ruiz-garcia et al-2008-journal of pharmaceutical sciences
DESCRIPTION
Pharmacokinetics and drugTRANSCRIPT
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Pharmacokinetics in Drug Disco
ANA RUIZ-GARCIA,1 MARIVAL BERMEJO,2 AARON MOSS,3 V
1 Am
ity o
ox 3
Received 19 October 2006; revised 28 January 2007; accepted 5 March 2007
biophysical models; biopharmaceutics classification system (BCS); bioequivalence;
REVIEW OF BASIC metabolites in serum, plasma, or whole blood,
,/
further subdivided into the study of distributions
Kperspective, we consider that the drug has beeneliminated when it is no longer in its originalJournal of Pharmaceutical Sciences, Vol. 97, 654690 (2008)
2007 Wiley-Liss, Inc. and the American Pharmacists Associationand elimination. The term elimination includemetabolism and excretion since, from the P
Correspondence to: Ana Ruiz-Garcia (Telephone: 206-265-7408; Fax: 206-217-0494; E-mail: [email protected])PHARMACOKINETIC CONCEPTS
Pharmacokinetics (PK) is the study of a drug and/or its metabolite kinetics in the body. It refersto the temporary evolution of a drug and its
tissue target and target organs over time.1 Thebody is a very complex system and a drugundergoes many steps as it is being absorbeddistributed through the body, metabolised, andor excreted (ADME). Pharmacokinetics hasbeen broadly divided into two categories ofstudy: absorption and disposition. Disposition isADME654 JOURNApharmacokinetics in Drug Discovery. The review is structured into four sections. Thefirst section is a general overview of what we understand by pharmacokinetics and thedifferent LADMET aspects: Liberation, Absorption, Distribution, Metabolism, Excre-tion, and Toxicity. The second section highlights the different computational or in silicoapproaches to estimate/predict one or several aspects of the pharmacokinetic profile ofa discovery lead compound. The third section discusses the most commonly usedin vitro methodologies. The fourth and last section examines the various approachesemployed towards the pharmacokinetic assessment of discovery molecules; including allthe LADME processes, discussing the different mathematical methodologies availableto establish the PK profile of a test compound; what the main differences are andwhat should be the criteria for using one or another mathematical approach. Themajor conclusion of this review is that the use of the appropriate preclinical assayshas a key role in the long-term viability of a pharmaceutical company since applyingthe right tools early in discovery will play a key role in determining the companysability to discover novel safe and effective therapeutics to patients as quickly aspossible. 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci97:654690, 2008
Keywords: absorption; toxicology; structureactivity relatioship (SAR); populationpharmacokinetics; pharmacokinetic/pharmacodynamic models; computational ADME;ABSTRACT: The aim of this current reine in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21009
view is to summarize the present status ofPublished onl1Pharmacokinetics and Drug Metabolism, Amgen, Inc, 120
2Pharmaceutics Department, College of Pharmacy, UniversBurjassot 46100, Valencia, Spain
3Department of Pharmaceutics, University of Washington, BL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUvery
ICENTE G. CASABO2
gen Court West, Seattle, Washington 98119
f Valencia, Avda Vicente Andres Estelles s/n,
57610, Seattle, Washington 98195, SpainARY 2008
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chemical structure. Restated, when any biotrans-formation of the parent compound takes place andeven if the resulting metabolites remain in thebody, it has been eliminated.
LADMET-R and Pharmacokinetics
When the studies are focused solely in one specificpharmacokinetic aspect (Absorption, Distribu-tion, Metabolism, or Excretion) by in vitro,in situ, in vivo, or in silico techniques it is usuallyreferred to as ADME studies whereas the namePharmacokinetics is normally reserved to in vivostudies where an integrated approach of allthe ADME processes together is taken. Foreither ADME or Pharmacokinetics, the truth ofthe matter is that under both approaches, it isnecessary to command a more or less sophisti-cated knowledge of algebra and calculus tocorrectly interpret the dataset. Although ADMEassays have been the gold standards in PK, thereare additional tests that should be incorporated,since they play a key role in Drug Discovery andfurther development. Liberation of the drug from
solutions. . .etc) determines the disposition of thedrug.812 Response and toxic effects are the othertwo key aspects to consider since they are themain reasons for Drug Discovery failure (seeFig. 1). In summary, when we refer to the differentindividual assays that should be performed tocharacterize the PK profile of a new drug in vitro,in situ, in vivo, or in silico we should also consider,besides the gold standard ADME, (1) releasefrom the pharmaceutical form, (2) toxicity, and (3)activity/response in the target site (LADMET-R).
