A modern in vivo pharmacokinetic paradigm: combining snapshot, rapid and full PK approaches to optimize and expedite early drug discovery
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Drug Discovery Today Volume 18, Numbers 1–2 January 2013 REVIEWS
A modern in vivo pharmacokinetic
paradigm: combining snapshot, rapid
Reviews POST SCREEN
and full PK approaches to optimize and
expedite early drug discovery
Chun Li, Bo Liu, Jonathan Chang, Todd Groessl, Matthew Zimmerman,
You-Qun He, John Isbell and Tove Tuntland
Department of Metabolism and Pharmacokinetics, Genomics Institute of the Novartis Research Foundation, Novartis Institute of Biomedical Research, San Diego,
CA, USA
Successful drug discovery relies on the selection of drug candidates with good in vivo pharmacokinetic
(PK) properties as well as appropriate preclinical efficacy and safety profiles. In vivo PK profiling is often a
bottleneck in the discovery process. In this review, we focus on the tiered in vivo PK approaches
implemented at the Genomics Institute of the Novartis Research Foundation (GNF), which includes
snapshot PK, rapid PK and full PK studies. These in vivo PK approaches are well integrated within
discovery research, allow tremendous flexibility and are highly efficient in supporting the diverse needs
and increasing demand for in vivo profiling. The tiered in vivo PK studies expedite compound profiling
and help guide the selection of more desirable compounds into efficacy models and for progression into
development.
High-throughput in vitro absorption, distribution, metabolism and testing and confirmations of in vitro ADME results in early drug
elimination (ADME) assays have been implemented in early drug discovery and, therefore, there is always a continuous demand for
discovery to identify and eliminate compounds with poor drug- in vivo PK studies.
like properties and to promote potential pharmaceutical candi- In vivo rodent PK studies are crucial to ensure compounds have
dates for more labor-intensive in vivo PK profiling [1–5]. Data from appropriate PK properties to be evaluated in preclinical pharma-
in vitro ADME assays often contribute to the understanding of cology and safety studies. In addition, characterization of in vivo
underlying mechanisms of drug absorption and disposition, PK of new chemical entities provides insight into complex in vivo
which have proven invaluable to establish structure–activity rela- biological systems and correlates drug concentration at the site of
tionships (SAR) that guide new chemical synthesis. However, action with pharmacological response. Despite their important
despite the advances in the in vitro technologies and in silico role in drug discovery, most in vivo animal PK studies are still
approaches [6–9] for prediction of in vivo PK parameters, the conducted in a traditional, low-throughput manner in many
predictive power of these approaches is not always reliable and pharmaceutical companies and, therefore, remain the bottlenecks
accurate. Complete reliance on in vitro assays in the absence of an of discovery projects.
in vitro–in vivo correlation (IVIVC) can sometimes mislead or slow In this review, we present three tiered in vivo rodent PK
down the pace of a drug discovery program [10,11]. The PK profile approaches that are highly efficient in supporting early drug dis-
of a compound is governed by many physicochemical and che- covery research. The study designs, strategies and applications of
mical properties of the molecule, such as its lipophilicity, solubi- each of the tiered assays are discussed and compared with other
lity, permeability and metabolic stability. The processes by which a commonly used in vivo rodent PK approaches in the pharmaceutical
compound is absorbed, distributed, metabolized and eliminated in industry. These tiered in vivo rodent PK approaches span from the
vivo through an intact animal or human are often far more com- simple and abbreviated study design of ‘snapshot’ PK [12], to the
plex than in isolated in vitro systems. It is essential to have in vivo more labor-intensive intravenous/per oral (IV/PO) PK study designs,
such as ‘rapid PK’ and the conventional ‘full PK’. Depending on the
Corresponding author: Tuntland, T. (ttuntland@gnf.org) needs and stage of a specific project, different study designs can be
1359-6446/06/$ - see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2012.09.004 www.drugdiscoverytoday.com 71
REVIEWS Drug Discovery Today Volume 18, Numbers 1–2 January 2013
used to answer specific PK questions. In combination, these tiered in and six blood samples are collected serially from each animal.
