a modern in vivo pharmacokinetic paradigm: combining snapshot, rapid and full pk approaches to...

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Drug Discovery Today � Volume 18, Numbers 1–2 � January 2013 REVIEWS

A modern in vivo pharmacokineticparadigm: combining snapshot, rapidand full PK approaches to optimize andexpedite early drug discoveryChun 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

elimination (ADME) assays have been implemented in early drug

discovery to identify and eliminate compounds with poor drug-

like properties and to promote potential pharmaceutical candi-

dates for more labor-intensive in vivo PK profiling [1–5]. Data from

in vitro ADME assays often contribute to the understanding of

underlying mechanisms of drug absorption and disposition,

which have proven invaluable to establish structure–activity rela-

tionships (SAR) that guide new chemical synthesis. However,

despite the advances in the in vitro technologies and in silico

approaches [6–9] for prediction of in vivo PK parameters, the

predictive power of these approaches is not always reliable and

accurate. Complete reliance on in vitro assays in the absence of an

in vitro–in vivo correlation (IVIVC) can sometimes mislead or slow

down the pace of a drug discovery program [10,11]. The PK profile

of a compound is governed by many physicochemical and che-

mical properties of the molecule, such as its lipophilicity, solubi-

lity, permeability and metabolic stability. The processes by which a

compound is absorbed, distributed, metabolized and eliminated in

vivo through an intact animal or human are often far more com-

plex than in isolated in vitro systems. It is essential to have in vivo

Corresponding author: Tuntland, T. ([email protected])

1359-6446/06/$ - see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.

testing and confirmations of in vitro ADME results in early drug

discovery and, therefore, there is always a continuous demand for

in vivo PK studies.

In vivo rodent PK studies are crucial to ensure compounds have

appropriate PK properties to be evaluated in preclinical pharma-

cology and safety studies. In addition, characterization of in vivo

PK of new chemical entities provides insight into complex in vivo

biological systems and correlates drug concentration at the site of

action with pharmacological response. Despite their important

role in drug discovery, most in vivo animal PK studies are still

conducted in a traditional, low-throughput manner in many

pharmaceutical companies and, therefore, remain the bottlenecks

of discovery projects.

In this review, we present three tiered in vivo rodent PK

approaches that are highly efficient in supporting early drug dis-

covery research. The study designs, strategies and applications of

each of the tiered assays are discussed and compared with other

commonly used in vivo rodent PK approaches in the pharmaceutical

industry. These tiered in vivo rodent PK approaches span from the

simple and abbreviated study design of ‘snapshot’ PK [12], to the

more labor-intensive intravenous/per oral (IV/PO) PK study designs,

such as ‘rapid PK’ and the conventional ‘full PK’. Depending on the

needs and stage of a specific project, different study designs can be

2012.09.004 www.drugdiscoverytoday.com 71

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http://dx.doi.org/10.1016/j.drudis.2012.09.004

REVIEWS Drug Discovery Today � Volume 18, Numbers 1–2 � January 2013

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used to answer specific PK questions. In combination, these tiered in

vivo PK approaches offer tremendous flexibility and continuous

support to projects at all stages of discovery process.

Study designs of the three tiered rodent in vivo PKapproachesSnapshot PK study designDetailed information about the snapshot PK study design has been

described in an earlier publication [12]. Briefly, compounds are

dosed discretely to two mice or rats via oral gavage, and blood or

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-

dardized and automated, and PK reports are generated automati-

cally and published to an internal database using web-publishing

tools [12].

Rapid PK study designA new in vivo rodent PK paradigm, namely ‘rapid PK’ is introduced

as the second tier PK approach at GNF. The in-life portion of the

study design is similar to that of a conventional standard full PK

study. A typical rapid PK study of a compound includes both IV

and PO arms with three animals in each dosing arm. Briefly,

compounds are formulated on the day of dosing using polyethy-

lene glycol 300 (PEG300):5% dextrose in distilled water (D5W) 3:1,

and the formulation is filtered before IV and PO dosing; alterna-

tively, a 0.5% methylcellulose/0.5% Tween 80 suspension formu-

lation is used for PO administration. Blood samples from six

different time points are collected for each animal up to 24 h

via serial blood sampling. For mouse rapid PK studies, blood

samples (50 mL) are taken via retro-orbital or alternatively, by

other serial sampling techniques, such as tail vein bleed [13–15]

or lateral saphenous vein puncture [16]. The blood samples from

the three mice are pooled and centrifuged to obtain pooled plasma

samples. For rat rapid PK studies, individual blood samples

(100 mL) are taken from each animal via the saphenous vein. After

centrifugation, 20 mL of the plasma samples are pooled across the

three rats within a dosing arm. A total of 12 plasma samples per PK

study are obtained after pooling of blood or plasma samples across

three IV group and three PO group animals.

