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Knock-out or humanized animals may also be used to assess specific metabolism
clearance mechanisms [155].
4.5.5.2 In Situ Perfused Organs Isolated perfused organs have been used to
investigate drug metabolism in numerous preclinical animal species. Isolated liver
models in the rat have been used most extensively and the procedure is described
elsewhere [23, 156–158]. The in situ perfusion of liver has several advantages
including intact tissue architecture, cell polarity, and bile flow. This model is able
to perform both metabolism and hepatic uptake and biliary secretion, and thus can be
used to determine the overall hepatic elimination.
4.5.5.3 Liver Slices Liver slices are prepared from liver by using a razor blade or a
precision-cutting device to cut small sections of liver tissue to a thickness of
200–500 mm [159]. The slices can then be incubated in buffer or cell culture medium
and used for drug metabolism experiments. As drug metabolism tools, liver slices
contain intact cells and have some of the same advantages as hepatocytes [160].
4.5.5.4 In Vitro Cell-Based Systems Hep atocyte suspension is a very popular
system for studying drug metabolism. Hepatocytes are intact cells and thus are
capable of performing both Phase I and Phase II metabolic reactions and also
sequential biotransformation reactions.
Hepatocytes are prepa red from intact liver by perfusing the liver with collagenase
to isolate the hepatocytes from the extracellular matrix. The isolated hepatocytes can
be incubated immediately with test compound in cell culture medium in an oxygenated atmosphere, or can be cryopreserved for future use.
4.5.5.5 In Vivo Subcellular Fractions Subcellular fractions isolated from animal
or human liver such as microsomes, S9 fraction, and cytosolic fraction are widely used
in drug discovery to study the rate of metabolism, reaction phenotyping, and
predicting metabolic profiles of drug candidates, and their potential DDI. Microsomes
preparations are the system most frequently used for high-throughput metabolic
screening as well as for more mechanistic drug metabolism studies in vitro.
Liver Microsomes Liver microsomes are prepared by further centrifuging liver S9
endoplasmic reticulum, which contains membrane-bound drug-metabolizing
enzymes, into vesicles called microsomes, which can be resuspended in buffer and
stored frozen. The microsomes contain the important CYP, FMO, and UDP-GT
enzymes, among others.
MICROSOMES VERSUS HEPATOCYTES Microsomes and hepatocytes can both be used for
determination of metabolic stability and intrinsic clearance. Each system has
advantages and disadvantages. Microsomes that are subcellular fractions lack cell
membranes and provide ready access of the test compound to the metabolizing
enzymes. They are used mostly for determining metabolism by CYP enzymes, which
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represent a viable screening strategy since CYP metabolism has the greatest overall
impact on metabolic stability. Microsomes can also be used to determine the
metabolic rates of other membrane-bound drug-metabolizing enzymes. However,
the addition of exogenous cofactors may be required, for example, UPD-glucuronic
acid fortified systems are used to facilitate formation of glucuronide conjugates by
UDP-GT enzymes. Hepatocyte incubations, by comparison, use cultured intact cells,
and thus have the advantage of structurally intact membrane-bound and cytosolic
enzymes. They are capable of catalyzing the entire complement of hepatic drugmetabolizing functions, and may thus provide a more relevant prediction of in vivo
metabolism. Hepatocyte systems sometimes yield artifactual low rates of metabolism
particularly for highly lipophilic compounds often encountered in early discovery
because of either poor solubility of test compound in the cell culture medium or
inability to enter the hepatocytes to gain access to the drug-metabolizing enzymes.
For lipophilic compounds, especially in screening mode, microsomal incubations
tend to provide higher rates of metabolism and are more useful for decision making
and rank ordering of compounds.
Liver S9 Fractions Liver S9 is a subcellular fraction prepared by homogenizing
liver, centrifuging at 9000 g and saving the postmitochondrial supernatant, which
can be stored frozen and used as a crude source of drug-metabolizing enzymes. An
advantage of liver S9 fraction is that it contains both cytosolic and membrane-bound
drug-metabolizing enzymes.
Liver Cytosolic Fractions Liver cytosolic fraction is prepared from the liver S9
fraction during the preparation of microsomes (see section “Liver Microsomes”).
