study, where intravenous
dose was compared to an oral or rectal dose [16]. Altogether, the
data in these 3 reports have been used to assess the 4Brain model
prediction performance and none of these clinical data has been
used in the model development.
To mimic the clinical study as closely as possible, total 610 vir-
tual subjects (proportion of females 36.1% (22/61), grouped into 10
trails) aged 18e84 years were generated using the North European
Caucasian library population included within the Simcyp Simulator
(Version 14 Release 1). The simulated systemic plasma concentra-
tion and spinal CSF concentration after a standard 1000 mg intra-
venous dose over 15 min were then compared to the clinical
observations [16,26,27].
Furthermore, the Simcyp Healthy Volunteer population was
used to generate 60 virtual male subjects aged 18e45 years old and
these virtual subjects were randomly grouped into 10 trials of 6
subjects in each trial to match the clinical study design in healthy
volunteers [16]. A thousand milligrammes of paracetamol admin-
istered as a solid oral dosage formulation was simulated using the
Simcyp ADAM model [15]. The simulation results were then
compared with the clinical observation [16].
2.5.3. Phenytoin case study
Phenytoin is selected as a model compound where penetration
across the BBB/BCSFB is controlled by both passive permeability
and various active transporters. The observed increased expression
of ATP-dependent efflux transporters on the BBB may be related to
the limited penetration of antiepileptic drugs into brain [28e31].
Rapid penetration of phenytoin from blood into brain was
observed in status epilepticus after intravenous phenytoin admin-
istration [32]. In this clinical study, plasma concentration and spinal
CSF concentration were reported in 6 adult patients receiving
individualized phenytoin therapy in terms of dose amount and
duration (Table 3).
The drug-related parameters in Table 2 are essential to simulate
the phenytoin brain penetration. However the transport clearances
for the BBB/BCSFB efflux/uptake transporters were lacking. For
simplification, it is assumed that all the efflux transporters in the
BBB can be represented by P-gp only and that there are no other
BBB uptake transporters or BCSFB efflux/uptake transporters
contributing to phenytoin penetration into brain. Hence P-gp is
representing the net effect of the active transport.
Sensitivity analysis was used to obtain estimates of the clear-
ance of BBB P-gp (CLBout in Fig. 1 and Table 3). The parameter
estimation (PE) module was used to scale down the tissue-to-

