Pharmacokinetics in Drug Discovery: Review of Basic Pharmacokinetic Concepts

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Pharmacokinetics in Drug Discovery

ANA RUIZ-GARCIA,1 MARIVAL BERMEJO,2 AARON MOSS,3 VICENTE G. CASABO2


1
Pharmacokinetics and Drug Metabolism, Amgen, Inc, 1201 Amgen Court West, Seattle, Washington 98119
2
Pharmaceutics Department, College of Pharmacy, University of Valencia, Avda Vicente Andres Estelles s/n,
Burjassot 46100, Valencia, Spain
3
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, Washington 98195, Spain

Received 19 October 2006; revised 28 January 2007; accepted 5 March 2007


Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21009

ABSTRACT: The aim of this current review is to summarize the present status of
pharmacokinetics in Drug Discovery. The review is structured into four sections. The
first section is a general overview of what we understand by pharmacokinetics and the
different LADMET aspects: Liberation, Absorption, Distribution, Metabolism, Excre-
tion, and Toxicity. The second section highlights the different computational or in silico
approaches to estimate/predict one or several aspects of the pharmacokinetic profile of
a discovery lead compound. The third section discusses the most commonly used
in vitro methodologies. The fourth and last section examines the various approaches
employed towards the pharmacokinetic assessment of discovery molecules; including all
the LADME processes, discussing the different mathematical methodologies available
to establish the PK profile of a test compound; what the main differences are and
what should be the criteria for using one or another mathematical approach. The
major conclusion of this review is that the use of the appropriate preclinical assays
has a key role in the long-term viability of a pharmaceutical company since applying
the right tools early in discovery will play a key role in determining the company’s
ability to discover novel safe and effective therapeutics to patients as quickly as
possible. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci
97:654–690, 2008
Keywords: absorption; toxicology; structure–activity relatioship (SAR); population
pharmacokinetics; pharmacokinetic/pharmacodynamic models; computational ADME;
biophysical models; biopharmaceutics classification system (BCS); bioequivalence;
ADME

REVIEW OF BASIC metabolites in serum, plasma, or whole blood,


PHARMACOKINETIC CONCEPTS tissue target and target organs over time.1 The
body is a very complex system and a drug
Pharmacokinetics (PK) is the study of a drug and/ undergoes many steps as it is being absorbed,
or its metabolite kinetics in the body. It refers distributed through the body, metabolised, and/
to the temporary evolution of a drug and its or excreted (ADME). Pharmacokinetics has
been broadly divided into two categories of
study: absorption and disposition. Disposition is
further subdivided into the study of distribution
and elimination. The term elimination includes
Correspondence to: Ana Ruiz-Garcia (Telephone: 206-265-
7408; Fax: 206-217-0494; E-mail: [email protected]) metabolism and excretion since, from the PK
Journal of Pharmaceutical Sciences, Vol. 97, 654–690 (2008)
perspective, we consider that the drug has been
ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association eliminated when it is no longer in its original

654 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008


PHARMACOKINETICS IN DRUG DISCOVERY 655

chemical structure. Restated, when any biotrans- solutions. . .etc) determines the disposition of the
formation of the parent compound takes place and drug.8–12 Response and toxic effects are the other
even if the resulting metabolites remain in the two key aspects to consider since they are the
body, it has been eliminated. main reasons for Drug Discovery failure (see
Fig. 1). In summary, when we refer to the different
individual assays that should be performed to
LADMET-R and Pharmacokinetics characterize the PK profile of a new drug in vitro,
When the studies are focused solely in one specific in situ, in vivo, or in silico we should also consider,
pharmacokinetic aspect (Absorption, Distribu- besides the ‘‘gold standard’’ ADME, (1) release
tion, Metabolism, or Excretion) by in vitro, from the pharmaceutical form, (2) toxicity, and (3)
in situ, in vivo, or in silico techniques it is usually activity/response in the target site (LADMET-R).
referred to as ADME studies whereas the name
Pharmacokinetics is normally reserved to in vivo
studies where an integrated approach of all DISCOVERY AND DEVELOPMENT
the ADME processes together is taken. For
either ADME or Pharmacokinetics, the truth of New drug development can be divided in two
the matter is that under both approaches, it is different stages: discovery and development.
necessary to command a more or less sophisti- Recently, Kola and Landis13 reviewed the major
cated knowledge of algebra and calculus to causes of attrition in development (see Fig. 1). In
correctly interpret the dataset. Although ADME their review, they showed how the root causes of
assays have been the gold standards in PK, there drug failure have evolved over time (1991–2000).
are additional tests that should be incorporated, In 1991, PK and bioavailability were the major
since they play a key role in Drug Discovery and reasons for drug failure (40%) dropping dramati-
further development. Liberation of the drug from cally to 10% in 2000. This significant change is
the pharmaceutical form is a key parameter in mainly due to the time and effort that Industry
bioequivalence studies (e.g., a sustained release has invested in the last decade toward a deeper
versus immediate release formulation)2–7 or, for and better understanding of PK, partially in an
intravenous formulations, where the rate of attempt to overcome poor bioavailability but also
release from the formulation (liposomes, micellar trying to look into more predictive kinetic

Figure 1. Main reasons for drug development failure. Adapted from Kola 200013 and
Tufts Center for the Study of drug Development.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
656 RUIZ-GARCIA ET AL.

behavior of the drug candidates to allow for more the sponsor. The project team needs to be aware of
efficient dose regimens. Lack of efficacy and safety the target product profile in order to make
were reported as the most relevant causes of why educated decisions about the direction that the
compounds undergo attrition in the clinic in 2000 project needs to evolve in order to reach the next
(30%). The Tufts Center for the Study of Drug milestone in development. Components of the
Development14 published in 2005 the three main target product profile are: disease indication,
reasons for terminating unpromising new drugs. minimum efficacy requirements, required safety
Again, safety and efficacy were listed among the profile, desirable dose regimen, dosage form,
main three. In summary, the identified issues in maximum cost of goods, planned date of regula-
that report have been the main focus of study in tory submission and expected approval date.18
recent years as well as a driving force determining In summary, when planning exploratory stu-
which strategy to follow in Drug Discovery. The dies in humans, under an Investigational New
composite of activity, safety, and acceptable Drug (IND) application, there is some preclinical
LADME properties, rather than a specific attri- data as well as chemistry, manufacturing and
bute, will dictate the success of the drug program. controls information that need to be generated.
In order to identify potential liabilities in dis- The approaches taken in generating this data can
covery and eliminate those molecules from further be optimized expediting the progress into devel-
consideration, high throughput screening (HTS) opment and increasing the chances of success of
of reliable and appropriate in vitro, and/or in situ the IND filing by compiling a good quality dataset
assays seem to be the fastest and more efficient in an efficient manner. Depending on the goals of
way to proceed,15–17 as shown in Figure 2. the proposed investigation, the amount of data
Generally, when a drug is granted to progress that need to be submitted can vary.19
into development, a project team is formed
with members of different areas of expertise
(i.e., including, but not restricted to, toxicology, DRUG DISCOVERY:
pharmacokinetics, clinical development, medic- HOW DOES THIS WORK?
inal chemistry, formulation, marketing and reg-
ulatory affairs), with the goal of establishing an The quality and quantity of preclinical data
early development plan. Successful drug develop- provided by discovery groups to support the
ment is a result of getting to this stage with development of a new drug has considerably
enough information about the previously men- improved in the last few years. This is due to the
tioned processes (LADMET-R) in conjunction with acknowledgement from Industry of the relevance
a worthwhile investment that provides value to of this information to the success of the drug as

Figure 2. Drug discovery.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
PHARMACOKINETICS IN DRUG DISCOVERY 657