DISCOVERY AND DEVELOPMENT
New drug development can be divided in twodifferent stages: discovery and development.Recently, Kola and Landis13 reviewed the majorcauses of attrition in development (see Fig. 1). Intheir review, they showed how the root causes ofdrug failure have evolved over time (19912000).In 1991, PK and bioavailability were the majorreasons for drug failure (40%) dropping dramati-cally to 10% in 2000. This significant change is
pmeopm
PHARMACOKINETICS IN DRUG DISCOVERY 655the pharmaceutical form is a key parameter inbioequivalence studies (e.g., a sustained releaseversus immediate release formulation)27 or, forintravenous formulations, where the rate ofrelease from the formulation (liposomes, micellar
Figure 1. Main reasons for drug develoTufts Center for the Study of drug DevelDOI 10.1002/jps JOURNnt failure. Adapted from Kola 200013 andent.mainly due to the time and effort that Industryhas invested in the last decade toward a deeperand better understanding of PK, partially in anattempt to overcome poor bioavailability but alsotrying to look into more predictive kineticAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
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behavior of the drug candidates to allow for moreefficient dose regimens. Lack of efficacy and safetywere reported as the most relevant causes of whycompounds undergo attrition in the clinic in 2000(30%). The Tufts Center for the Study of DrugDevelopment14 published in 2005 the three mainreasons for terminating unpromising new drugs.Again, safety and efficacy were listed among themain three. In summary, the identified issues inthat report have been the main focus of study inrecent years as well as a driving force determiningwhich strategy to follow in Drug Discovery. Thecomposite of activity, safety, and acceptableLADME properties, rather than a specific attri-bute, will dictate the success of the drug program.In order to identify potential liabilities in dis-covery and eliminate those molecules from furtherconsideration, high throughput screening (HTS)of reliable and appropriate in vitro, and/or in situassays seem to be the fastest and more efficient
1517
the sponsor. The project team needs to be aware ofthe target product profile in order to makeeducated decisions about the direction that theproject needs to evolve in order to reach the nextmilestone in development. Components of thetarget product profile are: disease indication,minimum efficacy requirements, required safetyprofile, desirable dose regimen, dosage form,maximum cost of goods, planned date of regula-tory submission and expected approval date.18
In summary, when planning exploratory stu-dies in humans, under an Investigational NewDrug (IND) application, there is some preclinicaldata as well as chemistry, manufacturing andcontrols information that need to be generated.The approaches taken in generating this data canbe optimized expediting the progress into devel-opment and increasing the chances of success ofthe IND filing by compiling a good quality datasetin an efficient manner. Depending on the goals of
improved in the last few years. This is due to the
Dru
656 RUIZ-GARCIA ET AL.way to proceed, as shown in Figure 2.Generally, when a drug is granted to progress
into development, a project team is formedwith members of different areas of expertise(i.e., including, but not restricted to, toxicology,pharmacokinetics, clinical development, medic-inal chemistry, formulation, marketing and reg-ulatory affairs), with the goal of establishing anearly development plan. Successful drug develop-ment is a result of getting to this stage withenough information about the previously men-tioned processes (LADMET-R) in conjunction witha worthwhile investment that provides value to
Figure 2.JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008acknowledgement from Industry of the relevanceof this information to the success of the drug as
g discovery.the proposed investigation, the amount of datathat need to be submitted can vary.19
DRUG DISCOVERY:HOW DOES THIS WORK?
The quality and quantity of preclinical dataprovided by discovery groups to support thedevelopment of a new drug has considerablyDOI 10.1002/jps
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PHARMACOKINETICS IN DRUG DISCOVERY 657mentioned above. A variety of in vitro assays havebeen automated through the use of robotics.In silico models are being used to assist in theselection of the right assay and the set ofcompounds undergoing further in vitro screening.With the emerging new computational models(in silico), a deeper understanding of the relation-ship between important LADME-T parametersand molecular descriptors and/or in vitro para-meters has been achieved, allowing for an earlyestimation of several LADME-T properties (seeTab. 1).
HTS facilitates a researcher to effectivelyconduct some biological test to a large numberof potential therapeutic moieties.20 Through thisprocess, rapid discrimination of active ingredientsversus undesirable compounds based on theresults of the particular assay can be achieved.The main difference of this strategy versus thetraditional pharmaceutical screening is thatless rigorous results are needed. There are fewsamples from many compounds as opposed to avery rich database from few compounds. HTSscreening is, in essence, a single goal to which allthe data may subsequently be applied. HTS isused at early stages of discovery to gatherLADME-T information that serve as key factorsfor candidate selection. As a result, there has beena recent focus on enhancing the efficiency ofobtaining absorption, disposition, and toxicitydata, which has permitted LADME-T scientiststo contribute more effectively to the drugdiscovery process.
Since the oral route is the preferred adminis-tration route for patients, and this fact assurescompliance of the drug therapy, a lot of time andeffort has been invested toward a good under-standing of the physicochemical properties thatplay a key role in bioavailability. Bioavailabilityhas been defined by the FDA as the rate and extentto which the active ingredient or active moiety isabsorbed from a drug product and becomesavailable at the site of action.21 Thus, for oralpharmaceutical forms, systemic exposure is goingto be highly dependent on the extent of absorptionin the gastrointestinal tract (GI). The Biophar-maceutics Classification System (BCS)22 as adrug development tool allows for the estimationof contributions of the three major factors thataffect drug absorption: dissolution, solubility, andintestinal permeability.2224 Based on in vitrosolubility and in vivo permeability values, drugscan be divided into four groups: class 1 (highpermeability, high solubility, HP:HS), class 2DOI 10.1002/jps JOURN(high permeability, low solubility, HP:LS), class 3(low permeability, high solubility, LP:HS), andclass 4 (low permeability, low solubility, LP:LS),see Figure 3.