The pooled plasma samples are diluted appropriately using a

generic dilution scheme to ensure that concentrations are within

the dynamic range of the standard curve (1–5000 ng/mL). Auto-

mated sample preparation and protein precipitation are carried

out, and up to eight compounds are prepared in a batch. Liquid

Chromatography Mass Spectrometry (LC/MS/MS) analysis is used

with a fast generic gradient elution method together with atmo-

spheric pressure chemical ionization (APCI) or electrospray (ESI) in

the positive or negative ion mode on an API-4000 triple quadruple

mass spectrometer. The analyte and internal standard are tuned

automatically using Automaton (now Discovery QuanTM) and the

analysis is conducted using multiple reaction monitoring (MRM).

Data collection and peak integration are performed using Ana-

lystTM 1.4.1 software.

Conventional full PK study designConventional full PK studies at GNF include IV/PO PK studies,

which are similar to those described in the rapid PK section above.

Compounds are dosed discretely with n = 3 in each dosing arm,

72 www.drugdiscoverytoday.com

and six blood samples are collected serially from each animal.

Individual plasma samples are analyzed and individual PK curves

are reported for each animal, such that inter subject variability can

be assessed. The bioanalytical methods are similar to those

described in the rapid PK section above, but are more comprehen-

sive. Each compound is tuned for optimum sensitivity and each

method includes two sets of standard curves and three quality-

control (QC) samples at low, medium and high concentrations

within the standard curve range. This is the gold standard in vivo

PK assay widely used by the pharmaceutical industry.

DiscussionTiered rodent in vivo PK approaches are adapted at GNF to address

diverse needs of discovery projects at different stages of the drug

discovery process. All the approaches are reviewed and approved

by the Institutional Animal Care and Use Committee (IACUC),

and are governed by the 3Rs principle (Replacement, Reduction,

and Refinement) to ensure the most appropriate and responsible

use of animals.

Snapshot PK assay performance and its applicationsSnapshot PK, the first tier in vivo PK study approach, utilizes

abbreviated blood sampling and sample pooling across the ani-

mals (n = 2). Average oral exposure (AUC0–5 h) is reported and test

compounds are categorized into low, moderate or high plasma

exposure based on the dose normalized AUC0–5 h. The comparison

of data from 177 compounds tested in both snapshot PK and

conventional full PK studies indicates that the snapshot PK assay

is efficient and reliable in categorizing their relative oral exposures

[12]. Recent data from an additional 224 compounds show an

identical trend in exposure comparisons between snapshot PK and

follow-up rapid PK studies. As shown in Fig. 1, 75% of compounds

are placed in the correct category and 98% of compounds are

placed in the correct or adjacent exposure categories.

A successful application of snapshot PK studies is exemplified in

the recent discovery of spiroindolones, a potent compound class for

the treatment of malaria [17,18]. In vitro screening at Novartis of a

large library of natural products and synthetic compounds with

structural features found in nature products generated 17 reliable

hits with reconfirmed submicromolar activity against falciparum

malaria. When 14 out of the 17 hits were profiled in snapshot PK

studies at GNF, most compounds exhibited negligible or no oral

exposures. One natural product belonging to the spiroazepinein-

dole class showed the best oral PK profile and became the starting

point for medicinal chemistry lead optimization [17]. Further synth-

esis and evaluations of approximately 200 derivatives yielded the

optimized spiroindolone analog NITD609 [18], which has improved

PK properties relative to the original hit and overall good drug-like

attributes. NITD609 has recently advanced to the initial phase of

clinical trials and is currently undergoing proof-of-concept testing

as an antimalarial agent with a novel mechanism of action.