During the final centrifugation step, the microsomes form the pellet and the
supernatant comprises the cytosolic fraction, which contains soluble drug-metabolizing enzymes, as opposed to the membrane-bound enzymes in the microsomes. The
cytosolic fraction is an excellent source of numerous Phase I drug-metabolizing
enzymes, including epoxide hydrolase, several reductases, alcohol dehydrogenase,
aldehyde oxidase, xanthine oxidase, and also Phase II enzymes capable of conjugating drugs with sulfate, glutathione, and methyl and acyl groups.
4.5.5.6 In Vivo Recombinant Drug-Metabolizing Enzymes Recombinant cDNAexpressed human drug-metabolizing enzymes are metabolism tools that are now
commercially available from numerous sources. A specific human drug-metabolizing
enzyme mRNA is used as the source for generation of a cDNA, which is recombined
with an expression vector and introduced into a cell culture system suitable for
production of a large quantity of human enzyme. The cells can be homogenized and
centrifuged to prepare microsomes or the enzymes can be further purified and
reconstituted.
4.5.5.7 In Silico Tools In silico models have been developed to predict drug
metabolism properties [161]. The simplest models are used for screening and utilize
physicochemical properties to predict metabolic stability. More sophisticated models
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have been developed for predicting hepatic clearance, metabolite formation, the
potential for metabolic DDI, and other metabolism properties [20, 162].
4.5.6 Applications of Drug Metabolism Tools
A variety of tools are available to investigate the role of metabolism in the overall
disposition of a test compound or drug candidate. The main uses of metabolism tools
are for the prediction of human exposure and safety, and generally fall into the
following four categories:
1. metabolic stability and the intrinsic clearance;
2. reaction phenotyping (identification of enzymes contributing to the metabolism
of a particular chemical entity);
3. metabolic profiling; and
4. prediction of potential metabolic DDI.
4.5.6.1 Metabolic Stability and Intrinsic Clearance The determination of metabolic stability of the newly synthesized compounds is usually done at a very early
stage of the drug discovery projects in a high-throughput screening mode aiming to
determine basic metabolic liabilities of the compounds, estimate their intrinsic
metabolic clearance, and establish so-called in vitro–in vivo correlations (IVIVC).
Metabolic Stability Liver microsomes and S9 fraction are the tools of choice for
determining metabolic stability, and fully automated, high-throughput systems for
these are applied at early stages of drug discovery. Test compounds are incubated
in vitro with the enzyme preparation and an NADPH regenerating system to supply
electrons for the CYP reactions. Consumption of parent compound from the incubation mixture is measured either at a fixed time point or using a time course to
determine the in vitro metabolic disappearance half-life. The resulting information is
used to advance compounds for further testing based on a pass/fail criterion or to
determine the rank order of a series of drug candidates.
Drugs that are eliminated principally by metabolism require a rate of metabolism
that is appropriate for the desired pharmacokinetic profile: short elimination half-life
for fast acting drugs, longer half-life suitable for BID or QD dosing regimen for most
chronically administered drugs. For drug candidates intended for chronic administration, rapid metabolism is often a key factor in poor pharmacokinetics. Several tools
and techniques are used at various discovery stages to screen out compounds
demonstrating undesirable high rates of metabolism.
Intrinsic Clearance Metabolic stability assays that measure an in vitro half-life are
used to determine intrinsic clearance, which essentially represents the fraction of
hepatic cleara nce driven by metabolism. The in vitro half-life is converted to an
in vitro intrinsic clearance value, and then physiological scaling factors are applied.
The scaled value is incorporated into a calculation of in vivo hepatic clearance, such as
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the well-stirred model (described previously in Section 4.3.3). The in vivo hepatic
clearance can be used for elucidation of the metabolic contribution to clearance and
also can be incorporated into the prediction of the in vivo elimination half-life [84].
In Vitro–In Vivo Clearance Correlation An important technique in the investigation
of the rate of drug metabolism is the determination of the correlation of in vitro
predicted hepatic clearance to that of in vivo hepatic clearance from animal pharmacokinetic studies. A lack of correlation such that the in vivo clearance is
significantly higher than the in vitro predicted value usually indicates that the
in vitro system represents only a part of the overall clearance mechanism. In the
case of underpredicted clearance using microsomes, either an S9 system fortified with
cofactors or a hepatocyte incubation system may provide the missing metabolic
contribution and a more accurate prediction. If metabolic intrinsic clearance
still underpredicts in vivo clearance, the results may indicate that either biliary
secretion or extrahepatic metabolism are significant factors in the elimination of the
compound.