This analysis points out conditions under whichno in vitroin vivo correlation may be expected forexample, rapidly dissolving low permeabilitydrugs. Furthermore, it is suggested that for veryrapidly dissolving high solubility drugs, forexample, 85% dissolution in less than 15 min, asimple one-point dissolution test is all that may beneeded to insure bioavailability. For slowlydissolving drugs, a dissolution profile is requiredwith multiple time points in systems which wouldinclude low pH, physiological pH, and surfactants,where the in vitro conditions should mimic thein vivo processes. The draft guidance documententitled Waiver of In Vivo Bioavailability andBioequivalence Studies for Immediate ReleaseSolid Oral Dosage Forms Containing CertainActive Moieties/Active Ingredients Based on aBiopharmaceutics Classification System pro-poses to further expand the regulatory applica-tions of BCS and also recommends methods forclassifying drugs and immediate release formula-tions.25 However, Wu and Benet26 suggest analternative classification attending to solubilityvalues and metabolism rather than permeabilityvalues, The Biopharmaceutics Drug DispositionClassification System (BDDCS). The authors,while recognizing that drug metabolism can differdepending on the drugs solubility and perme-ability characteristics, consider that switchingpermeability values to extent of elimination wouldbe less restrictive, expanding the Class I drugseligible for waiver of bioequivalence (BE).27
For drugs with low permeability, the rate atwhich they are being actively carried throughthe GI and reaching the systemic circulation ishighly dependent on the carrier-mediated sys-tems involved. Both influx and efflux under thesecircumstances will play an active role in the oralbioavailability of the compound. In light ofthis, Klopman et al.28 discussed the importanceof lipophilicity in membrane transport models.
Several experimental techniques have beendescribed to evaluate intestinal absorptionincluding physicochemical measurements (e.g.,solubility, lipophilicity, partition coefficients),subcellular fractions (brush border membranevesicles, basolateral membrane vesicles), cellculture-based models, artificial membranes, iso-lated tissues, and organ preparations. Thesetechniques are briefly described in this reviewAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008 DOI 10.1002/jps
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PHARMACOKINETICS IN DRUG DISCOVERY 659DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
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660 RUIZ-GARCIA ET AL.under in vitro screening. Examples of theirapplication have been tabulated in Table 2.
In silico models are extensively applied toabsorption descriptors properties and bioavail-ability predictions. For instance, the quanti-tative structurebioavailability relationship of232 structurally diverse drugs was studied toevaluate the feasibility of constructing a predic-tive model for the human oral bioavailability ofprospective new medicinal agents by Yoshidaet al.29 where they found lipophilicity to be asignificant factor influencing oral bioavailability(see Tab. 1).
However, for many compounds, oral bioavail-ability may be limited by extensive metabolismrather than poor absorption. Metabolism can bedefined as the chemical changes (biotransforma-tion) that take place in a given chemical substancewithin an organism. The biotransformation thatnormally takes place within the body leadstowards more hydrophilic or water-soluble moi-eties than the parent compound which facilitates/
Figure 3. Biopharmaceutics classification systemand biopharmaceutics drug disposition classificationsystem.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008accelerates the excretion from the body. Thus, thepotential of a compound containing a chemicalmoiety known to be extensively metabolized maybe predicted with reasonable accuracy on the basisof abundant historical data.30 Several in vitromethods are routinely used to establish themetabolic 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 arebased on the native metabolic enzyme they are anexcellent source for the experimental generationof metabolite profiles, which will help influencethe selection of the appropriate pre-clinical safetyspecies. Several in vitro experimental techniqueshave been briefly described later in this review(see Tab. 3).
At the level of Metabolism, there are also somepredictive models. Bursi et al.33 have derived astructurepharmacokinetic relationship for a dataset of 32 in-house steroidal androgens. The samegroup developed an electronic model for hydrogenabstraction in steroidal androgens in which theactivation energies of steroid radical systems couldbe used to predict relative rates of metabolism toguide the design and redesign process of metabo-lically more stable steroidal androgens. Many 3Dligand-based and structure-based computationalapproaches have been used to predict the metabo-lism catalyzed by the enzymes of the cytochromeP450 superfamily (P450s) responsible for 70% ofthe metabolism of all drugs. Computationalmethodologies have focused on a few P450s thatare directly involved in drug metabolism. Modelsderived for P450s help to explain and predict theinvolvement of P450s in the metabolism of specificcompounds and guide the drug-design process.34
See Table 1 for additional computational models forhuman P450s.