Comparison of snapshot PK with other reported approachesA few similar assays to snapshot PK have been reported and used by

other pharmaceutical companies. A cassette accelerated rapid rat

screen (CAARS) was first introduced by scientists at Schering–

Plough [19,20], and a rapid rat PK screening paradigm was pub-

lished by Han et al. at Pfizer [21]. Relying on an abbreviated blood


Drug Discovery Today

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Under-predictedby two categories

Under-predictedby one category

Predictedcorrectly

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Over-predictedby two categories

0.4% 4.5%

75.0%

18.3%

1.8%

No

. of

com

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Prediction outcome

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

usefulness of the approach to estimate oral exposures of discovery

compounds and served as efficient filters for selecting compounds

for further in vivo profiling.

Advantages and limitations of snapshot PK approachGiven that the oral route is the anticipated clinical route for most

small molecule discovery projects, oral exposure is an important

PK characteristic essential to ensure adequate target coverage and

in vivo efficacy in subsequent pharmacology studies. The snapshot

PK approach provides practical information that is useful in deci-

sion-making and has proven to be an effective in vivo PK tool in

early discovery. The key advantages of this tier 1 in vivo approach

are relatively high throughput, fast turn-around time and signifi-

cant reductions in animal usage. It has been shown that snapshot

PK studies can reliably characterize compounds into low, medium

or high exposure categories [12], and that most compounds in the

low oral exposure category are deprioritized or discontinued for

further in vivo PK profiling. The combination of in vitro biology, in

vitro ADME and snapshot PK data enables the project teams to

triage compounds effectively and to focus their efforts on selected

compounds in the high oral exposure category. Of over 1300

compounds studied in snapshot PK, only 27% were placed in

the high exposure category [12]. Eventually, only 14% of the

compounds, predominately those exhibiting moderate or high

oral exposure, were followed up in more detailed full PK studies. In

the long run, the strategy saves resources, animal use and follows

the principles of the 3Rs.

It is worth noting that oral exposure is a complex composite of

several key PK parameters, such as absorption, clearance, and

volume of distribution. A thorough understanding of processes

affecting ADME cannot be obtained from snapshot PK profile only,

so further examination is required to fully characterize the PK

behavior of lead drug candidates.

Rapid PK assay performance and its applicationsTraditionally, in vivo animal PK studies for full characterization of

PK parameters governing drug disposition (i.e. clearance and

volume of distribution) and oral bioavailability are resource inten-

sive and throughput is relatively low. A new method, called ‘rapid

PK’ serves as the second-tier in vivo PK study approach, and offers

much improved throughput. A crucial full set of averaged (pooled)

PK parameters, such as clearance (CL), volume of distribution (Vss),

mean residence time (MRT), half-life (T1/2), oral exposure (AUC,

Cmax) and oral bioavailability (%F) are readily obtained.

The rapid PK assay was initially validated using a diverse set of

15 compounds from different therapeutic areas. The resulting PK

parameters were compared with those from full PK studies, and

excellent correlations in the PK parameters were obtained (data

not shown). In the 3 years following its implementation, the rapid

PK assay has been used routinely and successfully to support drug

discovery projects at GNF. The performance of the rapid PK

method relative to the conventional full PK method was further

demonstrated in a larger set of diverse compounds. A total of 51

rapid IV PK studies (26 in mouse and 25 in rat) were compared with

full IV PK studies of the same compounds for disposition kinetics.

The performance of the rapid PK approach in terms of oral expo-

sure was also compared with that of conventional full PK studies. A

total of 41 studies were compared between rapid oral PK and full PK

studies (24 in mouse and 17 in rat). To avoid confounding results

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(a)

10026 Compounds in mouse25 Compounds in rat

R2 = 0.79 R2 = 0.95

R2 = 0.90 R2 = 0.88

CL

(mL

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/kg

) fr

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ose

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PK

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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 andfull PK studies.

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owing to dose-dependent PK or formulation effects, only studies

with similar doses and formulations were chosen for the compar-

ison. The correlation results are shown in Fig. 2. Good correlations

are found for the key PK parameters clearance (R2 = 0.79) and

volume of distribution (R2 = 0.95). The data comprise mainly

compounds having low clearance (�75% of compounds had

CL � 30% liver blood flow) because such compounds are likely

to be good development candidates, and can be selected for further

evaluation in the full PK studies. Most of the highly cleared

compounds were filtered out effectively using the rapid PK

approach. Overall, over 90% of the compounds tested in the rapid

and full PK studies showed comparable CL and Vss values that are

within a twofold difference. Oral exposures between rapid and full

PK studies also correlated well, as shown in Fig. 2c and d, the


correlation coefficients were 0.90 and 0.88 for dose-normalized

AUC and Cmax, respectively. The rapid and full PK studies typically

were conducted months to a year apart and often with unique

batches of test material, suggesting that the studies are highly

reproducible. Collectively, these data indicate that the rapid PK

study is a practical and valid approach for supporting discovery-

stage routine PK studies.