vivo PK approaches offer tremendous flexibility and continuous Individual plasma samples are analyzed and individual PK curves
support to projects at all stages of discovery process. are reported for each animal, such that inter subject variability can
be assessed. The bioanalytical methods are similar to those
Study designs of the three tiered rodent in vivo PK described in the rapid PK section above, but are more comprehen-
approaches sive. Each compound is tuned for optimum sensitivity and each
Snapshot PK study design method includes two sets of standard curves and three quality-
Detailed information about the snapshot PK study design has been control (QC) samples at low, medium and high concentrations
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described in an earlier publication [12]. Briefly, compounds are within the standard curve range. This is the gold standard in vivo
dosed discretely to two mice or rats via oral gavage, and blood or PK assay widely used by the pharmaceutical industry.
plasma samples at 0.5, 1, 3 and 5 hours post-dose are pooled across
animals. All the steps involved in the PK study process are stan- Discussion
dardized and automated, and PK reports are generated automati- Tiered rodent in vivo PK approaches are adapted at GNF to address
cally and published to an internal database using web-publishing diverse needs of discovery projects at different stages of the drug
tools [12]. discovery process. All the approaches are reviewed and approved
by the Institutional Animal Care and Use Committee (IACUC),
Rapid PK study design and are governed by the 3Rs principle (Replacement, Reduction,
A new in vivo rodent PK paradigm, namely ‘rapid PK’ is introduced and Refinement) to ensure the most appropriate and responsible
as the second tier PK approach at GNF. The in-life portion of the use of animals.
study design is similar to that of a conventional standard full PK
study. A typical rapid PK study of a compound includes both IV Snapshot PK assay performance and its applications
and PO arms with three animals in each dosing arm. Briefly, Snapshot PK, the first tier in vivo PK study approach, utilizes
compounds are formulated on the day of dosing using polyethy- abbreviated blood sampling and sample pooling across the ani-
lene glycol 300 (PEG300):5% dextrose in distilled water (D5W) 3:1, mals (n = 2). Average oral exposure (AUC0–5 h) is reported and test
and the formulation is filtered before IV and PO dosing; alterna- compounds are categorized into low, moderate or high plasma
tively, a 0.5% methylcellulose/0.5% Tween 80 suspension formu- exposure based on the dose normalized AUC0–5 h. The comparison
lation is used for PO administration. Blood samples from six of data from 177 compounds tested in both snapshot PK and
different time points are collected for each animal up to 24 h conventional full PK studies indicates that the snapshot PK assay
via serial blood sampling. For mouse rapid PK studies, blood is efficient and reliable in categorizing their relative oral exposures
samples (50 mL) are taken via retro-orbital or alternatively, by [12]. Recent data from an additional 224 compounds show an
other serial sampling techniques, such as tail vein bleed [13–15] identical trend in exposure comparisons between snapshot PK and
or lateral saphenous vein puncture [16]. The blood samples from follow-up rapid PK studies. As shown in Fig. 1, 75% of compounds
the three mice are pooled and centrifuged to obtain pooled plasma are placed in the correct category and 98% of compounds are
samples. For rat rapid PK studies, individual blood samples placed in the correct or adjacent exposure categories.
(100 mL) are taken from each animal via the saphenous vein. After A successful application of snapshot PK studies is exemplified in
centrifugation, 20 mL of the plasma samples are pooled across the the recent discovery of spiroindolones, a potent compound class for
three rats within a dosing arm. A total of 12 plasma samples per PK the treatment of malaria [17,18]. In vitro screening at Novartis of a
study are obtained after pooling of blood or plasma samples across large library of natural products and synthetic compounds with
three IV group and three PO group animals. structural features found in nature products generated 17 reliable
The pooled plasma samples are diluted appropriately using a hits with reconfirmed submicromolar activity against falciparum
generic dilution scheme to ensure that concentrations are within malaria. When 14 out of the 17 hits were profiled in snapshot PK
the dynamic range of the standard curve (1–5000 ng/mL). Auto- studies at GNF, most compounds exhibited negligible or no oral
mated sample preparation and protein precipitation are carried exposures. One natural product belonging to the spiroazepinein-
out, and up to eight compounds are prepared in a batch. Liquid dole class showed the best oral PK profile and became the starting
Chromatography Mass Spectrometry (LC/MS/MS) analysis is used point for medicinal chemistry lead optimization [17]. Further synth-
with a fast generic gradient elution method together with atmo- esis and evaluations of approximately 200 derivatives yielded the
spheric pressure chemical ionization (APCI) or electrospray (ESI) in optimized spiroindolone analog NITD609 [18], which has improved
the positive or negative ion mode on an API-4000 triple quadruple PK properties relative to the original hit and overall good drug-like
mass spectrometer. The analyte and internal standard are tuned attributes. NITD609 has recently advanced to the initial phase of
automatically using Automaton (now Discovery QuanTM) and the clinical trials and is currently undergoing proof-of-concept testing
analysis is conducted using multiple reaction monitoring (MRM). as an antimalarial agent with a novel mechanism of action.