Drug administration has the main goal ofachieving therapeutically effective drug concen-trations at the site of their clinical activity whileminimizing adverse effects (usually linked to non-target site drug concentrations).35 However, theability of drugs to reach the site of action dependson many pharmacokinetic aspects such us drugavailability and drug metabolism, both alreadymentioned, but also binding to plasma proteins.Drug tissue distribution is often correlated withtotal plasma/serum concentrations (bound andunbound fractions), which for drugs with a highpercentage of protein binding that act in periph-eral tissues becomes a questionable assumption.Several techniques have been developed to studyDOI 10.1002/jps
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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
PHARMACOKINETICS IN DRUG DISCOVERY 661
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662 RUIZ-GARCIA ET AL.Table 3. In Vitro Systems for Metabolism and To
System Origin
Liver microsomes Liver tissueIntestinal microsomes Intestinal tissueCytosolic fractions Liver tissueSupersomes Liver tissue
(Baculovirus-insect-cellexpressed)
S9 fractions Liver tissue
Isolated perfused liver Liver
Liver Slices Liver
Hepatocytes Liver
HepG2 Human hepatoma cell lin
HLE Human lens epithelial cedrug protein binding in plasma, with the twopredominant methods being ultrafiltration (UF)and equilibrium dialysis (ED).36,37
There is a clear need for companies to find waysto evaluate safety of drug candidates earlier in thedevelopment process. Animal toxicology studiesare the foundation of an IND. The principalsafety concerns are usually in the area of genetictoxicology, target organ toxicity and cardio-safety. The identification of HTS assays thatcan accelerate the advance of the drug candidateinto more relevant in vivo testing as soon aspossible is the main goal in the discovery stages.There are several toxicity studies that areroutinely performed depending on the nature ofthe drug. Genotoxicity, cytotoxicity, and targetorgan toxicity may be, at least in a first instance,evaluated through in vitro screening. The use ofpositive (known toxic reagents) and negativecontrols (non toxic reagents) is necessary to assure
BC2 Human hepatoma cell line
DNA microarray Rat liverRat liver
Genotoxicity COMET assayAmes test
Renal toxiciy DNA microarrayHepatotoxicityCytotoxicity MTT assay
SRB assayClonogenic assay
hERG potassium channels IKr assay
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008ty Studies
Comments Reference
Phase I metabolism 173175,183Phase I metabolism 176,177Phase II metabolism 178,179
Specific CYP/UGT mediatedmetabolism
32,183
Phase I and II metabolism 180,181Detecting DNA damage 182Hepatotoxicity 204206Metabolism 207,208Hepatotoxicity 184,185Metabolism 184,186Hepatotoxicity 199201,234236Metabolism 203Hepatotoxicity 188190Metabolism 187,191
e Hepatotoxicity 193,194Metabolism 192,193that the in vitro systems respond as the in vivotissue would. In vitro studies should be conductedwith concentrations and exposure times similar tothe in vivo conditions. The in vitro models mayallow high throughput screening, decreasing thenumber of chemicals tested in whole animals.
Mutagenicity screening is a regulatory require-ment for drug approval since they imply a toxicrisk in humans.38 The International Agencyfor Research on Cancer (IARC) discussed in aconsensus report39 the term genotoxicity, con-sidering that this term includes both direct andindirect effects in DNA. Direct effects are con-sidered inductions of mutations (gene, chromo-somal, genomial, and recombinational) that at themolecular level are similar to events known to beinvolved in carcinogenesis. Indirect DNA effectsinvolve surrogate events associated with muta-genesis (e.g., unscheduled DNA synthesis (UDS)and sister chromatid exchange (SCE), or DNA
Hepatotoxicity 196,197Metabolism 195,198Phase I metabolism 211Phase II metabolism 212Detecting DNA damage 44,45
41,42Changes in gene expression 273,274
275,276Cell proliferation 48,49,54,57
50,5152,53
Electrophysiology study 237,238
DOI 10.1002/jps
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Ta
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4.
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Sin
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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
PHARMACOKINETICS IN DRUG DISCOVERY 663
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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
664 RUIZ-GARCIA ET AL.damage (e.g., the formation of adducts) beingthe most relevant ones, which may eventuallylead to mutations. Mutations are permanenthereditary changes in the cell lines. Genotoxicitythus preceeds mutagenicity although most ofgenotoxicity is repaired and is never expressedas mutations.40 The Ames test and Comet assay(Single-Cell Gel, SCG) are in vitro cell-basedassays that are routinely used to assess genotoxi-city induced by xenobiotics4147 and they havebeen briefly described later in this review.