Rapid PK studies are used effectively by project teams at differ-

ent stages of drug discovery, from target validation with tool

compounds to lead optimization and candidate selection. This

second-tier PK is most suitable and utilized during the lead opti-

mization stage, where the medicinal chemistry effort is focused.

During this stage, establishment of in vitro to in vivo correlations is

important to enable effective use of high-throughput in vitro


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

simultaneously to same groups of

animals

Discrete dosing; plasma samples from

the same time points are pooled across

different studies with multiplecompounds

Discrete dosing; plasma samples from

same time points are pooled across

three animals within a study for a singlecompound

Pros Saving in-life resources and animals

(5 � reduction for 5-in-1); complete PK

profiles for each compound in

individual animals

Discrete dosing, no DDI concerns;

complete PK profiles for each

compound in individual animals

Discrete dosing, no DDI concerns; each

compound analyzed separately,

bioanalytical method development

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 limiteddose; complexity in selection of

compounds and formulating multiple

compounds together; complexity inbioanalysis of multiple analytes;

potential interference, signal

suppression from pooled compounds

or metabolites

No savings in in-life dosing, samplingand number of animals; complexity in

bioanalysis of multiple analytes,

potential interference, signalsuppression from pooled compounds

or metabolites; difficult to adapt for

mouse PK owing to small sample

volume; longer method developmenttime; sample dilution and higher LLOQ

No savings in in-life dosing, samplingand number of animals; no interanimal

variability data available

* 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

assays. In addition, rapid PK has a crucial role in guiding the

selection of compounds and dosing regimen for the resource-

intensive and often rate-limiting in vivo efficacy studies. By select-

ing compounds with favorable PK profiles and choosing appro-

priate dosing regimens, the likelihood of achieving efficacy and

demonstrating a PK/pharmacodynamics (PD) relationship is sig-

nificantly improved.

Comparison of rapid PK with other reported approachesDifferent strategies to improve the throughput and capacity of

conventional full PK studies have been described in the literature.

These strategies typically involve cassette dosing (or N-in-one dos-

ing) or sample pooling. Each of the strategies aiming to improve PK

capacity and efficiency has its own advantages and limitations. A

comparison of these strategies, including the pros and cons asso-

ciated with each method, is summarized in Table 1. The final choice

of strategy depends on several factors, such as the rate-limiting steps

in PK throughput, the experience of the PK and bioanalytical

scientists, and available resources and instrumentation.

Cassette dosing [22–25], an approach in which several com-

pounds are simultaneously co-administered to a single animal,

has the advantage of significant savings in both animal usage and

in-life resources. However, its use has been controversial and

debated, and a decline in the frequency of its use in drug discovery

setting has been reported [22]. The well-known disadvantages of

cassette dosing are potential drug–drug interactions (DDI) from

the co-administration of multiple compounds, complications in

the proper selection of compounds, difficulties in formulating

multiple compounds in the same vehicle, and potential bioana-

lytical interferences from co-administered compounds and their

metabolites. It is time consuming to identify and troubleshoot

problems encountered in cassette dosing, and studies often need

to be repeated in discrete dosing studies to verify or confirm the

results.

Another strategy for improving the throughput of routine PK

studies is to pool blood samples after discrete dosing of individual

compounds, thereby avoiding the practical limitations and poten-

tial risk of DDI associated with cassette dosing. One of the com-

monly used pooling approaches is cassette analysis [26–29], in

which equal volumes of plasma samples from different studies are

combined for simultaneous bioanalysis. The improved sensitivity

and specificity of modern LC/MS/MS systems made simultaneous

multicomponent analysis feasible and cassette pooling a viable

option [29]. By pooling samples across compounds, the PK profiles

of each individual compound in individual animals are obtained

and inter subject variability in PK can be assessed. In practice,

typically 3–5 compounds or studies are pooled for cassette analysis

[21,29], even though more are possible in theory. The bioanaly-

tical complexity increases with more pooled components, and

potential interferences and signal suppression from co-eluting

compounds and/or their circulating metabolites can result in

erroneous PK information. In addition, sample dilution owing

to pooling across studies is inherent and might be an issue,

especially for the terminal phase where pooled concentrations

might fall below the limit of quantification.