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10
Table 3
Phenytoin clinical design and scaling factor.
Patient Gender Age (years) Dose amount (mg/kg)
Patient 1 Not specified 52 9.3
Patient 2 Male 60 12.5
Patient 3 Male 48 11.6
Patient 4 Male 52 10.0
Patient 5 Male 32 10.9
Patient 6 Male 25 11.1
plasma partition coefficient values (Kp scalar in Table 3) to match
the systemic PK profile of each epileptic patient.
The North European Caucasian virtual population available
within the Simcyp Simulator database was used to generate 100
virtual patients to match each of the clinical 6 epileptic subjects in
terms of age, gender and dose regimen [32]. For example, when
simulating Patient 1, phenytoin is intravenously infused (9.3 mg/kg
over 7 min) in 100 virtual patients (50 males and 50 females) aged
52 years old, while in Patient 2 simulation, phenytoin is intrave-
nously infused at 12.5 mg/kg over 10 min in 100 virtual male pa-
tients aged 60 years old.
2.6. Sensitivity analysis
In order to investigate the impact of different model parameters
on the drug PK in brain/CSF and plasma, sensitivity analysis was
carried out and a range of ‘what-if’ scenarios were investigated as
follows:
 impact of brain transporter inhibition on the brain
concentration.
 impact of frequent lumbar puncture experiment
 impact of age-dependent CSF turnover on local drug
concentration
Full details of this investigation are given in Supplementary 5.
3. Result
3.1. The paracetamol case study
3.1.1. Intravenous dose
The simulated systemic plasma concentration and spinal CSF
concentration in 610 virtual subjects after 1000 mg paracetamol
administrated intravenously over 15 min are compared to the
clinical observations, as shown in Fig. 2.
The paracetamol whole body PBPK model predicts the clinical PK
profiles reasonably well (Fig. 2(a)). The predicted mean maximum
concentration, Cmax, is 40.6 mg/L, which is occurring at the end of
intravenous infusion and about 2-fold higher than the clinical
observation of 21.6 mg/L measured at the same time point. The area
under the concentrationetime profile over the simulated duration
(AUC0e12 h) is 55.3 mg/L h, which is comparable to the clinical
observation of 50 mg/L h [16,26]. The total clearance estimated
based on the given dose and AUC is 20.2 L/h, again in line with the
clinical observation of 20.7 L/h in Singla's study and 22.4 L/h
in Moreau's study [16,27]. Furthermore, as shown in Fig. 2(a), the
reported inter-individual variability in the clinical observations is
within the upper and lower percentile range of the simulated sys-
temic PK profiles.
The simulated spinal CSF concentration is comparable to the
clinical observations (Fig. 2(b)). The peak of the simulated mean
spinal CSF concentration Cmax,CSF is 6.1 mg/L, which is consistent
with the clinical observation 5.9 mg/L [16]. The simulated time at
which Cmax,CSF occurs, Tmax,CSF, is around 2.6 h, which is in line with
Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
5
Dose route Dose duration (min) Kp scalar Vss (L/kg) CLBout (L/h)
intravenously 7 0.30 0.94 1.8
intravenously 10 0.20 0.64 72
intravenously 15 0.40 1.23 216
intravenously 20 0.65 1.98 36
intravenously 15 0.65 1.98 72
intravenously 20 0.35 1.09 108
the clinical observation of 2 h with a range of 1e4 h [16]. The
simulated upper and lower percentiles cover the clinical inter-
individual variability well.
There is a time lag of about 140 min between the maximal
plasma and the maximal CSF concentration after the single intra-
venous dose administration (Fig. 2). This is consistent with the
clinical observations [16,26].
3.1.2. Oral dose
In this simulation, 60 virtual male subjects aged 18e45 years old
were generated using the Simcyp Healthy Volunteer population
library. These virtual subjects are randomly grouped into 10 trials of
6 subjects in each trial to mimic the clinical study design [16]. The
simulation results are shown in Fig. 3.
Fig. 3(a) shows that the simulated population mean systemic
plasma concentration and its percentiles are reasonably predicting
the clinical observations with the exception of variability up to 1 h
after dose. The simulated mean Cmax is 8.3 mg/L, which is compa-
rable with the clinical observation (8.6 mg/L). The simulated Tmax is
1.2 h, almost the same as the clinical observation of 1 h (range
0.5e2 h) [16].
Similarly, in Fig. 3(b) the simulated spinal CSF concen-
trationetime profile and its percentiles are in good agreement with
the clinical observations. The peak of the simulated mean spinal
CSF concentration Cmax,CSF is 3.9 mg/L, which is almost the same as
the clinical observation (3.7 mg/L). The simulated Tmax,CSF in the
spinal CSF PK is 3.9 h, consistent with the clinical observation of 4 h
(range 0.75e6 h).
From Figs. 2(a) and 3(a), giving the same dose (1000 mg), the
simulated maximum concentration of paracetamol in plasma is
about 6-fold lower after oral compared to intravenous dosing,
while this is around 2.5-fold for the clinical observations.
Comparing the spinal CSF concentration in Fig. 3(b) to that in
Fig. 2(b), the Cmax,CSF is also lower after oral compared with intra-
venous dose (3.9 vs. 6.1 mg/L in simulation and 3.7 vs. 5.9 mg/L in
observation, respectively), indicating that the systemic drug con-
centration has been propagated into the local CSF drug concen-
tration. In a similar manner, the earlier the Tmax in plasma
concentration the earlier the Tmax,CSF in the spinal CSF concentra-
tion. These are consistent with the clinical observations, in partic-
ular, a greater CSF penetration following the intravenous compared
to the oral dose and the significant correlation between the spinal
CSF concentration and the plasma concentration [16,33].
3.2. The phenytoin case study
3.2.1. Prediction of spinal CSF concentration
Simulated phenytoin concentration profiles were comparable to
the clinical observations in all 6 epileptic patients (Fig. 4). The large
inter-individual variability of systemic PKs observed in the clinic
has been recovered in the simulations.
Sensitivity analysis showed that the CLBout (the clearance of BBB
efflux transporter P-gp) should be in the same order as PSB (the
passive permeability-surface area product of BBB, which is 46.44 L/
h for phenytoin, Table 2) to recover the observed data. In order to
6 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10

Fig. 2. Paracetamol concentrations in plasma and spinal CSF (prediction vs. observation) after 1000 mg intravenous dose over 15 min, (a) the plasma concentration (b) the spinal
CSF concentration. The bold lines are the mean of the simulated population (n ¼ 610), while the dash lines are the upper (95%) and the lower (5%) percentiles. Clinical observations
are shown by symbols namely, triangle (Bannwarth et al., 1992, n ¼ 43), rectangle (Moreau et al., 1993, n ¼ 12) and circle (Singla et al., 2012, n ¼ 6), together with their reported
standard deviation.

simulate the observed mean CSF concentrationetime profiles for all
6 individuals, it was necessary to set the CLBout value in the range
1.8e216 L/h (Table 3) thus mimicking high inter-individual vari-
ability in the abundance or activity of the transporters involved in
phenytoin BBB penetration. Using these values, the 4Brain model is
able to simulate the mean spinal CSF concentrations observed in
the 6 clinical patients as well as their inter-individual variability
(Fig. 4).
individual variability in the spinal CSF concentration is relatively
high, compared to that in the plasma concentration. The maximum/
mean and minimum/mean concentration ratios for the plasma and
spinal CSF concentrations in the 100 virtual subjects (50 males and
50 females, aged 52 years old) mimicking Patient 1 in the clinical
study are shown in Supplementary 4 Figure 4.2. The simulated
inter-individual variability in spinal CSF concentration is higher
than that of the systemic concentration and consistent with the
clinical observations [32,34].