Cytotoxicity can be defined as the quality of axenobiotic to confer toxicity to cells. Cytotoxicityin different cell lines will provide useful informa-tion about potential target organs toxicities. MTT,SRB (Sulforhodamine B), and clonogenic assaysare used for measuring drug-induced cytotoxicityby measuring cellular proliferation in mammaliancell lines.4853 Moreover, a routine screening for awide range of pharmaceutical products is cuta-neous and ocular toxicity, where epidermalkeratocyte and corneal epithelial cultures havebecome relevant models that can provide valuableinformation about the mechanisms of cutaneousand ocular toxicities of test compounds.5457
Hepatotoxicity is one of the major reasons forwithdrawal of marketed drugs over the past twodecades.58,59 The pharmaceutical industry hasdedicated much emphasis on developing in vitroscreening systems to detect hepatotoxicity (iso-lated perfused liver, liver slices, primary hepato-cyte cultures, human liver-derived cell lines,etc).Toxins can be classified as hepatotoxins,which cause liver damage in the majority of thepopulation with or without metabolic activation.Further in this nomenclature, hepatotoxins canbe subdivided into cytotoxics if they injure thehepatocytes and cholestatics if they interfere withthe bile flow.60
The International Conference of Harmonisation(ICH) guideline S7B describes a nonclinicaltesting strategy for assessing the potential of atest substance to delay ventricular repolarization(VR). VR is determined by the duration of thecardiac action potential as a result of the activitiesof many membrane ion channels and transpor-ters. In this guideline are descriptions of in vitro(patch clamp) and in vivo electrophysiologystudies for competition binding protocols in whichtest substances are studied for their ability todisplace a hERG channel blocker (human Ether-a-go-go Related Gene).61,62 The hERG geneencodes a potassium ion channel responsible forthe repolarizing IKr current in the cardiac actionDOI 10.1002/jps
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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
PHARMACOKINETICS IN DRUG DISCOVERY 665
-
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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
PHARMACOKINETICS IN DRUG DISCOVERY 667
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Klopman et al.25 have developed a model for MDR
studied drug and proteins involved in LADMETprocesses (e.g., carrier-mediated systems such us
668 RUIZ-GARCIA ET AL.reversal agents (to overcome Multi-Drug Resis-tance) to estimate the MDR reversal activity ofcompounds. The same author discussed theimportance of lipophilicity values (representedas the logarithm of the n-octanol/water partitioncoefficient) and its correlation with their pharma-cological and toxic activities.29 Kazius et al.43 wereable to perform mutagenicity predictions of anindependent validation set of 535 compounds withan error percentage of 15%. The authors con-cluded that toxic properties can often be related tosubstructures, which are generally identified astoxicophores, and that these toxicophores can beapplied to risk assessment processes and canguide the design of chemical libraries for hit andlead optimization. Yoshida et al. have usedsome physicochemical descriptors (n-octanol/water partition coefficient, topological polar sur-face area, diameter, summed surface area ofatoms with partial charges) to carry out 2D-quantitative structureactivity relationship (2D-QSAR) studies on 104 hERG channel blockerswith diverse structures collected from the litera-ture, thus formulating interpretable models toguide chemical-modification studies and virtualscreening.64 Combination of predictive models hasalso been performed with great success. OBrianet al. combined hERG channel blocking andCYP450 2D6 inhibition computational modelswith better predicted values than their individualpredictions.17
IN SILICO LADMET
The line between in silico LADME-T and in vitro-in silico LADME-T is difficult to define since themajority of the in silico models use not onlyphysicochemical parameters but also some in vitrodata (see Tab. 1).
In recent years, the number of computationalmodels for the different LADMET processes hasconsiderably increased with the aim of promisingpotential. Abnormalities in this channel may leadto either Long QT syndrome (LQT2) (with loss offunction mutations) or Short QT syndrome (withgain of function mutations). Both are potentiallyfatal cardiac arrhythmia due to repolarizationdisturbances of the cardiac action potential.Erroneous drug binding to this channel may leadto acquired Long QT Syndrome.63
In silico models, described below, are also beingused for predicting activity as well as toxicity15.JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008p-gp, enzymes responsible of either phase I (e.g.,CYPs) or II biotransformation(e.g., Glutathionetranferases). Under molecular modelling, we candistinguish: Ligand-based models, Structure-based models, and Homology Models.
Ligand-Based Models
These attempts to link chemical structures withobserved activities based on information aboutactive site, shape, electronic properties and con-formation of substrates, inhibitors or metabolicproducts. The simplest one is QSAR: QuantitativeStructureActivity relationship. Three-dimensionalquantitative StructureActivity relationship (3D-QSAR) refers to the analysis of the quantitativerelationship between the biological activity of a setof compounds and their spatial properties usingstatistical methods.6669 3D-QSAR are often basedon Molecular Field Analysis, MFA. MFA employs acombination of reasonable molecular description,statistical analysis, and graphical display ofresults.67,70,71 Molecular structures are describedwith molecular interaction energies as steric andleads and the elimination of unsuitable ones.Although these models can never be accurateenough to replace real circumstances, many ofthem can be extremely useful if they are builtunder the correct assumptions and right set ofdata. When the in silico models have beencarefully developed and rigorously validated theinformation they provide can be valuable in earlyDrug Discovery. It is, therefore, not surprisingthat there is considerable interest in developingmathematical models capable of accurately pre-dicting some LADME-T key information for newdrug candidates. However, the misleading useand interpretation of in silico LADMET is oftenthe reason why these models have been exten-sively criticized by a large part of the scientificcommunity.65 These models are usually based inin vitro data and/or physico-chemical properties.Two different types of computational models arebeing used currently: molecular and data model-ing. A brief description of the fundaments of themost used computational models is presentedbelow and listed in Table 1.