The second sampling pooling strategy, as used at GNF for the

snapshot and rapid PK studies, is to pool plasma samples at the

same time point across different animals dosed with the same

compound within a particular study [12]. The bioanalytical

method development and data processing are greatly simplified

and amendable for automation and batch processing, thereby

resulting in significant savings in bioanalytical resources. Given

that mice are frequently used as pharmacology model species, it is

important to implement a reliable and efficient method that is

applicable for both mouse and rat PK studies. Given the small



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plasma sample volumes (�20 mL) collected in mice serial blood

sampling, pooling across different studies with different com-

pounds can be challenging. In our mouse rapid PK approach,

blood samples (50 mL) are pooled at the time of collection from

three individual mice in the same group. The sample processing

time is reduced, the sample plate is simplified and potential errors

are minimized. Although sample pooling strategies post-dosing

have no impact on reducing animal usage in the study, usage of

mice is greatly reduced by serial blood sampling [13–16] rather

than the traditional one animal per time point design, reflecting

the 3Rs principle.

Pros and cons of the rapid PK approachThe rapid PK approach is highly integrated and automated, and

uses a sample pooling strategy across animals within a particular

study to increase efficiency and maintain simplicity. As shown in

this review, the mean parameters from rapid PK studies correlate

well with those obtained from full PK studies, and the rapid PK

approach has proven to be effective and sufficient in supporting

drug discovery, especially in lead optimization stage.

The main disadvantage of this pooling strategy is the loss of

inter-animal variability, as samples across different animals are

Snapshot PK ‘One in one’ ‘

Two animals per compoundFour samples per animal over 5 h

Six aniSix sample

Pooling Pooling

One compound per sampleFour samples per compound

One set of standards

One co12 sam

One

0–5 h curve (po) without SD 0–24 h cu Advantages Advantages

• Savings in a• Obtain full s

Liabilities Liabilities• Truncated AUC0-5h • Labor intens

• No interanimal variability • No interanim

Used during ‘Hit to lead’ Used during • To triage compounds based on oralexposure; to select for efficacy

• When CL an progression t

Used during ‘ Lead optimization’ Used during• To investigate structure-activity relationships

• To get full s(CL, Vss , Cm

• Savings in number of animals andanalytical resources (~8x)

In-lifeportion

Sampleanalysis

Prosvs.

cons

Application

PK curve

A A A

0.5 h 1 h 3 h 5 h

0.5 h 1 h 3 h 5 h

PO n=2

IVn=3

0.5 h 1 h 3 h 5 hA

FIGURE 3

Comparison of tiered in vivo pharmacokinetic (PK) approaches used at the Genomdiscovery, including in-life study design, sample preparation, data output, pros an


pooled to result in one averaged profile per compound per dosing

route. However, a survey of rodent full PK studies conducted at

GNF showed that inter subject variability is small, so although

impact owing to outliers on averaged PK profiles from rapid PK is

possible, the likelihood is believed to be low, as clearly demon-

strated from the good correlations shown in Fig. 1. The correlation

values between our rapid PK and full PK approaches reflect not

only the validity of our pooling strategy, but also the excellent

reproducibility of separate in vivo studies that are typically con-

ducted months apart with unique batches of compound.

Applications of full PK studies and PK/PD studiesThe third-tier conventional full PK studies offer the gold standard

approach, and are mostly utilized during candidate selection stage

to profile fully the PK characteristics of selected drug candidates

with the most stable salt forms and optimized formulations sui-

table for development. Streamlining and automating the first two

tiers of rodent in vivo PK studies enable scientists to focus their

attention on the more complex full PK studies and in-depth PK/PD

or PK/efficacy studies. Several types of full PK study are conducted

during the drug discovery stage, including PK studies designed

to support formulation optimization; single dose escalation PK

Rapid PK Full PKOne in one’ ‘One in one’

mals per compounds per animal over 24 h

Six animals per compoundSix samples per animal over 24 h

No pooling

mpound per sampleples per compound

set of standards

One compound per sample 36 samples per compoundTwo sets of standards, QCs

rves (iv, po) without SD 0–24 h curves (iv, po) with SD

Advantagesnalytical resources (~3x) • Obtain full set of PK parameterset of PK parameters • Obtain interanimal variability