Data collection and peak integration are performed using Ana-
lystTM 1.4.1 software. Comparison of snapshot PK with other reported approaches
A few similar assays to snapshot PK have been reported and used by
Conventional full PK study design other pharmaceutical companies. A cassette accelerated rapid rat
Conventional full PK studies at GNF include IV/PO PK studies, screen (CAARS) was first introduced by scientists at Schering–
which are similar to those described in the rapid PK section above. Plough [19,20], and a rapid rat PK screening paradigm was pub-
Compounds are dosed discretely with n = 3 in each dosing arm, lished by Han et al. at Pfizer [21]. Relying on an abbreviated blood
72 www.drugdiscoverytoday.comDrug Discovery Today Volume 18, Numbers 1–2 January 2013 REVIEWS
180
75.0%
160
140
120
No. of compounds
100
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80
60
18.3%
40
20
4.5%
0.4% 1.8%
0
Under-predicted Under-predicted Predicted Over-predicted Over-predicted
by two categories by one category correctly by one category by two categories
Prediction outcome
Drug Discovery Today
FIGURE 1
Prediction of oral exposure category by snapshot pharmacokinetic (PK) studies. Oral exposures of 224 compounds were evaluated (182 in mouse and 42 in rat) first
in snapshot PK, then in rapid PK studies. Based on the dose-normalized area under the curve (AUC) values, compounds were classified into low, moderate or high
oral exposure categories [12]. Of the 224 compounds studied, 75.0% were categorized consistently by both assays and hence placed in the right box, 22.8% of
compounds were off by one category, where as only 2.2% were off by two exposure categories.
sampling and pooling strategy, both assays demonstrated the volume of distribution. A thorough understanding of processes
usefulness of the approach to estimate oral exposures of discovery affecting ADME cannot be obtained from snapshot PK profile only,
compounds and served as efficient filters for selecting compounds so further examination is required to fully characterize the PK
for further in vivo profiling. behavior of lead drug candidates.
Advantages and limitations of snapshot PK approach Rapid PK assay performance and its applications
Given that the oral route is the anticipated clinical route for most Traditionally, in vivo animal PK studies for full characterization of
small molecule discovery projects, oral exposure is an important PK parameters governing drug disposition (i.e. clearance and
PK characteristic essential to ensure adequate target coverage and volume of distribution) and oral bioavailability are resource inten-
in vivo efficacy in subsequent pharmacology studies. The snapshot sive and throughput is relatively low. A new method, called ‘rapid
PK approach provides practical information that is useful in deci- PK’ serves as the second-tier in vivo PK study approach, and offers
sion-making and has proven to be an effective in vivo PK tool in much improved throughput. A crucial full set of averaged (pooled)
early discovery. The key advantages of this tier 1 in vivo approach PK parameters, such as clearance (CL), volume of distribution (Vss),
are relatively high throughput, fast turn-around time and signifi- mean residence time (MRT), half-life (T1/2), oral exposure (AUC,
cant reductions in animal usage. It has been shown that snapshot Cmax) and oral bioavailability (%F) are readily obtained.
PK studies can reliably characterize compounds into low, medium The rapid PK assay was initially validated using a diverse set of
or high exposure categories [12], and that most compounds in the 15 compounds from different therapeutic areas. The resulting PK
low oral exposure category are deprioritized or discontinued for parameters were compared with those from full PK studies, and
further in vivo PK profiling. The combination of in vitro biology, in excellent correlations in the PK parameters were obtained (data
vitro ADME and snapshot PK data enables the project teams to not shown). In the 3 years following its implementation, the rapid
triage compounds effectively and to focus their efforts on selected PK assay has been used routinely and successfully to support drug
compounds in the high oral exposure category. Of over 1300 discovery projects at GNF. The performance of the rapid PK
compounds studied in snapshot PK, only 27% were placed in method relative to the conventional full PK method was further
the high exposure category [12]. Eventually, only 14% of the demonstrated in a larger set of diverse compounds. A total of 51
compounds, predominately those exhibiting moderate or high rapid IV PK studies (26 in mouse and 25 in rat) were compared with
oral exposure, were followed up in more detailed full PK studies. In full IV PK studies of the same compounds for disposition kinetics.