Molecular Modeling
The main objective of molecular modeling is toassess the potential interaction between theDOI 10.1002/jps
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overlay in 3D space in an attempt to describe the
the same drug have the same concentration time
PHARMACOKINETICS IN DRUG DISCOVERY 669physical, spatial, and chemical properties of theactive site.10,7382
Structure-Based Models
Included in this category are X-ray crystallogra-phy,83 nuclear magnetic resonance (NMR),84
spectroscopy and electron microscopy. Thesemodels determine the 3D structure of proteinsthrough a variety of means. However, it is oftenvery difficult to use these techniques due to thenature of the proteins (e.g., difficult to crystallize,poor solubility, large molecular size).
Homology Models
As a result of the difficulties mentioned abovewhen trying to elucidate the 3D structure ofproteins, these models were developed. Homologymodels are an alternative method to elucidate the3D structure of proteins. These models are basedon the fact that the 3D structure of a protein isrelated to its amino acid sequence since proteinswith similar amino acid sequence tend to adoptsimilar 3D structure.8590
Data Modeling
Data modeling uses statistical tools to searchfor correlations between a given property and aset of physicochemical descriptors. Quantitativestructureactivity relationship (QSAR),28,29,64,9195
quantitative structureproperty relationship(QSPR),9699 population Analysis by topology-based QSAR (PATQSAR)100 and Artificial NeuralNetworks (ANN) are some examples of datamodeling. ANN is an adaptive system that chang-es its structure based on external or internalinformation that flows through the network.electrostatic fields surrounding the molecules; thestatistics is computed by partial least square (PLS)regression analysis and the output is displayed ascontours superimposed on the molecules. Thecomparative MFA, (CoMFA) methodology assumesthat a suitable sampling of steric and electrostaticfields around a set of aligned molecules provides allthe information necessary for understanding theirbiological properties.72 If no structural informationis available, an alternative means to assesspotential interactions is to use pharmacophoremodels. These are ligand-based models wheredifferent structures of ligands or their propertiesDOI 10.1002/jps JOURNprofiles at the intestinal membrane surface, thenthey will have the same rate and extent ofabsorption. Two conditions are necessary for thisstatement to be true: the two drug products havethe same in vivo dissolution profile under allluminal conditions and none of the formulationcomponents affects the membrane permeability orintestinal transit time.
IVIVC has been defined by the Food andDrug Administration (FDA) as a predictivemathematical model describing the relationshipbetween an in vitro property of a dosage form andan in vivo response.21,27 Generally, the in vitroproperty is the rate or extent of drug dissolution orrelease while the in vivo response is the plasmaANN may include molecular modeling and datamodeling.101103
Li et al.104 have done excellent work describingthe most recent explored statistical learningapproaches such as neural networks (NN), sup-port vector machines (SVM), etc. The authorsconcluded that both classification-based andregression-based statistical learning methodshave consistently shown promising capabilityfor predicting chemical agents of diverse rangesof structures and of a wide variety of LADMETpoperties.
Table 1 contains a list of references of publishedin silico work in LADME-T.
In Vitro In Vivo Correlations (IVIVC)
Bioequivalent products are those whose rate andextent of absorption do not show significantdifferences when administered at the same dose.21
The bioequivalence of two drug products is usuallyevaluated through in vivo assays in humanvolunteers. However, under some particularconditions, it should not be necessary to carryout an in vivo pharmacokinetic study to assurebioequivalence; a well validated in vitro studyshould be able to assess that. From an ethicalpoint of view, if the assay with human volunteersis not essential to demonstrate the equivalencebetween two formulations, the assay should not beperformed, but, on the other hand, we need toassure that the in vitro surrogate is reliable. Thefactors that we have to analyze to establish thetheoretical basis for correlating in vitro dissolu-tion and in vivo absorption are the parametersthat control rate and extent of absorption. Thebasic concept is if two drug products containingAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
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content (genomics), protein expression (proteo-mics), and measurements of metabolites (meta-
caused by a biological perturbation.
670 RUIZ-GARCIA ET AL.bonomics). Hopefully, the combination of thesedifferent readouts will provide us a combination ofpotential biomarkers as well as better predictorsof a disease state.
Genomics
Genomics is the study of an organisms genomeand the identification of the genes involved inbiological processes. Genomics has the potential ofoffering new therapeutic methods for the treat-ment of some diseases, as well as new diagnosticmethods. As a consequence of the identification ofgenome sequences, it is possible to engineer DNAmicroarrays, which can measure gene expressionof thousands of genes simultaneously.60,107,108
New applications in the field of genomics areemerging in two major areas.109 The first onefocuses on understanding the mechanism of actioninvolved in disease states or compound-inducedphenotypic changes. The second one involvesdrug concentration or amount of drug absorbed.The main objective of developing and evaluatingan IVIVC is to establish the dissolution test as asurrogate for human bioequivalence studies,which may reduce the number of bioequivalencestudies performed during the initial approvalprocess as well as with certain scale-up and postapproval changes.105,106 Two step or one stepmethods can be applied to obtain these correla-tions. Using the two-step method, by deconvolu-tion or by a mass balance method, the in vivofunction is computed and from dissolution assays,the in vitro variable is calculated, then, in a secondstep, plasma concentration are predicted byconvolution based on the in vitro data. A one stepmethod involves a convolution step where plasmaconcentrations predicted from the model andthose observed are directly compared. Publishedwork in this matter has been referenced inTable 1.