mentioned above. A variety of in vitro assays have (high permeability, low solubility, HP:LS), class 3
been automated through the use of robotics. (low permeability, high solubility, LP:HS), and
In silico models are being used to assist in the class 4 (low permeability, low solubility, LP:LS),
selection of the right assay and the set of see Figure 3.
compounds undergoing further in vitro screening. This analysis points out conditions under which
With the emerging new computational models no in vitro–in vivo correlation may be expected for
(in silico), a deeper understanding of the relation- example, rapidly dissolving low permeability
ship between important LADME-T parameters drugs. Furthermore, it is suggested that for very
and molecular descriptors and/or in vitro para- rapidly dissolving high solubility drugs, for
meters has been achieved, allowing for an early example, 85% dissolution in less than 15 min, a
estimation of several LADME-T properties (see simple one-point dissolution test is all that may be
Tab. 1). needed to insure bioavailability. For slowly
HTS facilitates a researcher to effectively dissolving drugs, a dissolution profile is required
conduct some biological test to a large number with multiple time points in systems which would
of potential therapeutic moieties.20 Through this include low pH, physiological pH, and surfactants,
process, rapid discrimination of active ingredients where the in vitro conditions should mimic the
versus undesirable compounds based on the in vivo processes. The draft guidance document
results of the particular assay can be achieved. entitled ‘‘Waiver of In Vivo Bioavailability and
The main difference of this strategy versus the Bioequivalence Studies for Immediate Release
traditional pharmaceutical screening is that Solid Oral Dosage Forms Containing Certain
less rigorous results are needed. There are few Active Moieties/Active Ingredients Based on a
samples from many compounds as opposed to a Biopharmaceutics Classification System’’ pro-
very rich database from few compounds. HTS poses to further expand the regulatory applica-
screening is, in essence, a single goal to which all tions of BCS and also recommends methods for
the data may subsequently be applied. HTS is classifying drugs and immediate release formula-
used at early stages of discovery to gather tions.25 However, Wu and Benet26 suggest an
LADME-T information that serve as key factors alternative classification attending to solubility
for candidate selection. As a result, there has been values and metabolism rather than permeability
a recent focus on enhancing the efficiency of values, The Biopharmaceutics Drug Disposition
obtaining absorption, disposition, and toxicity Classification System (BDDCS). The authors,
data, which has permitted LADME-T scientists while recognizing that drug metabolism can differ
to contribute more effectively to the drug depending on the drug’s solubility and perme-
discovery process. ability characteristics, consider that switching
Since the oral route is the preferred adminis- permeability values to extent of elimination would
tration route for patients, and this fact assures be less restrictive, expanding the Class I drugs
compliance of the drug therapy, a lot of time and eligible for waiver of bioequivalence (BE).27
effort has been invested toward a good under- For drugs with low permeability, the rate at
standing of the physicochemical properties that which they are being actively carried through
play a key role in bioavailability. Bioavailability the GI and reaching the systemic circulation is
has been defined by the FDA as the rate and extent highly dependent on the carrier-mediated sys-
to which the active ingredient or active moiety is tems involved. Both influx and efflux under these
absorbed from a drug product and becomes circumstances will play an active role in the oral
available at the site of action.21 Thus, for oral bioavailability of the compound. In light of
pharmaceutical forms, systemic exposure is going this, Klopman et al.28 discussed the importance
to be highly dependent on the extent of absorption of lipophilicity in membrane transport models.
in the gastrointestinal tract (GI). The Biophar- Several experimental techniques have been
maceutics Classification System (BCS)22 as a described to evaluate intestinal absorption
drug development tool allows for the estimation including physicochemical measurements (e.g.,
of contributions of the three major factors that solubility, lipophilicity, partition coefficients),
affect drug absorption: dissolution, solubility, and subcellular fractions (brush border membrane
intestinal permeability.22–24 Based on in vitro vesicles, basolateral membrane vesicles), cell
solubility and in vivo permeability values, drugs culture-based models, artificial membranes, iso-
can be divided into four groups: class 1 (high lated tissues, and organ preparations. These
permeability, high solubility, HP:HS), class 2 techniques are briefly described in this review

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
658
RUIZ-GARCIA ET AL.

Table 1. In Silico LADMET Studies

Descriptor
Involved/Substrate Used Modeling Approach Comments Reference
Elimination CYP1A2 Pharmacophore 73

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91
CYP2C9 3-D QSAR 66
Pharmacophore 74,75,9
CYP2D6 Pharmacophore 76
Neuronal network and 17
Bayesian statistics Characterizing the sensitivity of a
CYP3A4 Pharmacophore compound to metabolism predicting 77–80
CYP450s Homology models its potential elimination profile 88–90
MAO A,B QSAR 92
3D-QSAR and CoMFA 67
Phenol sulfotransferase (PST) QSAR 93
Glucuronidation QSAR(DARC/PELCO) 94,95
UDP-glucuronosyltransferases
Distribution Bile acid carrier 3-D-QSAR and CoMFA 68
PEPT Correlation 245
CoMFA 70
P-gp CoMFA Characterizing the sensitivity of a 71
Catalyst Pharmacophore compound to be substrate of protein 81
Pharmacophore carriers which will affect its distribution 82
3-D-QSAR/PLS 69

DOI 10.1002/jps
Absorption Partition coefficient Biophysical models Absorption/permeability 246,124
and Molecular Weight predictions
PATQSAR 100
QSAR 28

DOI 10.1002/jps
Liposome partitioning Correlation 128
Polar surface data Correlation 142,247
Polar surface data Correlation Solubility predictions 142
Molecular descriptors ANN 101,103
Aminoacid sequence SVM 248
Everted intestinal rings Correlation Drug accumulation predictions 139
Partition coefficient, Molecular weight Biophysical models Bioavailability Predictions 249
In situ absorption rate constant values 249,250
(Rat small intestine)
Physico-chemical properties (HDM, Non linear regression 142
Caco-2 and 2/4/A1)
Liberation In vitro dissolution test IVIVC Bioavailability predictions 2–7,105,106
Physicochemical and structural factors QSAR/ORMUCS 29
Response Partition coefficient, Molecular weight PATQSAR In vitro evaluation for prediction 100
of in vivo response
Atom typing Naive Bayes classifier 25
Physicochemical descriptors 2D-QSAR 64
Toxicity Chemical structure (Ames test) Correlation In vitro evaluation for prediction 43
of toxic effects in vivo
Identification of potential hERG channel Neuronal Networks and 17
blockers (Dofetidine DisplacementTest) Bayesian statistics
Subcutaneous and ocular toxicity (MTT) Correlation 55,57
34
Adapted from de Groot and Ekins.80,251.
PHARMACOKINETICS IN DRUG DISCOVERY
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660 RUIZ-GARCIA ET AL.

accelerates the excretion from the body. Thus, the


potential of a compound containing a chemical
moiety known to be extensively metabolized may
be predicted with reasonable accuracy on the basis
of abundant historical data.30 Several in vitro
methods are routinely used to establish the
metabolic profile of a moiety31,32 such as micro-
somes, supersomes, cytosol, S-9 fraction, cell-
based models, primary hepatocytes, liver slices,
and perfused liver. Because these assays are
based on the native metabolic enzyme they are an
excellent source for the experimental generation
of metabolite profiles, which will help influence
the selection of the appropriate pre-clinical safety
species. Several in vitro experimental techniques
have been briefly described later in this review
(see Tab. 3).
At the level of Metabolism, there are also some
predictive models. Bursi et al.33 have derived a
structure–pharmacokinetic relationship for a data
set of 32 in-house steroidal androgens. The same
group developed an electronic model for hydrogen
abstraction in steroidal androgens in which the
activation energies of steroid radical systems could
be used to predict relative rates of metabolism to
guide the design and redesign process of metabo-
lically more stable steroidal androgens. Many 3D
ligand-based and structure-based computational
Figure 3. Biopharmaceutics classification system approaches have been used to predict the metabo-
and biopharmaceutics drug disposition classification lism catalyzed by the enzymes of the cytochrome
system. P450 superfamily (P450s) responsible for 70% of
the metabolism of all drugs. Computational
under in vitro screening. Examples of their methodologies have focused on a few P450s that
application have been tabulated in Table 2. are directly involved in drug metabolism. Models
In silico models are extensively applied to derived for P450s help to explain and predict the
absorption descriptor’s properties and bioavail- involvement of P450s in the metabolism of specific
ability predictions. For instance, the quanti- compounds and guide the drug-design process.34
tative structure–bioavailability relationship of See Table 1 for additional computational models for
232 structurally diverse drugs was studied to human P450s.
evaluate the feasibility of constructing a predic- Drug administration has the main goal of
tive model for the human oral bioavailability of achieving therapeutically effective drug concen-
prospective new medicinal agents by Yoshida trations at the site of their clinical activity while
et al.29 where they found lipophilicity to be a minimizing adverse effects (usually linked to non-
significant factor influencing oral bioavailability target site drug concentrations).35 However, the
(see Tab. 1). ability of drugs to reach the site of action depends
However, for many compounds, oral bioavail- on many pharmacokinetic aspects such us drug
ability may be limited by extensive metabolism availability and drug metabolism, both already
rather than poor absorption. Metabolism can be mentioned, but also binding to plasma proteins.
defined as the chemical changes (biotransforma- Drug tissue distribution is often correlated with
tion) that take place in a given chemical substance total plasma/serum concentrations (bound and
within an organism. The biotransformation that unbound fractions), which for drugs with a high
normally takes place within the body leads percentage of protein binding that act in periph-
towards more hydrophilic or water-soluble moi- eral tissues becomes a questionable assumption.
eties than the parent compound which facilitates/ Several techniques have been developed to study

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
DOI 10.1002/jps
Table 2. In Vitro Systems for Permeability and Protein Binding Studies

System Origin Comments Reference


Dissolution studies Simulated intestinal fluids Establish the dissolution profile 253–255
CaCo-2 Human colorectal adenocarcinoma Most well established cell model that expresses relevant 256,257,258
carrier-mediated systems
MDCK Canine kidney Ideal for transfections. Low instrinsic expression of 152,153
ABC family transporters
LLC-PK1 Pig kidney Ideal for transfections. Low instrinsic expression of 154,259
ABC family cassette transporters
2/4/A1 Rat fetal intestinal Ideal for studying paracellular route of absorption 260,261,262
TC-7 CaCo-2 clone Same than CaCo-2 263,264
HT-29 Human colorectal adenocarcinoma Contains mucus-producing globet cells 265,266
IEC-18 Rat Ileum Permeability and para-cellular transport 267
PAMPA Phospholipid-coated filter Permeability for passive absorption 20,125,268
IAM cell membrane phospholipids Permeability for passive absorption 156,158
immobilized to solid surfaces
HDMs Artificial Hexadecane Membranes Passive transcellular permeability 262,141,142
BBMV Brush border membrane vesicles Passive transcellular permeability and active 131–133
carrier-mediated systems
In situ intestinal Rat small intesine Passive transcellular permeability and active 23,138,162
perfusion carrier-mediated systems
Liposomes Bilayer vesicles Passive absorption 129,269
Partition coefficient N octanol/water Lipophilicity parameter 124,270
Ultracentrifugation Partitioning Unbound drug fraction in plasma 170,171
Microdyalisis Microdialysis probes Unbound drug fraction in the interstitial fluid 271,272
Adapted from Balimane 2005252.
PHARMACOKINETICS IN DRUG DISCOVERY
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662 RUIZ-GARCIA ET AL.