the long run, the strategy saves resources, animal use and follows The performance of the rapid PK approach in terms of oral expo-
the principles of the 3Rs. sure was also compared with that of conventional full PK studies. A
It is worth noting that oral exposure is a complex composite of total of 41 studies were compared between rapid oral PK and full PK
several key PK parameters, such as absorption, clearance, and studies (24 in mouse and 17 in rat). To avoid confounding results
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(a) (b)
100 20
R2 = 0.79 26 Compounds in mouse R2 = 0.95
26 Compounds in mouse
90 25 Compounds in rat
25 Compounds in rat
CL (mL/min/kg) from full PK
80
Vss (L/kg) from full PK
15
70
60
50 10
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40 Mouse
Mouse
30 Rat Rat
15 Line of unity
Line of unity
20
10
0 0
0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20
CL (mL/min/kg) from rapid PK Vss (L/kg) from rapid PK
(c) (d)
15000 5000
24 Compounds in mouse R2 = 0.90 24 Compounds in mouse
R2 = 0.88
AUC/Dose(h*nM/mg/kg) from full PK
Cmax /Dose(nM/mg/kg) from full PK
17 Compounds in rat 17 Compounds in rat
4000
10000
3000
Mouse Mouse
Rat Rat
Line of unity 2000 Line of unity
5000
1000
0 0
0 5000 10000 15000 0 1000 2000 3000 4000 5000
AUC/Dose(h*nM/mg/kg) from rapid PK Cmax /Dose(nM/mg/kg) from rapid PK
Drug Discovery Today
FIGURE 2
Comparison of performance between rapid pharmacokinetic (PK) and full PK studies. A total of 26 mouse intravenous (IV; 5 mg/kg) and 25 rat IV (3 mg/kg) rapid PK
studies were compared with separately conducted IV full PK studies with the same compounds. In addition, oral exposures of 24 mouse (20 mg/kg) and 17 rat oral
(10 mg/kg) rapid PK studies were compared with separately conducted oral full PK studies using similar formulation and doses. (a) Correlation of clearance (CL)
obtained from rapid PK versus full PK studies; (b) correlation of volume of distribution at steady-state (Vss) obtained from rapid PK and full PK studies; (c)
correlation of oral dose normalized area under the curve (AUC) between rapid and full PK studies; (d) correlation of oral dose-normalized Cmax between rapid and
full PK studies.
owing to dose-dependent PK or formulation effects, only studies correlation coefficients were 0.90 and 0.88 for dose-normalized
with similar doses and formulations were chosen for the compar- AUC and Cmax, respectively. The rapid and full PK studies typically
ison. The correlation results are shown in Fig. 2. Good correlations were conducted months to a year apart and often with unique
are found for the key PK parameters clearance (R2 = 0.79) and batches of test material, suggesting that the studies are highly
volume of distribution (R2 = 0.95). The data comprise mainly reproducible. Collectively, these data indicate that the rapid PK
compounds having low clearance (75% of compounds had study is a practical and valid approach for supporting discovery-
CL 30% liver blood flow) because such compounds are likely stage routine PK studies.
to be good development candidates, and can be selected for further Rapid PK studies are used effectively by project teams at differ-
evaluation in the full PK studies. Most of the highly cleared ent stages of drug discovery, from target validation with tool
compounds were filtered out effectively using the rapid PK compounds to lead optimization and candidate selection. This
approach. Overall, over 90% of the compounds tested in the rapid second-tier PK is most suitable and utilized during the lead opti-
and full PK studies showed comparable CL and Vss values that are mization stage, where the medicinal chemistry effort is focused.