Genomics, Proteomics, and Metabonomics
Lately, these terms have been incorporated intomany scientists vocabulary with not always aclear idea of the intended meaning.
In addition to the traditional information aboutthe disease state, we now posses a set of newdescriptors obtained by molecular profiling. Inother words, we have access to large scale ofsystematic readouts at various levels such as DNAJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008The integration of genomic, proteomic andmetabonomic data constitutes a very powerfuldata source for in silico analysis. Although we donot fully understand the complexity of thespecific biomarker identification for various pur-poses ranging from disease monitoring to diseaseprogression and prognosis.
Proteomics
Proteomics is a high throughput study of proteins,particularly their structures and functions. Pro-teomics is much more complicated than genomics.Most importantly, while the genome is a ratherconstant entity, the proteome differs from cell tocell and is constantly changing through itsbiochemical interactions with the genome andthe environment. The entirety of proteins inexistence in an organism throughout its life cycle,or on a smaller scale the entirety of proteins foundin a particular cell type under a particular type ofstimulation, are referred to as the proteome ofthe organism or cell type respectively.110112 Sinceproteins play a central role in the life of anorganism, proteomics is instrumental in thediscovery of biomarkers, such as markers thatindicate a particular disease.113117
Metabonomics
Metabonomics has been defined as the quantita-tive measurement and identification of thebiochemicals contained in a biological samplesuch as the metabolic response of living systems topathophysiological stimuli or genetic modifica-tion. This approach has been used in toxicology,disease diagnosis, and a number of other fields.This technology has been used to identify bio-markers for disease as well as to identify off-targetside effects in marketed drugs and new chemicalentities in development.15,60,118120
A similar and related concept that is worthdefining here is Metabolomics, which refers to thestudy of the chemical fingerprints that specificcellular processes may produce. The study ofmetabolite profiles will fall into this category.Although there is still no absolute agreement,there is a growing consensus that the differencebetween the two terms resides in the fact thatmetabolomics places a greater emphasis oncomprehensive metabolic profiling, while meta-bonomics is used to describe multiple (but notnecessarily comprehensive) metabolic changesDOI 10.1002/jps
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provides the basis for establishing in vitroin vivo
N-Octanol/Water Partition Coefficient
PHARMACOKINETICS IN DRUG DISCOVERY 671This parameter is often expressed by the Log(P)value. The Log(P) is basically a parameter oflipophilicity where the distribution between asolute dissolved in an aqueous buffer (aqueousphase) and n-octanol, as organic phase, ismeasured.124126 The measure of disappearancecorrelations (IVIVC) and for justifying biowaivers,or in other words, permission to use dissolution testdata as a surrogate for pharmacokinetic data.Hence, a dissolution test can be used as an in vitrobioequivalence study instead of an in vivo bioequi-valence study.
Absorptionbiological systems and pathologies, by gatheringthe various sources of information and analyzingthe data together, we will certainly see increas-ed contributions toward the establishment offingerprints for toxicity, metabolism and otherLADMET-related processes.
IN VITRO LADMET
The following in vitro assays are probably themost common screening tools for the PK aspectsalready commented on throughout this review.Tables 2 and 3 list these in vitro assays pointingout the parameter assayed and some relevantpublished work in that matter.
Dissolution Studies
Dissolution studies are routinely performed as apart of the quality protocol of solid dosage forms,because these studies help to ensure that themanufacturing process has not deviated signifi-cantly from the established standards. The use ofdissolution assays as a quality control indexrequires a simple dissolution media with simpledissolution conditions in order to minimizepractical problems, such as analytical complica-tions, and to keep the cost of the test at theminimum value.121123 However, if a test isrequired that provides more information aboutwhat will happen in vivo, the BCS can simplify therequirements of the test and provide guidanceregarding the inferences that we can obtain fromin vitro assays.23 As mentioned previously, BCS isa framework for classifying drug substances basedon their aqueous solubility and permeability andDOI 10.1002/jps JOURNof the solute from the aqueous phase indicateshow much solute travelled to the organic phase.Both phases, aqueous and organic, should besaturated in each other to avoid changes involume when they contact as that will introduceconsiderable error in the determination of thisparameter. The partition coefficient is defined asthe concentration ratio between the organic andaqueous phase as follows:
P CoCa
Qai Qaf =VoQaf=Va
Where Qai and Qaf represent the amount ofsolute in the aqueous phase before and afterbeing in contact for a specific period of timewith the organic phase in continuous agitation.The pH value of the aqueous phase along withthe temperature used for the partitioning arethe variables that determine the value of thisparameter.