4.5.6.2 Reaction Phenotyping An important discovery stage activity is the
determination of which drug-metabolizing enzymes are responsible for the elimination of a drug candidate. This knowledge is used to predict if the drug candidate will
be eliminated by a single metabolic pathway, resulting in a potential metabolic DDI
risk (especially for CYP3A4). It is also implemented to predict whether the drug
candidate will be metabolized by an enzyme that is polymorphically expressed in
human and could lead to high variability of drug exposures, which could result in
clinical safety issues [163]. The polymorphically expressed human drug-metabolizing enzymes of most concern are CYP2D6 and CYP2C19 [164]. Depending on which
enzymes and how many enzymes contribute to metabolism, different techniques are
employed.
In vitro incubation with different subcellular liver fractions can be used to assess
the location of the enzyme (microsomal membrane-bound or cytosolic fraction).
Addition or subtraction of required cofactors and/or specific chemical inhibitors to
in vitro incubations can be used to assess the type of enzyme (CYP,UDP-GT,etc.). For
CYP enzymes, the battery of assays used to determine the fraction of metabolism
catalyzed by the various CYPs is referred to as reaction phenotyping [163] and utilizes
pooled human liver microsome incubations in combination with isof orm-selective
inhibitors and additional experiments with recombinant human CYP enzymes.
4.5.6.3 Metabolic Profiling The aim of the metabolic profiling of drug candidates
at early stages of the discovery projects is to determine the structural elements of the
molecules causing high metabolic instability of the compounds. Those functional
groups (“soft spots”) can then be modified or replaced with different functional groups
in order to reduce the rate of the metabolism of the drug candidates.
At later stages of discovery projects, metabolic profiling is typically used to
compare the metabolites formed in the preclinical animal species with those that can
be formed in the humans to ensure the safety of human subjects in clinical trials.
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Identification of Metabolites and Metabolic “Soft Spots” Metabolic soft spot
identification is utilized in early discovery to identify metabolically labile sites that
cause unacceptably high rates of metabolism. For a compound of interest or a few
compounds in a chemical series, major metabolites are putatively identified using
LC–MS/MS (liquid chromatography–electrospray tandem mass). The metabolic
profiles are examined to determine whether a single site can be blocked or if multiple
metabolic routes exist and thus metabolism will be more difficult to reduce by
strategic modifications. Liver microsome, S9 or hepatocyte incubations, as well as
animal plasma and urine can all be used as the source of metabolites for assessment of
highly labile sites, although in early discovery screening, microsomes are the tool
used most often.
In vitro metabolizing systems, particularly liver microsomes, are used as a source
of metabolites for the assessment of active metabolites in early discovery pharmacology screens. Test compounds are incubated in vitro with liver microsomes to
generate metabo lites. The incubations are then subjected to a simple purification
procedure and the resulting supernatants or resuspended extracts are tested for in vitro
potency. In later stages of discovery, major metabolites (>10% in circulation
compared to parent compound) may be purified or synthesized and submitted to
pharmacology testing to more accurately evaluate the contribution of the metabolite
to the overall in vivo pharmacological activity.
Animal and Human Metabolites in Safety Testing Prediction of the human metabolic profile is required in order to predict the elimination mechanisms in humans
and to understand the likely role of metabolism in human clearance. An additional
important consideration is that all predicted human metabolites should be adequately
represented in preclinical animal species during safety studies. The only relevant tools
for prediction of the human metabolic profile in the discovery stages are human
in vitro systems, due to species differences in metabolism, most significantly for CYP
enzymes.
Hepatocyte incubations are preferred as they provide the best overall picture of
metabolism. For low turnover compounds, liver microsomes and occasionally
recombinant expressed human CYP enzymes may be used to generate and identify
putative human metabolites. In the development stage, when human plasma and urine
become available from clinical Phase I studies, metabolite scouting can be performed,
while definitive human metabolite identification is typically performed later in
development using radiolabeled compound.
Cross-species metabolite identification is performed in the late discovery stage to
determine if the predicted human metabolite profile is qualitatively similar to the
metabolic profiles in the preclinical animal species (usually rat and dog or monkey)
used for toxicology testing. Identification of the formation of a metabolite that is
unique to human represents a drug development risk because the metabolite cannot be
generated by the toxicity species and thus cannot be evaluated prior to clinical trials.