within a twofold difference. Oral exposures between rapid and full During this stage, establishment of in vitro to in vivo correlations is
PK studies also correlated well, as shown in Fig. 2c and d, the important to enable effective use of high-throughput in vitro
74 www.drugdiscoverytoday.comDrug Discovery Today Volume 18, Numbers 1–2 January 2013 REVIEWS
TABLE 1
Comparison of different strategies used to increase in vivo PK efficiency
Strategies Cassette dosing (N-in-one) [22–25] Cassette analysis [26–29] Rapid PK*
Study design Multiple compounds dosed Discrete dosing; plasma samples from Discrete dosing; plasma samples from
simultaneously to same groups of the same time points are pooled across same time points are pooled across
animals different studies with multiple three animals within a study for a single
compounds compound
Pros Saving in-life resources and animals Discrete dosing, no DDI concerns; Discrete dosing, no DDI concerns; each
(5 reduction for 5-in-1); complete PK complete PK profiles for each compound analyzed separately,
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profiles for each compound in compound in individual animals bioanalytical method development
individual animals simplified; amendable for automation;
especially suitable for mouse PK; no
sample dilution and low LLOQ
(1 ng/mL)
Cons Potential in vivo DDI, must use limited No savings in in-life dosing, sampling No savings in in-life dosing, sampling
dose; complexity in selection of and number of animals; complexity in and number of animals; no interanimal
compounds and formulating multiple bioanalysis of multiple analytes, variability data available
compounds together; complexity in potential interference, signal
bioanalysis of multiple analytes; suppression from pooled compounds
potential interference, signal or metabolites; difficult to adapt for
suppression from pooled compounds mouse PK owing to small sample
or metabolites volume; longer method development
time; sample dilution and higher LLOQ
*
Li, C. et al. (2011) Rapid PK: an efficient in vivo preclinical pharmacokinetic approach to support drug discovery. Poster #295, presented at the 17th ISSX Conference (Atlanta, GA), 16–20
October, 2011.
ADME data, such as microsomal stability and in vitro permeability to be repeated in discrete dosing studies to verify or confirm the
assays. In addition, rapid PK has a crucial role in guiding the results.
selection of compounds and dosing regimen for the resource- Another strategy for improving the throughput of routine PK
intensive and often rate-limiting in vivo efficacy studies. By select- studies is to pool blood samples after discrete dosing of individual
ing compounds with favorable PK profiles and choosing appro- compounds, thereby avoiding the practical limitations and poten-
priate dosing regimens, the likelihood of achieving efficacy and tial risk of DDI associated with cassette dosing. One of the com-
demonstrating a PK/pharmacodynamics (PD) relationship is sig- monly used pooling approaches is cassette analysis [26–29], in
nificantly improved. which equal volumes of plasma samples from different studies are
combined for simultaneous bioanalysis. The improved sensitivity
Comparison of rapid PK with other reported approaches and specificity of modern LC/MS/MS systems made simultaneous
Different strategies to improve the throughput and capacity of multicomponent analysis feasible and cassette pooling a viable
conventional full PK studies have been described in the literature. option [29]. By pooling samples across compounds, the PK profiles
These strategies typically involve cassette dosing (or N-in-one dos- of each individual compound in individual animals are obtained
ing) or sample pooling. Each of the strategies aiming to improve PK and inter subject variability in PK can be assessed. In practice,
capacity and efficiency has its own advantages and limitations. A typically 3–5 compounds or studies are pooled for cassette analysis
comparison of these strategies, including the pros and cons asso- [21,29], even though more are possible in theory. The bioanaly-
ciated with each method, is summarized in Table 1. The final choice tical complexity increases with more pooled components, and
of strategy depends on several factors, such as the rate-limiting steps potential interferences and signal suppression from co-eluting
in PK throughput, the experience of the PK and bioanalytical compounds and/or their circulating metabolites can result in
scientists, and available resources and instrumentation. erroneous PK information. In addition, sample dilution owing
Cassette dosing [22–25], an approach in which several com- to pooling across studies is inherent and might be an issue,
pounds are simultaneously co-administered to a single animal, especially for the terminal phase where pooled concentrations
has the advantage of significant savings in both animal usage and might fall below the limit of quantification.