Table 3. In Vitro Systems for Metabolism and Toxicity Studies

System Origin Comments Reference


Liver microsomes Liver tissue Phase I metabolism 173–175,183
Intestinal microsomes Intestinal tissue Phase I metabolism 176,177
Cytosolic fractions Liver tissue Phase II metabolism 178,179
Supersomes Liver tissue
(Baculovirus-insect-cell Specific CYP/UGT mediated 32,183
expressed) metabolism
S9 fractions Liver tissue Phase I and II metabolism 180,181
Detecting DNA damage 182
Isolated perfused liver Liver Hepatotoxicity 204–206
Metabolism 207,208
Liver Slices Liver Hepatotoxicity 184,185
Metabolism 184,186
Hepatocytes Liver Hepatotoxicity 199–201,234–236
Metabolism 203
HepG2 Human hepatoma cell line Hepatotoxicity 188–190
Metabolism 187,191
HLE Human lens epithelial cell line Hepatotoxicity 193,194
Metabolism 192,193
BC2 Human hepatoma cell line Hepatotoxicity 196,197
Metabolism 195,198
DNA microarray Rat liver Phase I metabolism 211
Rat liver Phase II metabolism 212
Genotoxicity COMET assay Detecting DNA damage 44,45
Ames test 41,42
Renal toxiciy DNA microarray Changes in gene expression 273,274
Hepatotoxicity 275,276
Cytotoxicity MTT assay Cell proliferation 48,49,54,57
SRB assay 50,51
Clonogenic assay 52,53
hERG potassium channels IKr assay Electrophysiology study 237,238

drug protein binding in plasma, with the two that the in vitro systems respond as the in vivo
predominant methods being ultrafiltration (UF) tissue would. In vitro studies should be conducted
and equilibrium dialysis (ED).36,37 with concentrations and exposure times similar to
There is a clear need for companies to find ways the in vivo conditions. The in vitro models may
to evaluate safety of drug candidates earlier in the allow high throughput screening, decreasing the
development process. Animal toxicology studies number of chemicals tested in whole animals.
are the foundation of an IND. The principal Mutagenicity screening is a regulatory require-
safety concerns are usually in the area of genetic ment for drug approval since they imply a toxic
toxicology, target organ toxicity and cardio- risk in humans.38 The International Agency
safety. The identification of HTS assays that for Research on Cancer (IARC) discussed in a
can accelerate the advance of the drug candidate consensus report39 the term ‘‘genotoxicity,’’ con-
into more relevant in vivo testing as soon as sidering that this term includes both direct and
possible is the main goal in the discovery stages. indirect effects in DNA. Direct effects are con-
There are several toxicity studies that are sidered inductions of mutations (gene, chromo-
routinely performed depending on the nature of somal, genomial, and recombinational) that at the
the drug. Genotoxicity, cytotoxicity, and target molecular level are similar to events known to be
organ toxicity may be, at least in a first instance, involved in carcinogenesis. Indirect DNA effects
evaluated through in vitro screening. The use of involve surrogate events associated with muta-
positive (known toxic reagents) and negative genesis (e.g., unscheduled DNA synthesis (UDS)
controls (non toxic reagents) is necessary to assure and sister chromatid exchange (SCE), or DNA

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
Table 4. Online Information and Software Resources for Absorption as Well as Other LADME Aspects

URL Description Name


http://www.accelrys.com/products/ This is a package of six predictive ADME/Tox models—human intestinal absorption,

DOI 10.1002/jps
cerius2/ aqueous solubility, human CYP 2d6, blood–brain barrier penetration, serum protein
cerius2products/c2adme.html binding, and human hepatotoxicity.
http://www.compudrug.com/ Since the early 1960s, lipophilicity has proven to be very important molecular description, Pallas
often well-correlated with the bioactivity of chemical entities. Lipophilicity and hydrophobicity
are measured by lipophilic and hydrophobic indices, such as the logarithm of a partition
coefficient, which reflects the equilibrium partitioning of a molecule between a nonpolar and
polar (aqueous) phase. A new artificial neural network using atomic fragmental descriptors
has been developed to predict the octanol-water partition coefficient (logP). The fragmental
descriptors were obtained from the Atomic2005 linear logP calculation method implemented in
Pallas PrologP program. Using a numerically optimized weighted average of the older methods
and the current one, the result is significantly more accurate than the previous method, and
provides an exceedingly accurate prediction. The new logP prediction method was implemented
into the Pallas 3.4 version.
http://www.simulations-plus.com/ In an effort to help pharmaceutical companies bring new drugs to market faster and for less cost, Gastro-Plus
products.html Simulations Plus has developed GastroPlusTM, a unique software program that simulates the
dissolution and absorption of a drug in the human gastrointestinal tract.
http://www.simulations-plus.com/ ADMET Predictor is an advanced computer program that enables pharmaceutical researchers to ADMET
products.html estimate ADME properties (such as permeability, solubility, lipophilicity, diffusivity, etc.) of new Predictor
chemical entities (NCE’s) from their molecular structure.
http://www.simulations-plus.com/ (Dose Disintegration and Dissolution) is a new tool for forulation scientists that simulates DDDPlus
products.html the in vitro disintegration and dissolution of dosage forms in USP Paddle, Basket, and
Flow-Through experiments.
http://www.pasteur.fr/recherche/unites/ Database describing the properties and the protein composition of experimentally investigated ABCISSE
pmtg/abc/database.iphtml ATP-binding cassette (ABC) systems. In addition, we report complete inventories of the
predicted ABC systems for organisms whose complete genome sequence is known. In the
latter case, ABC proteins and their interacting partners were identified by comparing the
proteomes of these organisms to the full content of ABCISSE. Systems were reconstructed and
sorted in families and subfamilies according to the phylogenetic and functional classification,
which was described in our publications. We predict the polarity of transport, the substrate
specificity, and the biological role of these systems.
http://133.9.194.61/tp-search/index.html TP-Search is a comprehensive database on drug transporters, which are thought to play an TP-Search
important role in the pharmacokinetics of drugs. All the infomation is extracted from a large
number of published papers. With this database, users can obtain various kinds of basic
information on drug transporters.
http://nutrigene.4t.com/humanabc.htm Human ATP-binding cassette transporters
PHARMACOKINETICS IN DRUG DISCOVERY

(Continued)
663

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664 RUIZ-GARCIA ET AL.

damage (e.g., the formation of adducts) being


Name the most relevant ones, which may eventually
lead to mutations. Mutations are permanent
hereditary changes in the cell lines. Genotoxicity
thus preceeds mutagenicity although most of
genotoxicity is repaired and is never expressed
as mutations.40 The Ames test and Comet assay
(Single-Cell Gel, SCG) are in vitro cell-based
assays that are routinely used to assess genotoxi-
city induced by xenobiotics41–47 and they have
been briefly described later in this review.
Cytotoxicity can be defined as the quality of a
xenobiotic to confer toxicity to cells. Cytotoxicity
in different cell lines will provide useful informa-
tion about potential target organs toxicities. MTT,
SRB (Sulforhodamine B), and clonogenic assays
are used for measuring drug-induced cytotoxicity
functions, similarities, substrates/ligands, tissue distributions,

by measuring cellular proliferation in mammalian


cell lines.48–53 Moreover, a routine screening for a
associated proteins. It contains information about known
A database for facilitating the search for drug Absorption,

wide range of pharmaceutical products is cuta-


Description

neous and ocular toxicity, where epidermal


keratocyte and corneal epithelial cultures have
targets. Associated references are also included.

become relevant models that can provide valuable


information about the mechanisms of cutaneous
and ocular toxicities of test compounds.54–57
Human membrane transporter database