Liposomes
Liposomes are lipid bilayer vesicles used asmodels for biological lipid bilayer membranesfor the study of drug partitioning from aqueousphase into the liposome.127 Several authors havesuggested that this parameter correlates betterwith human drug absorption than n-octanol-water partition coefficient.128,129
Membrane Vesicles
The most commonly used are Brush BorderMembrane Vesicles (BBMV) and Basolateralmembrane vesicles (BLMV), both subcellularfractions. Its preparation involves tissue homo-genation and differential sedimentation, fractio-nation, and differential precipitation. For BLMVthere is an additional subfractionation step.Basically, these systems are used for transcellularabsorption studies130 as well as active andfacilitated transport mechanisms.131133 The tis-sues may be of human origin but most frequentlyare derived from different animal species such usrabbits, pigs, and rats. BBM contain a variety ofhydrolytic enzymes, which are valuable tools instudying drug stability. The distribution of theseenzymes is well known134136 enabling rationalapproaches to assessing protection of the drug byformulation or synthetic techniques. BBM matrixis especially useful in studying the specificity oftargeted prodrug reconversion at the intestinalwall.137AL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008
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Dialysis works on the principle of the diffusion of
672 RUIZ-GARCIA ET AL.Everted Intestinal Rings
Everted Intestinal rings provide a relativelyquick technique for measuring uptake of druginto tissue.138,139 The viability of the tissue overthe time course of the experiment may becompromised.140
Filter-Immobilized Hexadecane Membrane (HDM)
This technique consists of a hexadecane liquidlayer immobilized between two aqueous compart-ments.141,142 An HTS technique used to studypassive transport pathways.
Parallel Artificial Membrane PermeabilityAssay (PAMPA)
PAMPA is a phospholipid-based parallel artificialmembrane that lacks active transport and alsoparacellular pathways.20,125,142144
Cell Culture-Based Permeability Models
There exists a significant variety of cell linesnow routinely cultivated as a monolayer onpermeable filters. Caco-2 cell monolayers (humanepithelial colon adenocarcinoma cell line) areextensively used because it is an excellent systemfor the study of transcellular transport and alsoactive transport. Dipeptide carrier145,146 andP-glycoprotein147,148 (P-gp) are some of thetransporters expressed in Caco-2 cells. Other celllines are extensively used due to their lowintrinsic expression of ATP-binding cassettetransporters superfamily149151 (ABC) such usMadinDarby Canine Kidney (MDCK) and LLC-PK1 which make them ideal for transfec-tions.72,152154 Another cell line that is beingroutinely used is 2/4/A1 which lacks functionalexpression of several important active drugtransporters and does not present tight junctions,rendering this system ideal for studying para-cellular permeability.142
Immobilized Artificial Membranes (IAM)
The IAM system was developed by Pidgeonet al.155 and described in several papers.156159
In essence, IAMs are a chromatographic modelof lipidic membranes for studying passiveabsorption.
In Situ Intestinal Perfusion
The in situ perfusion technique provides enhanc-ed tissue viability as well as several samplingJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 2, FEBRUARY 2008solutes along a concentration gradient across asemipermeable membrane. Usually, the devicecontains two chambers divided by a semiperme-able membrane with a specific molecular weightcut-off, which means that only molecules with amolecular weight smaller than the cut-off size willpermeate through the membrane. In a typicalexperiment, plasma containing the test article ison one side of the membrane and a buffer is placedin the other dialysis chamber. A variant of ED iscomparative equilibrium dialysis (CED), whereplasma is placed on either side of the dialysismembrane.36 Microdialysis, as a specific type ofdialysis system, allow you to continuously samplethe unbound fraction on the interstitial spacefluid(ISF), which may be the actual targetcompartment for many drugs. Microdialysis isan in vivo probe-based sampling method linked toan analytical device for measurement of drugconcentration profiles. When a physiologicalsalt solution is slowly pumped through themicrodialysis probe, the solution equilibrates withthe ISF which then contains a representativeproportion of the tissue fluids molecule.35,166168sites.160,161 The technique typically involves iso-lation of an intestinal segment or the whole smallintestine, which remains in situ and is perfused(close or open loop) with a solution containing aknown concentration of the test moiety. Samplesof the intestinal perfusate are taken at specifictime measuring the disappearance of the studiedmolecule.126,160163
Isolated Sections of Intestinal Tissue
In this setting, sections of intestinal tissue aremounted in a chamber as the barrier between twocompartments.164 Both the serosal and mucosalsurfaces are bathed with oxygenated buffersolution and the passage of the compound insolution across the tissue is measured by standardanalytical techniques. The integrity of themembrane is monitored by measurement oftransepithelial electrical resistance across thetissue.126,164 Using chambers can be also utilizedhaving as a barrier cultured cell monolayer asoppose to epithelial tissue.165
Protein Binding
Equilibrium Dialysis (ED)DOI 10.1002/jps
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PHARMACOKINETICS IN DRUG DISCOVERY 673Human Recombinant Enzymes are basicallymicrosomes prepared from baculovirus-infectedinsect cells that express specific human CYPsand Uridine Diphosphoglucuronosyl Transferase(UGTs). This system is useful for