in-life resources. However, its use has been controversial and The second sampling pooling strategy, as used at GNF for the
debated, and a decline in the frequency of its use in drug discovery snapshot and rapid PK studies, is to pool plasma samples at the
setting has been reported [22]. The well-known disadvantages of same time point across different animals dosed with the same
cassette dosing are potential drug–drug interactions (DDI) from compound within a particular study [12]. The bioanalytical
the co-administration of multiple compounds, complications in method development and data processing are greatly simplified
the proper selection of compounds, difficulties in formulating and amendable for automation and batch processing, thereby
multiple compounds in the same vehicle, and potential bioana- resulting in significant savings in bioanalytical resources. Given
lytical interferences from co-administered compounds and their that mice are frequently used as pharmacology model species, it is
metabolites. It is time consuming to identify and troubleshoot important to implement a reliable and efficient method that is
problems encountered in cassette dosing, and studies often need applicable for both mouse and rat PK studies. Given the small
www.drugdiscoverytoday.com 75REVIEWS Drug Discovery Today Volume 18, Numbers 1–2 January 2013
plasma sample volumes (20 mL) collected in mice serial blood pooled to result in one averaged profile per compound per dosing
sampling, pooling across different studies with different com- route. However, a survey of rodent full PK studies conducted at
pounds can be challenging. In our mouse rapid PK approach, GNF showed that inter subject variability is small, so although
blood samples (50 mL) are pooled at the time of collection from impact owing to outliers on averaged PK profiles from rapid PK is
three individual mice in the same group. The sample processing possible, the likelihood is believed to be low, as clearly demon-
time is reduced, the sample plate is simplified and potential errors strated from the good correlations shown in Fig. 1. The correlation
are minimized. Although sample pooling strategies post-dosing values between our rapid PK and full PK approaches reflect not
have no impact on reducing animal usage in the study, usage of only the validity of our pooling strategy, but also the excellent
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mice is greatly reduced by serial blood sampling [13–16] rather reproducibility of separate in vivo studies that are typically con-
than the traditional one animal per time point design, reflecting ducted months apart with unique batches of compound.
the 3Rs principle.
Applications of full PK studies and PK/PD studies
Pros and cons of the rapid PK approach The third-tier conventional full PK studies offer the gold standard
The rapid PK approach is highly integrated and automated, and approach, and are mostly utilized during candidate selection stage
uses a sample pooling strategy across animals within a particular to profile fully the PK characteristics of selected drug candidates
study to increase efficiency and maintain simplicity. As shown in with the most stable salt forms and optimized formulations sui-
this review, the mean parameters from rapid PK studies correlate table for development. Streamlining and automating the first two
well with those obtained from full PK studies, and the rapid PK tiers of rodent in vivo PK studies enable scientists to focus their
approach has proven to be effective and sufficient in supporting attention on the more complex full PK studies and in-depth PK/PD
drug discovery, especially in lead optimization stage. or PK/efficacy studies. Several types of full PK study are conducted
The main disadvantage of this pooling strategy is the loss of during the drug discovery stage, including PK studies designed
inter-animal variability, as samples across different animals are to support formulation optimization; single dose escalation PK
Snapshot PK Rapid PK Full PK
‘One in one’ ‘One in one’ ‘One in one’
PO IV PO IV PO
In-life n=3 n=3 n=3 n=3
n=2
portion
Two animals per compound Six animals per compound Six animals per compound
Four samples per animal over 5 h Six samples per animal over 24 h Six samples per animal over 24 h
0.5 h 1h 3h 5h A B C D E F A B C D E F
A B C D E F A B C D E F
0.5 h 1h 3h 5h A B C D E F
A B C D E F
Pooling Pooling No pooling
Sample A B C D E F
A B C D E F
analysis 0.5 h 1h 3h 5h A B C D E F
A B C D E F
One compound per sample One compound per sample One compound per sample
Four samples per compound 12 samples per compound 36 samples per compound
One set of standards One set of standards Two sets of standards, QCs
PK
curve
0–5 h curve (po) without SD 0–24 h curves (iv, po) without SD 0–24 h curves (iv, po) with SD
Advantages Advantages Advantages
• Savings in number of animals and • Savings in analytical resources (~3x) • Obtain full set of PK parameters
Pros
analytical resources (~8x) • Obtain full set of PK parameters • Obtain interanimal variability
vs.
Liabilities Liabilities Liabilities
cons
• Truncated AUC0-5h • Labor intensive in-life portion • Labor intensive in-life portion
• No interanimal variability • No interanimal variability • Time-consuming bioanalysis
Used during ‘Hit to lead’ Used during ‘Hit to lead’ Used during ‘Lead optimization’
• To triage compounds based on oral • When CL and Vss are crucial for • To customize and optimize formulation
exposure; to select for efficacy progression to lead optimzation stage to enable further studies
Application
Used during ‘Lead optimization’ Used during ‘Lead optimization’ Used during ‘Candidate selection’
• To investigate structure-activity • To get full set of PK parameters • To get full set of PK parameters and
relationships (CL, Vss, Cmax, AUC, T1/2 and F%) interanimal variability
Drug Discovery Today
FIGURE 3
Comparison of tiered in vivo pharmacokinetic (PK) approaches used at the Genomics Institute of the Novartis Research Foundation (GNF) for supporting drug
discovery, including in-life study design, sample preparation, data output, pros and cons, as well as their applications.