3.2.2. Simulation of inter-individual variability

In the 4Brain model, based on the meta-analysis of the collated 4. Discussion
data, various coefficients of covariant (CV) have been used to
generate the brain parameters for each virtual subject. As shown in Due to the importance of the brain, the human body has
Fig. 4, in all the 6 simulations (representing patient 1e6), the inter- developed various anatomical and physiological mechanisms to

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10 7

Fig. 3. Paracetamol concentrations in plasma and spinal CSF (prediction vs. observation) after 1000 mg oral dose, (a) the plasma concentration (b) the spinal CSF concentration. The
bold lines are the simulated population mean, while the dashed lines are the upper (95%) and the lower (5%) percentiles of the simulated population (n ¼ 60). The circles represent
the clinical observations from Singla et al., 2012 (n ¼ 6), together with their standard deviation.

protect it from external and/or internal disturbance to ensure its
normal function. The tight junctions and transporters on the BBB
and BCSFB form a protective barrier to the brain, but at the same
time these mechanisms make it challenging to deliver therapeutic
drugs. The presence of the BBB can be the sole reason for clinical
failure of even a highly potent neurotherapeutic agent [35].
From a model-based drug development (MBDD) perspective,
there is a need for mechanistic and reliable brain PBPK models to
predict the distribution of drug candidates within the brain and
CSF, especially when these models can be based on in vitro data
[10]. Various factors including the local metabolism, active trans-
port at the BBB and BCSFB, local changes in brain barrier func-
tionality due to disease or ageing, and co-medication may affect the
local brain distribution of the drug. A brain PBPK model will be
helpful for systemic investigation of relationships between the
drug plasma concentration and the local CSF and brain
concentrations.
The hurdle of predicting the brain exposure using in vitro data in
a brain PBPK model can be overcome if a right balance is struck
between the model complexity, flexibility and usability. The model
should be based on the brain anatomy and physiology and capable
of describing drugs pharmacokinetics in different parts of the brain,
it should be underpinned by sufficient systems and drug data. A
simple perfusion-limited brain model cannot account for the
contribution of tight junctions, transporters and CSF circulation to
the local PK. However, if a brain model is described by too many

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
8 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10

Fig. 4. Phenytoin concentrations in plasma and spinal CSF (simulation vs. observation) after various intravenous doses in 6 patients with status epilepticus. Each of the 6 clinical
epileptic patients is simulated using 100 virtual subjects. The bold line is the mean value of 100 simulated virtual subjects. The plasma concentration for each of the simulated
individual is also shown on each graph by individual lines and clinical observations are shown by rectangles (left figures, plasma) and circles (right figures, spinal CSF). Subjects with
extremely high spinal concentrations in simulations for patients 1, 2, 4 and 5 are characteristic of high QSin and QSout indicating these subjects have high CSF shuttle flow rates
between the cranial and the spinal CSF compartments.