Also, predicted human major metabolites may be subject to required preclinic al
toxicity assessment in addition to the testing required for the parent compound as
described in the FDA Guidance for Industry called MIST (Metabolites in Safety
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Testing) [165]. Cross-species metabolite identification is performed, minimally, by
incubating the test compound in vitro with hepatocytes from human and all relevant
toxicity species, and then determining and comparing the metabolic profiles. Additional profiling of in vivo plasma and/or urine from the toxicity species can help to
understand the relevance of the in vitro metabolite profile to the in vivo situation and
strengthen the human prediction.
4.5.6.4 Prediction of Metabolic Drug–Drug Interactions A variety of human
in vitro systems are used for the prediction of a potential metabolic DDI due to the
inhibition or the induction of the CYP enzymes.
CYP Inhibition and Potential DDI CYP inhibition studies are conducted to assess
the potential for new chemical entities or drug candidates to potently inhibit any of the
major human CYP isoforms, typically CYP1A2, CYP2C9, CYP2C19, CYP 2D6, and
CYP3A4. Different techniques are used in differentstages of discovery but most assays
use pooled human liver microsomes as the CYP enzyme source and either a single
concentration or varying concentrations of test compounds to generate IC
50
values.
Early stage discovery assessment of CYP inhibition employs high-throughput
assays to screen large numbers of compounds and facilitate building structure–
inhibitor relationships that can be used for lead compound design [147].
In later stages of discovery, emphasis shifts to the determination of K
i
values and
investigation of possible atypical kinetics and determination of the type of inhibition,
especially nonreversible time-dependent inhibition [166]. In addition, rigorous,
validated assays have been developed for the definitive determination of CYP
inhibition during development [167]. Understanding of the potency and type of
CYP inhibition, combined with knowledge of the fraction metabolized by individual
CYP isoforms, can be used effectively to predict the potential for human metabolic
DDI using commercially available prediction software.
CYP Induction and Potent ial DDI Several in vitro models can be utilized to predict
the likelihood of a metabolic DDI due to induction. The main focus of these models is
to assess the potential for induction of CYP3A4 via binding to the nuclear receptor
PXR, which is the mechanism for most of the important examples of clinical
metabolic DDI caused by induction.
In early discovery, a CYP3A4 reporter gene assay can be used to screen a large
number of compounds. The cell line most often used is the HepG2 cell line [168]. In
this assay, compounds can be rank-ordered and compared to known clinical inducers
to categorize them as potential inducers. For definitive assessment of induction
potential, either the immortalized hepatocyte cell line Fa2N-4 can be used, or
preferably,primary cultured human hepatocytes. In either system, CYP3A4 induction
can be measured by increases in CYP3A4 mRNA, CYP3A4 protein, and CYP3A4
functional enzyme activity using a probe substrate. These increases can be compared
to established potent inducers such as rifampicin. The Fa2N-4 system has the
advantage of cell availability and batch-to-batch reproducibility, but has disadvantages such as low expression of some hepatic uptake transporters.
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Cultured human hepatocytes have been proposed as the preferred system for
definitive in vitro determination of human induction potential [149] (http://www.fda.
gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/
ucm072101.pdf) and is considered to be most predictive. However, this system has the
disadvantage of limited availability and batch to batch variability of induction
response [169].
4.5.7 Tools for Studying Drug Excretion
Tools for determining the role of excretion in the elimination of a drug candidate can
be employed at various stages of the drug discovery process and include in vivo
pharmacokinetic studies, in situ models, and cell-based or membrane vesicle transporter systems.
As opposed to the myriad of tools for assessing drug metabolism, excretion tools
are more limited and in many cases utilized only after it has been determined that
metabolism is not the major mechanism of elimination.
4.5.7.1 In Vivo Animal Studies Due to the complexity of the excretion proce ss
that often involves a combination of passive and active transport processes, in vivo
animal models are useful tools for investigating the role of excretion in drug
elimination.
Renal Excretion In Vivo Pharmacokinetic studies in preclinical animal species can
be used to determine the renal clearance of test compounds from urine concentration
data (Section 5.2.4.1). For compounds that are hydrophilic or charged at physiological
pH, renal excretion may represent the main route of elimination. Renal clearance in
human is generally well predicted by preclinical animal species [170, 171], especially
for passive glomerular filtration. Renal clearance data from pharmacokinetics studies
can be effectively used to screen discovery compounds.