Hepatotoxicity is one of the major reasons for


Distribution, Metabolism, Excretion

withdrawal of marketed drugs over the past two


decades.58,59 The pharmaceutical industry has
drug ADME associated proteins,

P-glycoprotein drug interactions

dedicated much emphasis on developing in vitro


and other properties of the

screening systems to detect hepatotoxicity (iso-


lated perfused liver, liver slices, primary hepato-
cyte cultures, human liver-derived cell lines,
ABC-transporter genes

etc).Toxins can be classified as hepatotoxins,


which cause liver damage in the majority of the
population with or without metabolic activation.
Further in this nomenclature, hepatotoxins can
be subdivided into cytotoxics if they injure the
hepatocytes and cholestatics if they interfere with
the bile flow.60
The International Conference of Harmonisation
http://xin.cz3.nus.edu.sg/group/admeap/

(ICH) guideline S7B describes a nonclinical


http://www.mhc.com/PGP/index.html

testing strategy for assessing the potential of a


http://lab.digibench.net/transporter/

test substance to delay ventricular repolarization


(VR). VR is determined by the duration of the
cardiac action potential as a result of the activities
http://www.gene.ucl.ac.uk/

of many membrane ion channels and transpor-


Table 4. (Continued )

genefamily/abc.html

ters. In this guideline are descriptions of in vitro


(patch clamp) and in vivo electrophysiology
nomenclature/

studies for competition binding protocols in which


admeap.asp

test substances are studied for their ability to


displace a hERG channel blocker (human Ether-
a-go-go Related Gene).61,62 The hERG gene
URL

encodes a potassium ion channel responsible for


the repolarizing IKr current in the cardiac action

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
Table 5. Online Information and Software Resources Mainly Focused in Metabolism

DOI 10.1002/jps
URL Description Name
http://drnelson.utmem.edu/ Cytochrome P450 Homepage
CytochromeP450.html
http://www.cypalleles.ki.se/ Home Page of the Human Cytochrome P450 (CYP) Allele Nomenclature Committee
http://www.icgeb.org/p450srv/ The goal of this www Directory is to facilitate access to electronic resources
world-wide for all researchers working in the field of P450 proteins and
P450-containing systems.
http://medicine.iupui.edu/flockhart/ Cytochrome P450 drug interactions table
P450 inhibiting drugs
http://www.accelrys.com/products/cerius2/ This is a package of six predictive ADME/Tox models—human intestinal absorption, C2 adme
cerius2products/c2adme.html aqueous solubility, human CYP 2D6, blood–brain-barrier penetration, serum protein
binding, and human hepatotoxicity
http://www.accelrys.com/products/chem_ This database has been compiled from and based on two noteworthy journals of the Metabolism
databases/databases/metabolism.html Royal Society of Chemistry (RSC): ’Biotransformations’ and ’Metabolic Pathways of
Agrochemicals’. It covers the metabolic pathways of drugs, agrochemicals and industrial
chemicals in various species. On this strong foundation, new entries are added based
on the expert abstraction of pertinent and relevant literature references
http://www.compudrug.com/ The knowledge base of the MetabolExpert module of Pallas has been extended with new MetabolExpert
metabolic reactions. The extension includes a set of special metabolic reactions collected
from the scientific literature focusing on the metabolisms of toxic and drug-like organic
compounds. Thanks to the inserted reactions, the new version of MetabolExpert will
manage a series of special metabolisms, including ring opening and closing reactions
http://www.lhasalimited.org/ Meteor is a computer program that helps scientists who need information about the METEOR
index.php?cat¼2& sub_cat¼68 metabolic fate of chemicals and want to be more efficient, more effective and make
better decisions. The program uses expert knowledge rules in metabolism to predict
the metabolic fate of chemicals and the predictions are presented in metabolic trees.
The only information needed by the program to make its prediction is the molecular
structure of the chemical
http://multicase.com/products/products.htm An expert system capable, when coupled with appropriate dictionaries, to predict the META
metabolic transformations that may be produced when the molecules are ingested
or dumped in the environment. The program is totally interfaced with MCASE
and permits a complete evaluation of the potential toxicological effect of a
molecule and its metabolites
http://mhc.com/Cytochromes/ P450, UGT, and P-gp drug interactions
PHARMACOKINETICS IN DRUG DISCOVERY
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666

Table 6. Online Resources in Toxicology.

URL Description Name


RUIZ-GARCIA ET AL.

http://www.accelrys.com/products/ This is a package of six predictive ADME/Tox models—human intestinal absorption,


cerius2/cerius2products/c2adme.html aqueous solubility, human CYP 2D6, blood–brain-barrier penetration, serum protein
binding, and human hepatotoxicity
http://www.compudrug.com/ Elimination of cytotoxic compounds in the early phases of drug discovery can save Pallas proLogP
substantial amounts of research and development costs. A new artificial neural
network based approach using atomic fragmental descriptors has been developed to
categorize compounds according to their in vitro human cytotoxicity. Fragmental
descriptions were obtained from the linear logP calculation method implemented in
Pallas PrologP program
http://multicase.com/products/ Knowledge-based systems are computer programs aimed at organizing relevant CASE/MCASE
products.htm experimental data for the purpose of helping a user make a decision about real world

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problems. Our group, over the years, has developed two such programs, CASE
(Computer Automated Structure Evaluation) and its successor, MCASE/MC4PC,
designed for the specific purpose of organizing biological/toxicological data obtained
from the evaluation of diverse chemicals. These programs can automatically identify
molecular substructures that have a high probability of being relevant or responsible for
the observed biological activity of a learning set comprised of a mix of active
and inactive molecules of diverse composition. New, untested molecules can then be
submitted to the program, and an expert prediction of the potential activity of the new
molecule is obtained. MCASE differs from CASE in a great many ways, but the major
algorithmic difference is the use of hierarchy in the selection of descriptors, leading to
the concept of biophores and modulators

DOI 10.1002/jps
DOI 10.1002/jps
Table 7. In Silico Online information and Databases

URL Description Name


http://iwrwww1.fzk.de/biostruct/ Application of STUN to in silico database screening
RLD/mtx.htm
http://redpoll.pharmacy.ualberta.ca/drugbank/ Bioinformatics and cheminformatics resource that combines detailed drug (i.e., DRUGBANK
chemical, pharmacological and pharmaceutical) data with comprehensive
drug target (i.e., sequence, structure, and pathway) information
http://www.psc.edu/general/software/packages/ GenBank is the NIH genetic sequence database, an annotated collection GENBANK
genbank/genbank.html of all publicly available DNA sequences. A new release is made every two
months. GenBank is part of the International Nucleotide Sequence Database
Collaboration, which is comprised of the DNA DataBank of Japan (DDBJ), the
European Molecular Biology Laboratory (EMBL), and GenBank at the National
Center for Biotechnology Information.Each GenBank entry includes a concise
description of the sequence, the scientific name and taxonomy of the source
organism, and a table of features that identifies coding regions and other sites of
biological significance, such as transcription units, sites of mutations or modifications,
and repeats. Protein translations for coding regions are included in the feature table
http://iwrwww1.fzk.de/biostruct/RLD/ Flexscreen performs fully automated in-silico screening of a large 3D database of
flexscreen.htm ligands against a structurally resolved protein receptor. Each ligand of the database is
docked against the receptor with the stochastic tunneling method using an all-atom
representation of both ligand and receptor. Both ligand and receptor can change their
conformation in the docking process. Using an all-atom scoring function based on
physical principles, the affinity of the each ligand to the receptor is evaluated. The
ligands with the best affinity are selected as lead candidates for drug development.
Using our secure, high-performace computational platform, we presently screen
the open ligand database of the National Cancer Institute (USA) which contains
about 250000 ligands, but other databases can also be used
http://www.accelrys.com/products/mstudio/ QSAR Plus builds on the base tools available in QSAR and extends them to QSAR PLUS
modeling/visualizationandstatistics/qsarplus.html include a neural networks model building method and accurate quantum
mechanical descriptors. QSAR Plus enables you to identify compounds
with optimal physicochemical properties
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668 RUIZ-GARCIA ET AL.