sub-compartments, to represent the various brain or CSF segments,
it is not always possible to provide all the necessary system and
drug data to parameterize the model, at least at the present time.
The current 4Brain model consists of 4-compartments separated
by BBB and BCSFB and linked by CSF circulation. As shown in the
paracetamol example, all the drug-related parameters required can
be generated from in vitro systems. The 4Brain model is also
adequately flexible as demonstrated in the application example of
phenytoin, where the clearance of efflux transporters was esti-
mated from clinical data. Based on this estimation, it is possible to
carry on sensitivity analysis and investigate various ‘what-if’ cases
to assist with understanding factors influencing the local drug
concentration in the brain, as detailed in Supplementary 5.
Recently, in an independent study, Sprague and co-workers re-
ported the application of this 4Brain model in simulating the brain
distribution of atomoxetine and duloxetine [36]. Ball and co-
workers also discussed the advantage of a permeability-limited
brain model within an IVIVE-linked PBPK framework after
comparing the translational population-PBPK to the bottom-up
IVIVE PBPK with a perfusion-limited brain model [37].
Combining the bottom-up and top-down approaches is a
practical paradigm to integrate in vitro and clinically observed data
to improve models' performance [38,39], and thence it is useful to
have fitting and sensitivity analysis features readily available in
addition to the mechanistic model. These generally help in
estimating unknown model parameters, for example in our case
the total net transporter clearance of efflux transporters at the BBB.
Recently, protein expression levels of these transporters are re-
ported in human brain microvessels [40,41] which pave the way
for IVIVE of transporters at the BBB. These data are all from pa-
tients (none from healthy subjects) and usually from studies with a
very small sample size. In order to obtain transporter clearances
using IVIVE techniques, scaling factors are also required. These
factors would ideally account for the differences in the activity and
abundance of transporters in in vitro and in vivo systems. Accurate
data in this area is still lacking, nonetheless encouraging progress
has been made recently [10,40].
Due to obvious ethnical restrictions it is not usually possible to
get brain concentrations in healthy volunteers or patients. Con-
centrations of lumbar CSF, cisternal CSF, and ventricular CSF are
often used as surrogates for the brain concentration in the clinic
[42,43]. However, spatial difference within the brain, due to brain
anatomy and physiology, CSF circulation from ventricle to sub-
arachnoid space and active transporters on BBB/BCSFB, contributes
to the observed differences between the CSF and brain concen-
trations. Individual paracetamol or phenytoin concentrations in
the 4 compartments are very different from each other in all the
simulations. Sensitivity analysis (Supplementary 5) demonstrated
that the spinal CSF concentration itself can be affected by various
internal and external factors and therefore may not be a useful

Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
 L. Gaohua et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e10
surrogate for the concentrations in the brain mass and cranial CSF,
which is consistent with the concern raised by de Lange on the
utility of CSF sampling [43].
The CSF hydrodynamics could be disturbed either acutely by the
lumbar puncture process [44] or slowly over the passage of time by
ageing [45]. Due to missing data, not all the disease-induced dy-
namic changes in system-related parameters (including the cere-
bral blood flow, CSF protein and pH, BBB permeability and so on)
have been explicitly included in the current 4Brain PBPK model,
however, when this becomes available these can be incorporated.
Nevertheless, sensitivity analysis using the 4Brain model has
shown uncertainty or change in the brain transporter (expression
or activity) or brain hydrodynamics can significantly introduce
inter-individual variability to the local PK in the brain.
While the 4Brain model is developed under an IVIVE-PBPK
framework, various questions related to the IVIVE side remain to
be answered. For example, which and how should the in vitro data
for passive permeability or active transport be generated in order to
apply the model in the drug discovery settings? Is it acceptable in
humans to use the same permeability value from in vitro systems
for both the BBB and BCSFB in vivo and correct it only for the
assumed surface area? Is it enough to use in vitro measurements to
simulate in vivo local drug concentration while the IVIVE approach
for transporters has not been fully established to date? Developing
such models facilitates identifying the gaps in the current knowl-
edge and as the required data become available the model appli-
cation and confidence in its performance will increase.
Overall, the 4Brain model can be used at different stage of drug
discovery and development to simulate the local brain concentra-
tion of compounds penetrating into the brain via passive and/or
active transport. The development of this model is a step towards
better understanding of time varying drug concentration in the
brain and CSF. It can be used by researchers in the field to gain
insight into the influence of various system and drug parameters on
brain exposure.
5. Conclusions
A brain physiologically-based multiple-compartmental
permeability-limited (4Brain) model has been developed and the
model structure and parameters are explained. The model perfor-
mance was evaluated using paracetamol in vitro data and the
simulation results were compared with clinical data obtained by
lumbar puncture after intravenous and oral doses. Further the
model performance in simulating the local phenytoin concentra-
tion in the brain was investigated. Sensitivity analyses were carried
out to explore various ‘what-if’ scenarios.
Conflict of interest
Lu Gaohua, Sibylle Neuhoff, Trevor N. Johnson, Amin Rostami-
Hodjegan and Masoud Jamei are employees of Simcyp (a Certara
Company).
Acknowledgements
This work was funded by Simcyp Limited (a Certara Company)
and partially supported by the European Commission, 7th FP
project NEUROBID (grant agreement No. 241778). The Simcyp
Simulator is freely available, following completion of the training
workshop, to approved members of academic institutions and
other non-for-profit organizations for research and teaching pur-
poses. The authors thank Dr. Scott G. Summerfield (GlaxoSmithK-
line R&D) for providing PS data for paracetamol and phenytoin. We
Please cite this article in press as: Gaohua L, et al., Development of a permeability-limited model of the human brain and cerebrospinal fluid
(CSF) to integrate known physiological and biological knowledge: Estimating time varying CSF drug concentrations and their variability
using in vitro data, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.03.005
9
acknowledge Ms. Eleanor Savill (Simcyp Limited) for her assistance
with collecting the references and preparation of the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dmpk.2016.03.005.
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