Biliary Excretion In Vivo The most useful tool for evaluating the contribution of
biliary secretion to drug elimination is the bile-duct cannulated animal model, and rat
is the species most widely used for this purpose. In this model, animals are surgically
cannulated so that bile can be continuously collected from the bile ducts in the liver.
Following intravenous administration of compound, a time course of bile samples can
be collected, concentrations determined, and biliary secretion calculated. Knowledge
of the rate of biliary secretion can also be used to investigate the role of hepatobi liary
recirculation in the disposition of compound.
In Situ and Ex Vivo Animal Models The use of the in situ excretion models is fairly
limited and is not very frequently used in the discovery settings.
Renal Excretion In Situ The isolated perfused kidney (IPK) model can be used to
study specific aspects of renal excretion [172, 173], and the effect of drug on various
renal functions can be determined by adding the drug to the recirculating perfusion
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medium. In addition to excretion, kidney metabolism and toxicity may also be
investigated usin g this model. The renal clearance of drug candidates may also be
estimated by measuring drug uptake in kidney slices [174].
Renal Excretion Ex Vivo The renal clearance of drug candidates can be estimated
by measuring the drug uptake by using kidney slices [174].
4.5.7.2 In Vitro Cell-Based Excretion Models The use of the in vitro cell-based
systems for studying drug excretion is a rapidly growing area of science and
technology driven by the need to assess the mechanism of the excretion processes
mediated by a broad variety of the transporters.
Cell-based transporter systems can be utilized to provide information pertaining to
the involvement of individual transporters in the excretion process. Specific transporters can be transfected into cells with low background activity.The transporters are
expressed in the cell membrane and the transport of compound into the cell can be
measured quantitatively. The most important application of this technology is to
determine if transport is likely to be saturable, which represents a safety liability.
Primary Cell Cultures Primary cell systems can be used to investigate transporters
that are important for renal or biliary excretion. An advantage of primary cell systems
is that they express the full complement of transporters and may best represent the
in vivo situation. A disadvantage is that these systems exhibit limited cell viability and
are also limited by the availability of tissue.
Sandwich-cultured rat and human hepatocytes have been developed and used as
tools to evaluate the in vivo biliary clearance of drugs [175]. The sandwich-culture d
cells are able to form intact bile canalicular networks and maintain functional
expression levels of uptake and efflux transporters for several days. This system
may be useful for predicting the huma n biliary secretion of drug candidates.
Immortalized Cells Immortalized cell systems are capable of expressing high levels
of specific transporters and can be grown in large quantities. However, expression of
some normal transport systems may be lacking, and transporter expression and
activity may become unstable in culture over time.
Transfected Cell Lines Numerous heterologous expression (trans fected) systems
have been developed to investigate the function of renal and biliary excretion
transporters. Transfected cell lines can express specific transporters of interest.
Although the transporters are expressed in a nonnative cell background, which may
affect the transport kinetics, these systems are especially useful tools for screeni ng
because large quantities can be easily produced.
Membrane Vesicle Transporter Systems For polar or charged compounds that
cannot enter cells via passive permeability, membrane vesicle systems can be used
to study efflux transport. Membrane vesicles are prepared from cells resulting in an
inverted transporter configuration, such that the test compound has access to the
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transporter, and uptake of the compound into the vesicle indicates functional efflux
transport.
4.6 STRATEGIES FOR ASSESSING ADME PROPERTIES
Designing the right ADME properties into a molecule starts with an understanding of
the pharmacokinetic properties needed to deliver the targeted pharmacodynamics
(PK–PD). Biomarker identification as an efficacy surrogate is also becoming more
important for the success of discovery programs. As nonclinical models are used to
predict efficacy in human disease, attention to the consistency of biology system
drivers in preclinical models is paramount. Otherwise, predicting efficacious human
doses may be quite difficult. Designing in the correct ADME properties can be easier
for precedented targets and mechanisms, where benchmark compounds are likely
available for comparison. However, unprecedented targets provide more significant
challenges to the discovery scientist. The tools applied in the assessment of ADME
properties within a screening paradigm will vary by stage of a program and can vary
with time as greater knowledge is gained. In general, the logic of applying ADME
tools starts with an understanding of the defined in vivo targets and using the
appropriate in silico (presynthesis) and in vitro (postsynthesis) tools to predict the
desired in vivo behavior. The judicious use and cross-validation of in silico, in vitro,
and in vivo tools, with an appreciation for their limitations, should provide a
framework for efficient drug design. The primary focus of screening should be on
early identification of rate-limiting ADME prope rties and subsequent optimization.