potential. Abnormalities in this channel may lead leads and the elimination of unsuitable ones.
to either Long QT syndrome (LQT2) (with loss of Although these models can never be accurate
function mutations) or Short QT syndrome (with enough to replace real circumstances, many of
gain of function mutations). Both are potentially them can be extremely useful if they are built
fatal cardiac arrhythmia due to repolarization under the correct assumptions and right set of
disturbances of the cardiac action potential. data. When the in silico models have been
Erroneous drug binding to this channel may lead carefully developed and rigorously validated the
to acquired Long QT Syndrome.63 information they provide can be valuable in early
In silico models, described below, are also being Drug Discovery. It is, therefore, not surprising
used for predicting activity as well as toxicity15. that there is considerable interest in developing
Klopman et al.25 have developed a model for MDR mathematical models capable of accurately pre-
reversal agents (to overcome Multi-Drug Resis- dicting some LADME-T key information for new
tance) to estimate the MDR reversal activity of drug candidates. However, the misleading use
compounds. The same author discussed the and interpretation of in silico LADMET is often
importance of lipophilicity values (represented the reason why these models have been exten-
as the logarithm of the n-octanol/water partition sively criticized by a large part of the scientific
coefficient) and its correlation with their pharma- community.65 These models are usually based in
cological and toxic activities.29 Kazius et al.43 were in vitro data and/or physico-chemical properties.
able to perform mutagenicity predictions of an Two different types of computational models are
independent validation set of 535 compounds with being used currently: molecular and data model-
an error percentage of 15%. The authors con- ing. A brief description of the fundaments of the
cluded that toxic properties can often be related to most used computational models is presented
substructures, which are generally identified as below and listed in Table 1.
toxicophores, and that these toxicophores can be
applied to risk assessment processes and can
guide the design of chemical libraries for hit and Molecular Modeling
lead optimization. Yoshida et al. have used
The main objective of molecular modeling is to
some physicochemical descriptors (n-octanol/
assess the potential interaction between the
water partition coefficient, topological polar sur-
studied drug and proteins involved in LADMET
face area, diameter, summed surface area of
processes (e.g., carrier-mediated systems such us
atoms with partial charges) to carry out 2D-
p-gp, enzymes responsible of either phase I (e.g.,
quantitative structure–activity relationship (2D-
CYPs) or II biotransformation(e.g., Glutathione
QSAR) studies on 104 hERG channel blockers
tranferases). Under molecular modelling, we can
with diverse structures collected from the litera-
distinguish: Ligand-based models, Structure-
ture, thus formulating interpretable models to
based models, and Homology Models.
guide chemical-modification studies and virtual
screening.64 Combination of predictive models has
Ligand-Based Models
also been performed with great success. O’Brian
et al. combined hERG channel blocking and These attempts to link chemical structures with
CYP450 2D6 inhibition computational models observed activities based on information about
with better predicted values than their individual active site, shape, electronic properties and con-
predictions.17 formation of substrates, inhibitors or metabolic
products. The simplest one is QSAR: Quantitative
Structure–Activity relationship. Three-dimensional
IN SILICO LADMET quantitative Structure–Activity relationship (3D-
QSAR) refers to the analysis of the quantitative
The line between in silico LADME-T and in vitro- relationship between the biological activity of a set
in silico LADME-T is difficult to define since the of compounds and their spatial properties using
majority of the in silico models use not only statistical methods.66–69 3D-QSAR are often based
physicochemical parameters but also some in vitro on Molecular Field Analysis, MFA. MFA employs a
data (see Tab. 1). combination of reasonable molecular description,
In recent years, the number of computational statistical analysis, and graphical display of
models for the different LADMET processes has results.67,70,71 Molecular structures are described
considerably increased with the aim of promising with molecular interaction energies as steric and

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PHARMACOKINETICS IN DRUG DISCOVERY 669

electrostatic fields surrounding the molecules; the ANN may include molecular modeling and data
statistics is computed by partial least square (PLS) modeling.101–103
regression analysis and the output is displayed as Li et al.104 have done excellent work describing
contours superimposed on the molecules. The the most recent explored statistical learning
comparative MFA, (CoMFA) methodology assumes approaches such as neural networks (NN), sup-
that a suitable sampling of steric and electrostatic port vector machines (SVM), etc. The authors
fields around a set of aligned molecules provides all concluded that both classification-based and
the information necessary for understanding their regression-based statistical learning methods
biological properties.72 If no structural information have consistently shown promising capability
is available, an alternative means to assess for predicting chemical agents of diverse ranges
potential interactions is to use pharmacophore of structures and of a wide variety of LADMET
models. These are ligand-based models where poperties.
different structures of ligands or their properties Table 1 contains a list of references of published
overlay in 3D space in an attempt to describe the in silico work in LADME-T.
physical, spatial, and chemical properties of the
active site.10,73–82
In Vitro In Vivo Correlations (IVIVC)
Structure-Based Models
Bioequivalent products are those whose rate and
Included in this category are X-ray crystallogra- extent of absorption do not show significant
phy,83 nuclear magnetic resonance (NMR),84 differences when administered at the same dose.21
spectroscopy and electron microscopy. These The bioequivalence of two drug products is usually
models determine the 3D structure of proteins evaluated through in vivo assays in human
through a variety of means. However, it is often volunteers. However, under some particular
very difficult to use these techniques due to the conditions, it should not be necessary to carry
nature of the proteins (e.g., difficult to crystallize, out an in vivo pharmacokinetic study to assure
poor solubility, large molecular size). bioequivalence; a well validated in vitro study
should be able to assess that. From an ethical
point of view, if the assay with human volunteers
Homology Models
is not essential to demonstrate the equivalence
As a result of the difficulties mentioned above between two formulations, the assay should not be
when trying to elucidate the 3D structure of performed, but, on the other hand, we need to
proteins, these models were developed. Homology assure that the in vitro surrogate is reliable. The
models are an alternative method to elucidate the factors that we have to analyze to establish the
3D structure of proteins. These models are based theoretical basis for correlating in vitro dissolu-
on the fact that the 3D structure of a protein is tion and in vivo absorption are the parameters
related to its amino acid sequence since proteins that control rate and extent of absorption. The
with similar amino acid sequence tend to adopt basic concept is if two drug products containing
similar 3D structure.85–90 the same drug have the same concentration time
profiles at the intestinal membrane surface, then
they will have the same rate and extent of
absorption. Two conditions are necessary for this
Data Modeling
statement to be true: the two drug products have
Data modeling uses statistical tools to search the same in vivo dissolution profile under all
for correlations between a given property and a luminal conditions and none of the formulation
set of physicochemical descriptors. Quantitative components affects the membrane permeability or
structure–activity relationship (QSAR),28,29,64,91–95 intestinal transit time.
quantitative structure–property relationship IVIVC has been defined by the Food and
(QSPR),96–99 population Analysis by topology- Drug Administration (FDA) as a ‘‘predictive
based QSAR (PATQSAR)100 and Artificial Neural mathematical model describing the relationship
Networks (ANN) are some examples of data between an in vitro property of a dosage form and
modeling. ANN is an adaptive system that chang- an in vivo response’’.21,27 Generally, the in vitro
es its structure based on external or internal property is the rate or extent of drug dissolution or
information that flows through the network. release while the in vivo response is the plasma

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670 RUIZ-GARCIA ET AL.

drug concentration or amount of drug absorbed. specific biomarker identification for various pur-
The main objective of developing and evaluating poses ranging from disease monitoring to disease
an IVIVC is to establish the dissolution test as a progression and prognosis.
surrogate for human bioequivalence studies,
which may reduce the number of bioequivalence
studies performed during the initial approval Proteomics
process as well as with certain scale-up and post Proteomics is a high throughput study of proteins,
approval changes.105,106 Two step or one step particularly their structures and functions. Pro-
methods can be applied to obtain these correla- teomics is much more complicated than genomics.
tions. Using the two-step method, by deconvolu- Most importantly, while the genome is a rather
tion or by a mass balance method, the in vivo constant entity, the proteome differs from cell to
function is computed and from dissolution assays, cell and is constantly changing through its
the in vitro variable is calculated, then, in a second biochemical interactions with the genome and
step, plasma concentration are predicted by the environment. The entirety of proteins in
convolution based on the in vitro data. A one step existence in an organism throughout its life cycle,
method involves a convolution step where plasma or on a smaller scale the entirety of proteins found
concentrations predicted from the model and in a particular cell type under a particular type of
those observed are directly compared. Published stimulation, are referred to as the proteome of
work in this matter has been referenced in the organism or cell type respectively.110–112 Since
Table 1. proteins play a central role in the life of an
organism, proteomics is instrumental in the
discovery of biomarkers, such as markers that
Genomics, Proteomics, and Metabonomics indicate a particular disease.113–117
Lately, these terms have been incorporated into
many scientists vocabulary with not always a Metabonomics
clear idea of the intended meaning.
Metabonomics has been defined as the quantita-
In addition to the traditional information about
tive measurement and identification of the
the disease state, we now posses a set of new
biochemicals contained in a biological sample
descriptors obtained by molecular profiling. In
such as the metabolic response of living systems to
other words, we have access to large scale of
pathophysiological stimuli or genetic modifica-
systematic readouts at various levels such as DNA
tion. This approach has been used in toxicology,
content (genomics), protein expression (proteo-
disease diagnosis, and a number of other fields.
mics), and measurements of metabolites (meta-
This technology has been used to identify bio-
bonomics). Hopefully, the combination of these
markers for disease as well as to identify off-target
different readouts will provide us a combination of
side effects in marketed drugs and new chemical
potential biomarkers as well as better predictors
entities in development.15,60,118–120
of a disease state.
A similar and related concept that is worth
defining here is Metabolomics, which refers to the
Genomics
study of the chemical fingerprints that specific
Genomics is the study of an organism’s genome cellular processes may produce. The study of
and the identification of the genes involved in metabolite profiles will fall into this category.
biological processes. Genomics has the potential of Although there is still no absolute agreement,
offering new therapeutic methods for the treat- there is a growing consensus that the difference
ment of some diseases, as well as new diagnostic between the two terms resides in the fact that
methods. As a consequence of the identification of ‘‘metabolomics’’ places a greater emphasis on
genome sequences, it is possible to engineer DNA comprehensive metabolic profiling, while ‘‘meta-
microarrays, which can measure gene expression bonomics’’ is used to describe multiple (but not
of thousands of genes simultaneously.60,107,108 necessarily comprehensive) metabolic changes
New applications in the field of genomics are caused by a biological perturbation.
emerging in two major areas.109 The first one The integration of genomic, proteomic and
focuses on understanding the mechanism of action metabonomic data constitutes a very powerful
involved in disease states or compound-induced data source for in silico analysis. Although we do
phenotypic changes. The second one involves not fully understand the complexity of the