76 www.drugdiscoverytoday.comDrug Discovery Today Volume 18, Numbers 1–2 January 2013 REVIEWS
Candidate
Hit-to-lead Lead optimization
selection
Tier 1: In Vitro Tier 2: In Vitro
• Solubility • Protein binding
In Vitro • Metabolite profiling
ADME • Metabolic stability
• Permeability • CYP inhibition (reversible inhibition of 5
major isozymes, time-dependent CYP3A4)
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• CYP3A4, 2D6, 2C9 inhibition
Tier 3: In Vivo
• Full PK (IV, PO)
Tier 1: In Vivo • Formulation PK
Snapshot PK (PO) • Mechanistic PK
In Vivo • PK/PD and PK/Efficacy
PK • Rat dose escalation
Tier 2: In Vivo
Rapid PK (IV, PO)
In Vivo
Efficacy In Vivo efficacy Safety
& Safety
Evaluation evaluation
Drug Discovery Today
FIGURE 4
Schematic illustration of how each of the tiered in vivo pharmacokinetic (PK) approaches, snapshot PK, rapid PK and full PK are integrated and applied in the drug
discovery paradigm. Arrows indicate compound progression.
studies to examine dose linearity; tissue distribution PK studies; exposure versus PD response relationships). In combination, the
multiple dose PK studies; and mechanistic studies designed to three-tiered in vivo PK approaches expedite compound profiling
examine the clearance mechanisms or barriers to oral bioavail- and help guide the selection of more desirable compounds into
ability. Significant efforts and resources are also dedicated to efficacy models and for progression into development. The pro-
support PK/PD or PK/efficacy studies. The design and complexity cesses enable tremendous flexibility and are highly efficient in
of these studies vary significantly and their applications are largely supporting the diverse needs and increasing demand for in vivo
dependent on project needs. Detailed discussion on these studies is profiling, and generally work efficiently for most discovery pro-
beyond the scope of this review. A recent publication by Amore jects.
et al. at Amgen [30] covered some aspects and applications of The integration and application of each of these in vivo PK
mechanistic PK studies and PK studies in support of in vivo phar- approaches in the overall drug discovery paradigm are illustrated
macology to understand PK/PD relationships. The importance of in Fig. 4.
characterizing PK/PD relationships in the changing paradigms of
drug discovery was discussed in a review by Summerfield and Concluding remarks
Jeffrey at Glaxo SmithKline [31]. Three-tiered in vivo rodent PK approaches (snapshot, rapid and full
PK studies) have been described and discussed for their applica-
Integrating the tiered in vivo PK approaches with drug discovery tions in supporting drug discovery. In all three approaches the
The three-tiered rodent in vivo PK approaches, differing in compound is dosed and analyzed discretely, thereby eliminating
throughputs, capacities and the resources required, are designed any DDI concerns and analysis complications typically associated
to address the varying needs of drug discovery projects at different with cassette dosing or cassette analysis. In combination, these
stages of project progression. A comparison of these tiered assays is tiered in vivo PK assays offer complementary approaches for addres-
summarized in Fig. 3, which includes study design, bioanalytical, sing different PK needs at different stages of drug discovery. These
PK information, pros and cons of each approach and their applica- approaches greatly facilitate the optimum compound selection
tions. Generally, compounds are first profiled in tier-1 snapshot PK and profiling processes for further drug development.
studies for the estimation of oral exposures; promising compounds
are examined further in tier-2 rapid PK studies to obtain a full Acknowledgements
description of drug disposition and oral bioavailability; selected We thank the GNF Pharmacology Animal Resource group, Liang
candidates with good in vivo PK properties and overall favorable Wang, Barbara Saechao and Mike Shapiro in the bioanalytical
profiles are then further advanced in tier-3 conventional full PK group, and Perry Gordon and Wendy Richmond in the
studies and other special PK studies (e.g. studies to characterize formulation group for their contributions.
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