Liabilities ive in-life portion • Labor intensive in-life portion

al variability • Time-consuming bioanalysis

‘Hit to lead’ Used during ‘Lead optimization’

d Vss are crucial foro lead optimzation stage

• To customize and optimize formulationto enable further studies

‘Lead optimization’ Used during ‘Candidate selection’ et of PK parametersax, AUC, T1/2 and F%)

• To get full set of PK parameters and interanimal variability

A B C D E FA B C D E FA B C D E F

B C D E FB C D E FB C D E F

POn=3

IVn=3

PO n=3

A B C D E FA B C D E FA B C D E F

B C D E F


ics Institute of the Novartis Research Foundation (GNF) for supporting drugd cons, as well as their applications.


CandidateselectionLead optimization Hit-to-lead

Tier 1: In Vivo Snapshot PK (PO)

Tier 1: In Vitro • Solubility• Metabolic stability• Permeability• CYP3A4, 2D6, 2C9 inhibition

Tier 3: In Vivo • Full PK (IV, PO)• Formulation PK• Mechanistic PK• PK/PD and PK/E fficacy• Rat dose escalation

Safetyevaluation

In VitroADME

In VivoPK

Tier 2: In Vivo Rapid PK (IV, PO)

In Vivo efficacyEvaluation

In VivoEfficacy& Safety

Tier 2: In Vitro • Protein binding• Metabolite profiling• CYP inhibition (reversible inhibition of 5 major isozymes, time-dependent CYP3A4)


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 drugdiscovery paradigm. Arrows indicate compound progression.


studies to examine dose linearity; tissue distribution PK studies;

multiple dose PK studies; and mechanistic studies designed to

examine the clearance mechanisms or barriers to oral bioavail-

ability. Significant efforts and resources are also dedicated to

support PK/PD or PK/efficacy studies. The design and complexity

of these studies vary significantly and their applications are largely

dependent on project needs. Detailed discussion on these studies is

beyond the scope of this review. A recent publication by Amore

et al. at Amgen [30] covered some aspects and applications of

mechanistic PK studies and PK studies in support of in vivo phar-

macology to understand PK/PD relationships. The importance of

characterizing PK/PD relationships in the changing paradigms of

drug discovery was discussed in a review by Summerfield and

Jeffrey at Glaxo SmithKline [31].

Integrating the tiered in vivo PK approaches with drug discoveryThe three-tiered rodent in vivo PK approaches, differing in

throughputs, capacities and the resources required, are designed

to address the varying needs of drug discovery projects at different

stages of project progression. A comparison of these tiered assays is

summarized in Fig. 3, which includes study design, bioanalytical,

PK information, pros and cons of each approach and their applica-

tions. Generally, compounds are first profiled in tier-1 snapshot PK

studies for the estimation of oral exposures; promising compounds

are examined further in tier-2 rapid PK studies to obtain a full

description of drug disposition and oral bioavailability; selected

candidates with good in vivo PK properties and overall favorable

profiles are then further advanced in tier-3 conventional full PK

studies and other special PK studies (e.g. studies to characterize

exposure versus PD response relationships). In combination, the

three-tiered in vivo PK approaches expedite compound profiling

and help guide the selection of more desirable compounds into

efficacy models and for progression into development. The pro-

cesses enable tremendous flexibility and are highly efficient in

supporting the diverse needs and increasing demand for in vivo

profiling, and generally work efficiently for most discovery pro-

jects.

The integration and application of each of these in vivo PK

approaches in the overall drug discovery paradigm are illustrated

in Fig. 4.

Concluding remarksThree-tiered in vivo rodent PK approaches (snapshot, rapid and full

PK studies) have been described and discussed for their applica-

tions in supporting drug discovery. In all three approaches the

compound is dosed and analyzed discretely, thereby eliminating

any DDI concerns and analysis complications typically associated

with cassette dosing or cassette analysis. In combination, these

tiered in vivo PK assays offer complementary approaches for addres-

sing different PK needs at different stages of drug discovery. These

approaches greatly facilitate the optimum compound selection

and profiling processes for further drug development.

AcknowledgementsWe thank the GNF Pharmacology Animal Resource group, Liang

Wang, Barbara Saechao and Mike Shapiro in the bioanalytical

group, and Perry Gordon and Wendy Richmond in the

formulation group for their contributions.



Review

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OSTSCREEN

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