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PHARMACOKINETICS IN DRUG DISCOVERY 671

biological systems and pathologies, by gathering of the solute from the aqueous phase indicates
the various sources of information and analyzing how much solute travelled to the organic phase.
the data together, we will certainly see increas- Both phases, aqueous and organic, should be
ed contributions toward the establishment of saturated in each other to avoid changes in
fingerprints for toxicity, metabolism and other volume when they contact as that will introduce
LADMET-related processes. considerable error in the determination of this
parameter. The partition coefficient is defined as
the concentration ratio between the organic and
IN VITRO LADMET
aqueous phase as follows:
The following in vitro assays are probably the Co ðQai  Qaf Þ=Vo
most common screening tools for the PK aspects P¼ ¼
Ca Qaf =Va
already commented on throughout this review.
Tables 2 and 3 list these in vitro assays pointing
Where Qai and Qaf represent the amount of
out the parameter assayed and some relevant
solute in the aqueous phase before and after
published work in that matter.
being in contact for a specific period of time
with the organic phase in continuous agitation.
Dissolution Studies The pH value of the aqueous phase along with
the temperature used for the partitioning are
Dissolution studies are routinely performed as a the variables that determine the value of this
part of the quality protocol of solid dosage forms, parameter.
because these studies help to ensure that the
manufacturing process has not deviated signifi- Liposomes
cantly from the established standards. The use of
dissolution assays as a quality control index Liposomes are lipid bilayer vesicles used as
requires a simple dissolution media with simple models for biological lipid bilayer membranes
dissolution conditions in order to minimize for the study of drug partitioning from aqueous
practical problems, such as analytical complica- phase into the liposome.127 Several authors have
tions, and to keep the cost of the test at the suggested that this parameter correlates better
minimum value.121–123 However, if a test is with human drug absorption than n-octanol-
required that provides more information about water partition coefficient.128,129
what will happen in vivo, the BCS can simplify the
requirements of the test and provide guidance Membrane Vesicles
regarding the inferences that we can obtain from The most commonly used are Brush Border
in vitro assays.23 As mentioned previously, BCS is Membrane Vesicles (BBMV) and Basolateral
a framework for classifying drug substances based membrane vesicles (BLMV), both subcellular
on their aqueous solubility and permeability and fractions. Its preparation involves tissue homo-
provides the basis for establishing in vitro–in vivo genation and differential sedimentation, fractio-
correlations (IVIVC) and for justifying ‘‘biowaivers,’’ nation, and differential precipitation. For BLMV
or in other words, permission to use dissolution test there is an additional subfractionation step.
data as a surrogate for pharmacokinetic data. Basically, these systems are used for transcellular
Hence, a dissolution test can be used as an in vitro absorption studies130 as well as active and
bioequivalence study instead of an in vivo bioequi- facilitated transport mechanisms.131–133 The tis-
valence study. sues may be of human origin but most frequently
are derived from different animal species such us
Absorption rabbits, pigs, and rats. BBM contain a variety of
hydrolytic enzymes, which are valuable tools in
N-Octanol/Water Partition Coefficient
studying drug stability. The distribution of these
This parameter is often expressed by the Log(P) enzymes is well known134–136 enabling rational
value. The Log(P) is basically a parameter of approaches to assessing protection of the drug by
lipophilicity where the distribution between a formulation or synthetic techniques. BBM matrix
solute dissolved in an aqueous buffer (aqueous is especially useful in studying the specificity of
phase) and n-octanol, as organic phase, is targeted prodrug reconversion at the intestinal
measured.124–126 The measure of disappearance wall.137

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672 RUIZ-GARCIA ET AL.

Everted Intestinal Rings sites.160,161 The technique typically involves iso-


lation of an intestinal segment or the whole small
Everted Intestinal rings provide a relatively
intestine, which remains in situ and is perfused
quick technique for measuring uptake of drug
(close or open loop) with a solution containing a
into tissue.138,139 The viability of the tissue over
known concentration of the test moiety. Samples
the time course of the experiment may be
of the intestinal perfusate are taken at specific
compromised.140
time measuring the disappearance of the studied
molecule.126,160–163
Filter-Immobilized Hexadecane Membrane (HDM)
This technique consists of a hexadecane liquid
layer immobilized between two aqueous compart- Isolated Sections of Intestinal Tissue
ments.141,142 An HTS technique used to study In this setting, sections of intestinal tissue are
passive transport pathways. mounted in a chamber as the barrier between two
compartments.164 Both the serosal and mucosal
Parallel Artificial Membrane Permeability surfaces are bathed with oxygenated buffer
Assay (PAMPA) solution and the passage of the compound in
PAMPA is a phospholipid-based parallel artificial solution across the tissue is measured by standard
membrane that lacks active transport and also analytical techniques. The integrity of the
paracellular pathways.20,125,142–144 membrane is monitored by measurement of
transepithelial electrical resistance across the
Cell Culture-Based Permeability Models tissue.126,164 Using chambers can be also utilized
having as a barrier cultured cell monolayer as
There exists a significant variety of cell lines oppose to epithelial tissue.165
now routinely cultivated as a monolayer on
permeable filters. Caco-2 cell monolayers (human
epithelial colon adenocarcinoma cell line) are
extensively used because it is an excellent system Protein Binding
for the study of transcellular transport and also
Equilibrium Dialysis (ED)
active transport. Dipeptide carrier145,146 and
P-glycoprotein147,148 (P-gp) are some of the Dialysis works on the principle of the diffusion of
transporters expressed in Caco-2 cells. Other cell solutes along a concentration gradient across a
lines are extensively used due to their low semipermeable membrane. Usually, the device
intrinsic expression of ATP-binding cassette contains two chambers divided by a semiperme-
transporters superfamily149–151 (ABC) such us able membrane with a specific molecular weight
Madin–Darby Canine Kidney (MDCK) and LLC- cut-off, which means that only molecules with a
PK1 which make them ideal for transfec- molecular weight smaller than the cut-off size will
tions.72,152–154 Another cell line that is being permeate through the membrane. In a typical
routinely used is 2/4/A1 which lacks functional experiment, plasma containing the test article is
expression of several important active drug on one side of the membrane and a buffer is placed
transporters and does not present tight junctions, in the other dialysis chamber. A variant of ED is
rendering this system ideal for studying para- comparative equilibrium dialysis (CED), where
cellular permeability.142 plasma is placed on either side of the dialysis
membrane.36 Microdialysis, as a specific type of
Immobilized Artificial Membranes (IAM) dialysis system, allow you to continuously sample
the unbound fraction on the interstitial space
The IAM system was developed by Pidgeon
fluid(ISF), which may be the actual target
et al.155 and described in several papers.156–159
compartment for many drugs. Microdialysis is
In essence, IAMs are a chromatographic model
an in vivo probe-based sampling method linked to
of lipidic membranes for studying passive
an analytical device for measurement of drug
absorption.
concentration profiles. When a physiological
salt solution is slowly pumped through the
In Situ Intestinal Perfusion
microdialysis probe, the solution equilibrates with
The in situ perfusion technique provides enhanc- the ISF which then contains a representative
ed tissue viability as well as several sampling proportion of the tissue fluid’s molecule.35,166–168

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PHARMACOKINETICS IN DRUG DISCOVERY 673

Ultrafiltration (UF) Lactate dehydrogenase release, protein synthesis,


K content, glutathione, glutathione S-tranferases,
It is the most common methodology for determi-
DNA adduct formation, etc.60,184,185 Liver slices
nation of unbound plasma concentrations.169–171
are a valuable model for studying the regulation
Ultrafiltration is a variety of membrane filtration
of a larger number of enzymes by single com-
in which hydrostatic pressure forces a liquid
pounds.184,186
against a semipermeable membrane. Partitioning
occurs without dilution, change in physiologic
pH, ion composition, or unbound microsolute Cell-Based Cultures
concentration. Protein leakages and adsorption
Liver cell lines do not present all families of
to the UF device are the major drawbacks of this
metabolic enzymes. Cultures of human cell lines
technique.172
are preferred versus animal liver cell lines. When
used for cell-based hepatotoxicity, these cultures
are relevant for elucidating the mechanism of
Metabolism action of the studied toxin. The most commonly
Liver and Intestinal Microsomes used human liver cell lines are HepG2,187–191
HLE,192–194 and BC2.195–198 (see Tab. 3)
Microsomes are small vesicles from fragmented
hepatocyte or enterocytes endoplasmic reticulum
prepared by differential centrifugation. Micro- Hepatocytes
somes contain phase I enzymes. The basic Primary hepatocytes are a well-controlled, rela-
processes included in phase I reactions are tively easy to handle in vitro system that is well-
oxidation, reduction, and/or hydrolysis mainly accepted for investigating xenobiotic biotransfor-
catalyzed by the CYP system requiring NADPH as mation, enzyme induction, and inhibition, and
cofactor.173–177 (biotransformation-mediated) hepatotoxicity.199–
201
However, it has been observed that a number
Liver Cytosol Fraction of liver-specific functions are progressively lost
The cytosolic fraction of the liver contains soluble with time when hepatocytes are isolated and
phase II enzymes. It is obtained by differential cultivated.202 Hepatocytes are the closest in vitro
centrifugation of whole-liver homogenate. Addi- model to the human liver, and they are the only
tion of co-factor is necessary.178,179 model which can produce a metabolic profile of a
drug which is very similar to that found in vivo.
Liver S9 Fraction Hepatocytes contain phase I and II metabolic
enzymes as opposed to microsomes that lack
S9 fraction contains both microsomal and cytosolic phase II metabolism. Phase II reactions generally
fractions. Co-factors are required.180,181 Liver inactivate the drug if it is not already inactive
fractions can be used in combination with the following phase I metabolism and makes the drug
Ames test (see below) for predicting potential more polar or water soluble facilitating its
mutagenicity of a compound.182 elimination.203