This is the topic of further discussion in Section 4.6.1.
4.6.1 Assessing ADME Attributes at Different Stages of Discovery Projects
A typical discovery project involves four major stage gates: Target Identification,
Lead Selection, Lead Optimization, and Preclinical Development (Figure 4.12). An
example of a similar process can be found in the literature [176, 177].
4.6.1.1 Target Identification The Target Identification stage focuses primarily on
the biology of a selected target. In addition, an initial assessment is made of the
ADME space to which the chemical space of interest belongs. This can provide an
early read of the deliverability of targeted molecules.
Animal testing, primarily in rodents, may be utilized for preliminary evaluation of
PK properties of screening hits and/or benchmark compounds described in the
literature. It is also used for selecting an appropriate dose and route of administration
to deliver sufficient exposures in the animal efficacy models to build confidence in the
rationale of the biology of the target.
4.6.1.2 Lead Selection The primary purpose of the Lead Selection stage of the
discovery project is to (1) identify the spectrum of ADME properties of several
chemical templates (series) and (2) determine the drivers of efficacy (and biomarkers)
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by analyzing the relationships between the extent and the duration of in vivo exposure,
efficacy and target modulation. Certain tissue distribution studies (e.g., brain penetration) and receptor occupancy assessments may provide valuable information
as to target site availability. Initial screening PK is used at this stage to assess
clearance mechanisms and IVIVC. Initial in vivo mechanistic studies aimed at
evaluating the primary routes of elimination may be conducted, and may include
bile-duct cannulated (BDC) rats or even dogs. Another important goal at this stage is
to begin setting up in silico, in vitro, and in vivo relationships so that these tools can be
appropriately incorporated into the screening paradigm paying particular attention to
model interrelationships. The Lead Selection stage of the project is particularly
important in establishing the program as the working chemical space is narrowed
significantly.
4.6.1.3 Lead Optimization Selection of the lead series is a significant milestone
for the discovery project, which now moves to the Lead Optimization stage. At this
point, much effort is applied to specifically fixing any revealed ADME problems and
to identify the best compounds for further advancement. In vitro and in silico tools for
ADME assessment are widely used at this stage of the project. In particular, predictive
in silico tools provide an effective way to rationally design and enrich the pool of
compounds that likely will possess the proper in vivo ADME properties.
4.6.1.4 Preclinical Development In the Preclinical Development stage, typically
a front running candidate is chosen from a handful of leads for eventual clinical
development. More definitive ADME-related studies are typically conducted in vitro
and in vivo to revise early assessments from screening studies. More extensive safety
evaluations are run to assess exposure multiples between projected human doses and
Lead
Optimization
Preclinical
Development
Lead
Selection
Target
Identification
Characterize ADME
space of initial hits
Characterize ADME
of benchmark
compounds if
available
Early exploration of
routes of
administration and
dose for efficacy
assessment
..
Early PKPD and
biomarker identification
.
Identify ADME issues
with lead chemical series
.
Correlate extent and
duration of exposures
with efficacy and target
modulation (efficacy
drivers)
.
Target tissue distribution
to ensure exposure at site
of action
.
Routes of elimination
.
In silico, in vitro, and in
vivo correlatation
identified
Optimize ADME
properties in the context
of efficacy and safety
projections
.
Support of efficacy and
biomarker studies
.
Routes of elimination for
in vitro–in vivo
correlation
.
Early dose projections
.
Established in silico, in
vitro, and in vivo
correlation
.
PKPD and biomarker
response understood
.
Clearance mechanisms
understood
.
Expsosure multiple
assessment from efficacy
and toxicology studies
.
Multispecies PK to
project human PK
.
In vitro inhibition and
induction DDI
assessment
.
Enabling formulation
selection based on PK
.
In vitro human
metabolite profile
.
In vivo metabolite profile
in toxicolo
gy sp
ecies
.
.
.
Figure 4.12 The stages of the discovery process and primary purposes of ADME studies.
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