Human Recombinant Enzymes


Isolated Perfused Liver
Human Recombinant Enzymes are basically
microsomes prepared from baculovirus-infected Pefusion of a freshly isolated liver is considered
insect cells that express specific human CYPs the closest system to in vivo experiments. It
and Uridine Diphosphoglucuronosyl Transferase maintains the three dimensional architecture, the
(UGTs). This system is useful for isozyme-specific bile flow and the liver’s hemodynamics.60,204–208
drug biotransformation and drug–drug interac- Livers are connected to a temperature-controlled
tion studies.32,183 perfusion system (open or closed system) and the
perfusion medium is continuously gassed with a
mixture of CO2 and O2. The test article is added to
Liver Slices
the circulated perfusate. Several parameters are
These pieces of liver tissue possess active phase I monitored during the length of the experiment to
and II xenobiotic metabolism. Toxicity can be determine the impact of the test article in the
measured by MTT, changes in ATP levels, functionality of the liver (liver enzymes, oxygen

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674 RUIZ-GARCIA ET AL.

consumption, bile content, ATP, perfusion flow (e.g., rat liver –S9 mix) is added to simulate the
rate,...etc). effect of metabolism as some compounds, like
benzopyrene, are not mutagenic themselves but
their metabolic products are. The bacteria are
DNA Microarray Assay (DNA chips) spread on a histidine-free agar plate in the middle
DNA Microarray assays consist of a collection of which the mutagen to be tested is added. The
of microscopic DNA fragments attached to a plates are then incubated for 48 h. The muta-
support material forming an array for the purpose genicity of a substance is proportional to the
of expression profiling, monitoring expression number of colonies observed.
levels for thousands of genes simultaneously. It
allows the evaluation of expression of the mRNA COMET Assay
transcripts for a large number of genes by a single
experiment (HTS).209,210 Basically, a cell is embedded in agar and exposed
Measuring gene expression using DNA micro- to a DNA-damaging agent such as UV radiation or
arrays is relevant in metabolism studies when we a chemical mutagen. The cell is then permeabi-
are investigating whether the gene expression of lized by adding detergent and an electric field
drug enzymes involved either in phase I211 or applied. If the cell’s genomic DNA has been broken
Phase II212 metabolism is affected by a potential into small fragments then these fragments move
drug. The measurement of gene expression out of the cell by electrophoresis and form a streak
levels upon exposure to a xenobiotic may provide or ‘‘tail’’ leading away of the cell. This looks a bit
information about its mechanism of action/elim- like a comet, hence the name of the assay.
ination forming a sort of genetic signature.
Microarray applications include the identifica- Hepatotoxicity
tion of disease-associated genes,213–215 drug
target validation,216 biological pathways dissec- Hepatotoxicity can be studied through some of the
tion,217–219 discovery of gene functions,220 experi- in vitro metabolism assays described above, such
mental annotation of the human genome,221 as: liver slices, cell-based cultures, hepatocytes,
compound toxicity and safety assessment and perfused liver. Mitochondrial dysfunction is
studies,222,223 tumor classifications,224–226 diag- often detected in liver cultures234–236 since the
nostic and prognostic predictions,227–230 and other liver is the main organ in contact with xenobiotics
biomarker identification.231–233 due to its detoxifying body function and the fact
that is a very well-perfused tissue. Table 3 lists
recent published in vitro toxicity work.

Toxicity
MTT Assay
Ames Test
A colorimetric assay based on the tetrazolium salt
In the Ames test, several strains of Salmonella MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
typhimurium that carry mutations in genes tetrazolium bromide) that measures only living
involved in histidine synthesis are used. The cells and can be read on a scanning multi-well
bacteria require histidine for growth. The variable spectrophotometer.
being tested is the mutagen’s ability to cause a
reversion to growth on a histidine-free medium.
SRB Assay
The tester strains are specially constructed to
have both frame shift and point mutations in the The goal of using Sulforhodamine B is to measure
genes required to synthesize histidine, which drug-induced cytotoxicity and cell proliferation for
allows for the detection of mutagens acting via large-scale drug-screening applications. Its prin-
different mechanisms. Some compounds are quite ciple is based on the ability of the protein dye
specific, causing reversions in just one or two Sulforhodamine B to bind electrostatically in a
strains. The tester strains also carry mutations pH-dependent manner to proteins and basic
in the genes responsible for lipopolysaccharide amino acid residues of trichloroacetic acid-fixed
synthesis, making the cell wall of the bacteria cells. Under mildly acidic conditions, it binds and
more permeable, and in the excision repair system can be extracted from cells and solubilized for
to make the test more sensitive. Rat liver extract measurement.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
PHARMACOKINETICS IN DRUG DISCOVERY 675

Clonogenic Assay substances at the molecular, cellular, tissue, and


individual animal levels.239 Gene expression
This is a microbiology technique for studying the
profiling is now being used in screening for
effect of specific agents on the proliferation of cells.
toxicity of new and existing chemical struc-
The term ‘‘clonogenic’’ refers to the fact that these
tures.240 The Health and Environmental Sciences
cells are clones of one another.
Institute (HESI) of the International Life Sciences
Institute (ILSI) has coordinated an international
study involving several pharmaceutical compa-
Ventricular Repolarization Assays: hERG Assay nies and governmental and academic institutions
to evaluate the harmonization of gene expression
The hERG assay is used to characterize the data an analyses showing that (1) patterns of
potential of a test substance to delay ventricular gene expression relating to biological pathways
repolarization. In these studies, test substances are robust enough to allow insight into mechan-
are studied for their ability to displace a hERG isms of toxicity, (2) gene expression data can
channel blocker (human Ether-a-go-go Related provide meaningful information on the physical
Gene). We can differentiate in vitro (patch clamp) location of the toxicity, (3) dose-dependent
and in vivo (electrophysiology) studies. For in vitro changes can be observed, and (4) concerns about
studies IKr assay, or patch clamp, is commonly used. oversensitivity of the technology may be
This technique traditionally uses a glass pipette unfounded.240 See Table 3 for some examples of
with a very small and smooth rounded surface tip gene markers of toxicity.
diameter (1 micrometer). This type of electrode is
known as a ‘‘patch clamp electrode.’’ The interior of
the pipette is filled with a solution that approx-
imates the intracellular fluid. A metal electrode in PHARMACOKINETICS: DIFFERENT
contact with this solution conducts the electrical APPROACHES, DIFFERENT GOALS
changes to a voltage clamp amplifier. During the
experiment, the researcher can manipulate the Pharmacokinetics is a discipline that uses mathe-
contents of this solution or add drugs to study matical models to describe and predict the time-
the ion channels under different conditions. The course of drug concentrations in body fluids. In
patch clamp electrode is pressed against a cell this respect, different methodologies or mathe-
membrane and suction is applied to the inside of the matical approaches have been described. Basi-
electrode to pull the cell’s membrane inside the tip cally, we can classify PK strategies under two
of the electrode. The suction causes the cell to form categories: Non-compartmental analysis (NCA)
a tight seal with the electrode (also-called ‘‘gigaohm and Modeling.241 Under this last category, we
seal,’’ since the electrical resistance of that seal is in have the classical compartmental analysis, Phy-
excess of a gigaohm). The patch clamp recording siologically-Based PK analysis (PBPK), and
uses a single electrode to voltage clamp a cell. Hybrid models where the classical compartmental
This allows a researcher to keep the voltage analysis and physiological based models are
constant while observing changes in current. mixed.
Alternately, the cell can be current clamped,
keeping current constant while observing changes
in membrane voltage.237,238 For the in vivo test, The
QT interval of the electrocardiogram (ECG) is the Non-Compartmental Analysis
most common endpoint. Additionally, blood pres- The NCA method uses algebraic equations and
sure, heart rate, PR interval, QRS duration, and provides a descriptive knowledge of the test
arrhythmias can be simultaneously monitored. compound. Very minimal assumptions are
made (the principal one regarding first order
exponential terminal phase), which minimizes
bias by eliminating the assumption required in
DNA Microarray Assay
any modeling. It is the method of choice for
DNA Microarray technology is becoming very Bioequivalence studies and the only analysis
popular in the toxicological field. Information on required by the FDA for a new drug application.
the global gene expression profile may provide However, no simulations can be performed using
clues to understanding biological actions of toxic this analysis.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
676 RUIZ-GARCIA ET AL.

Modeling what assumptions we can make, what is/are the


answer(s) that we are looking for and what is
Modeling uses differential equations. The model
the set of data that we have to work with.
can be totally empirical where the compartments
Depending on what PK analysis we perform, we
are black boxes (the drug scheme is commonly
will be able to answer different questions which
generalized into one- two- or three-compartment
will drive the conclusions that will be extracted
structures) or have some physiological meaning
out of that analysis. In summary, we need to
assigned to the compartments defined in the
understand the different PK mathematical
model (PBPK).242 The selection of the best fit
approaches to make a smart choice of which
model is done by using some sort of statistical
methodology to use, and this depends upon what
criterion along with diagnostic plots. However, in
information is required from the study. In general,
pharmacokinetics there is no such thing as ‘‘the
for early discovery up to lead optimization a NCA
model.’’ A good pharmacokinetic analysis will
as a ‘‘high throughput analysis’’ would be suffi-
provide a mathematical model that will be able to
cient in the majority of the cases. Even for filing
fit the data, simulate, and predict various case
an NDA, NCA is all that is required. Supporting
scenarios with a certain degree of comfort. This
an NDA with modeling would be recommendable
will not necessarily be the best model; it should be
in certain cases where nonlinearities due to
the simplest model that successfully describes the
metabolism or carried-mediated systems are
dataset.
significant in the dose range that is considered
The main purpose of a pharmacokinetic analy-
clinically relevant.
sis is to obtain a set of parameters that describe
the kinetic behavior of the drug in the body upon
administration. Thus, with the PK analysis, we
tend to extrapolate the results obtained in one SUMMARY
study to the whole population: the potential target
for the test article in study. According to the FDA, The analysis of the factors that impact
the term population pharmacokinetics (popPK) is drug discovery and development is extremely
‘‘the study of the sources and correlates of relevant for the success of new chemical entities.
variability in drug concentrations among indivi- More than half of the compounds that reached the
duals who are the target patient population ‘‘first in humans’’ stage failed due to toxicity
receiving clinically relevant doses of a drug of and safety concerns (see Fig. 1). Therefore, it is
interest.’’243,244 However, this definition is very imperative that the pharmaceutical industry
vague; popPK involves the analysis of data from a remains vigilant for faster, more efficient
group (population) of individuals, with all their methods of screening in order to survive in
data analyzed simultaneously to provide informa- the highly competitive pharmaceutical market-
tion about the variability of the model’s para- place. Expediting the discovery phase has proven
meters. The richness of this information will to be a crucial element of success in Drug
depend on the mathematical approach performed Development.
and the dataset available. Although the phrase In summary, there exists an extensive variety
‘‘population analysis’’ is being used to refer to one- of tools which LADMET scientists should take
stage analysis, it is equally applicable to naı̈ve advantage of in Drug Discovery. Good under-
pooled data (NPD), naı̈ve averaged data (NAD), standing of these systems allows us to wisely
standard two stage approach (S2S), Three stage apply them in an integrated fashion, always
analysis (3S), Bayesian Estimation, and one-stage keeping in mind their caveats and limitations,
analysis (linear and nonlinear mixed effects to extract more meaning from the data. We believe
modeling). this global overview and collection of methodol-
Table 8 summarizes some of the most relevant ogies will benefit Drug Discovery research in
internet resources for Pharmacokinetics/Pharma- taking the right approach to meet each specific
codynamics, including online PK courses, defini- goal that arises. Using the tools presented here is
tions, and examples. one step towards saving time while making smart
The intent of this review is not to conclude that choices in the design and direction of the research,
one approach is better than another. The goal is to through out the Drug Discovery/Development
understand when to use one or another based on continuum.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
Table 8. Online Information and Software Resources in Pharmacokinetics/Pharmacodynamics

URL Description Name


http://www.globomax.net/products/ NONMEM: Globomax NONMEM

DOI 10.1002/jps
nonmem.cfm
ftp://ftp.globomaxnm.com/Public/nonmem/ Shared resource for files and information for the NONMEM community
http://www.pharsight.com/main.php Pharsight facilitates strategic decision making in drug development. Pharsight WINONLIN/WINONMIX
software improves the value and availability of preclinical and clinical program
data for PK/PD modeling, analysis, drug attribute visualization, reporting and
trial simulation
http://xpose.sourceforge.net/ Xpose is an R-based model building aid for population analysis using NONMEM. XPOSE
It facilitates data set checkout, exploration and visualization, model diagnostics,
candidate covariate identification and model comparison
http://www.mrc-bsu.cam.ac.uk/bugs/ The BUGS (Bayesian inference Using Gibbs Sampling) project is concerned with BUGS
flexible software for the Bayesian analysis of complex statistical models using
Markov chain Monte Carlo (MCMC) methods
http://www.exprimo.com/ Exprimo is a European consulting company with the emphasis of its activities Exprimo NV
focused towards the application of quantitative, model-based approaches at
all stages of pharmaceutical development
http://www.emf-consulting.com/ EMF Consulting is a group of pharmaceutical consultants who provide EMF Consulting
drug development services, especially related to study design, analysis and
development planning
http://www.lapp.nl/ LAP&P provides interdisciplinary support on Pharmacokinetic and LAP&P Consultants
Pharmacodynamic aspects during preclinical and clinical Drug Development
http://www.boomer.org/pkin/ The purpose of this page is to provide links to information about the discipline of
Pharmacokinetics and Pharmacodynamics
http://depts.washington.edu/rfpk/ The Resource Facility for Population Kinetics is a computer resource facility
sponsored by the National Institute of Biomedical Imaging and Bioengineering
at the National Institutes of Health
http://anesthesia.stanford.edu/pkpd/ On this server you will find PK/PD software, computer controlled drug
administration programs, and simulation programs. This server is maintained
by Steve Shafer of the Department of Anesthesia at Stanford
http://www.lapk.org/ The Laboratory of Applied Pharmacokinetics (LAPK) of the School of Medicine LAPK
at the University of Southern California is a resource for optimal study and
control of pharmacokinetic systems and for individualized drug therapy
supported in part by the National Library of Medicine (NLM) and National
Center for Research Resources
http://www.drudevo.com/ More than 20þ years experience in modeling & simulation with a special DruDev0
focus on optimizing drug development and software development
http://members.aol.com/rdppweb/index.htm Research and development for population pharmacokinetics RDPP
PHARMACOKINETICS IN DRUG DISCOVERY

(Continued)
677

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008


678

Table 8. (Continued )

URL Description Name


RUIZ-GARCIA ET AL.

http://www.aster-cephac.com/index.asp Leading European contract research organization (CRO) ASTER.CEPHAC


providing clinical and bioanalytical services to pharmaceutical
companies
http://www.metrumrg.com/ It is a research-based organization focused on the investigation and Metrum Research
application of innovative mathematical modeling and simulation Group LLC
methods in clinical pharmacology and related biomedical sciences.
MetrumRG provides quality research and consulting services to
the pharma/biotech industries and non-profit research centers
http://www.nextlevelsolns.com/ Next level solutions offers both traditional and rapid analysis Next Level Solutions
solutions to population pharmacokinetics/pharmacodynamics
models

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008


http://accp1.org/pharmacometrics/ The objective of this free for all site is to aid in pharmacometrics American College of
index.html training Clinical Pharmacology
http://www.icp.org.nz/ By Dr Matt Doogue, Christchurch Hospital ONLINE text books or
courses
http://www.curvefit.com/index.htm By Harvey Motulsky, GraphPad Software, Inc.
http://web.vet.cornell.edu/public/ By The College of Veterinary Medicine at Cornell University
pharmacokinetics/pharmacokinetics.html
http://www.4um.com/tutorial/science/ By Pat Neligan
pharmak.htm
http://www.pharmacy.ualberta.ca/ By Dr. F. Jamali, Univ. of Alberta
pharm415/
http://www.boomer.org/c/p4/index.php?Loc¼ By David W.A. Bourne
Advance

DOI 10.1002/jps
PHARMACOKINETICS IN DRUG DISCOVERY 679

GLOSSARY ACKNOWLEDGMENTS

3-D-QSAR three dimensional quantitative We wish to thank Dr. Larry Wienkers (Amgen,
structure-affinity relationship Inc, Seattle, WA) for his insightful comments and
analysis helpful advice during the writing of this review.
3S three stage analysis His expertise contributed immensely to the orga-
ANN artificial neural network nization of the appropriate references and overall
BBMV brush border membrane vesicles clarity of this work
BCS biopharmaceutics classification
system
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