Section 73 Analytical Methods in Drug Metabolism Once the radioactive drug has been synthesized,it is used in metabolism studies in pre- 411 clinical species usually first n rats,mice.or guinea pigs.then indo monkey Typically. the rine and feces are collected from the animals,and the major radioactive moundre isolated and their structures determined (see Section 7.3.C.p.413).After demonstration of drug safety in animals following chronic dosing at elevated dosesand satisfactoryrevery of the radioactive dose(in the rine and feces:some of the radioactivity may be CO2,detected in the breath),then the drug can be tested for safety and tolerability in phase Iclinical trials with healthy human subjects.Once the safety is assured.the radioactive drug can be administered to humans during late phase Ior early phase ll clinical trials toobtain the human metabolic profile.In fact,[aceryl-14C]7.7 was used in a phase I human metabolism study The Food and Drug Administration approves a maximum absorbed doseof3remof radioactivity to a specific organ in a healthy adult volunteer for drug metabolism studies These radioactive levels are estimated from a determination of the absorbed dose in animal models,then 10-100 times lower amounts are used in the human studies.On rare occasions. other fluids such as saliva,cerebrospinal fluid,eye fluids.perspiration,or breath may be exam- ined as well as various organs and tissues.Generally the toxicological animal model species used are considered adequate if all of the major metabolites observed in humans are also observed in the animal models,even if more metabolites are observed in the animals.If a human metabolite is not formed in the animal toxicological model,a more relevant toxico- logical animal model has to be identified or additional toxicological studies need to be carried out with the metabolites unique to humans.2 If most of the radioactivity administered is not excreted from the animal,then it is dis- sected to determine the location of the radioactive compounds.3 A newer methodologyto determine tissue distribution of radiolabeled compounds in whole animals without dissection is quantitative whole-body autoradiography.4 From the above discussion it appears that drug metabolism studies are straightforward: however,until relatively recently these studies were difficult,at best,to carry out.The ready commercial availability of radioactively labeled precursors made the synthetic work much less tedious.The advent of high-performance liquid chromatography (HPLC)and the advance- ments in column packing materials permitted the separation of many metabolites very similar in structure.Metabolites that were previously overlooked can now be detected and identi- fied.Structure elucidation by various types of mass spectrometry(MS)(see the next section) and by various techniques of nuclear magnetic resonance spectrometry has been relatively routine.As a result of these advances in instrumentation,more information can be gleaned from drug metabolism studies than ever before,and this can result in the discovery of new leads or in a basis for prodrug design(see Chapter 8).This,also,means that the Food and Drug Administration can demand that many more metabolites be identified and their pharma- cological and toxicological properties be determined prior to drug approval(which is good news for the consumer,but bad news for the drug companies).The final step in the process to prove the identity of a metabolite is to synthesize it and demonstrate that its spectral and pharmacological properties are identical to those of the metabolite. 7.3 Analytical Methods in Drug Metabolism The four principal steps in drug metabolism studies are isolation(extraction),separation(chro- matography),identification(spectrometry),and quantification of the metabolites.Detection
412 Chapter 7 Drug Metabolism systems are sensitive enough to allow the isolation and identification of submicrogram quan tities of metabolites.Often the isolation step can be omitted,and the urine sample or other biological sample injected directly into the HPLC or gas chromatograph for separation. For eleaner results,though,sample preparation is recommended.Most pharmaceuti cal groups now rely most heavily on direct HPLC/electrospray(or atmospheric pressure chemical ionization)mass spectral analysis to identify drug metabolites,as described in Section 7.3.C,p.413. 7.3.A Isolation As discussed in Section 7.1.animals,including humans,usually convert drugs into more polar conjugates forexcretion.Enzymatic hydrolysis(B-glucuronidase and arylsulfatase)of the con- jugates releases the less polar drug metabolites foreasierextraction and structure identification. A clean sample for analysis is preferred,especially with in vivo drug metabolism stud- ies.Extensive older isolation methodologies,such as ion-pair extraction,used to remove hydrophilic ionizable compounds from aqueous solution:salt-solvent pair extraction,7 to separate metabolites into an ethyl acetate-soluble neutral and basic fraction,ethyl acetate- soluble acidic fraction,and a water-soluble fraction;and various ion-exchange resins such as the anion exchange resin DEAE-Sephadex,18 the cation exchange resin Dowex 50,19 and the nonionic resin Amberlite XAD-2,1201 used to separate acidic,basic,and neutral metabo- lites.respectively,from body fluids,have been replaced by high-throughput methodologies. With the advent of HPLC/MS analyses of metabolites described in Section 7.3.C.p.413,often the isolation step can be eliminated using a fast-flow on-line extraction method.I2 Biological samples are injected directly into the liquid chromatography/mass spectrometer(LC/MS).A narrow-bore HPLC column packed with large particle size material extracts small molecule analytes but allows large molecules (such as proteins)to flow to the waste.The adsorbed analytes are then eluted through a column-switching valve onto an analytical column for LC/MS/MS analysis.For many assays simple protein precipitation or liquid extraction is sufficient]Solid-phase extraction]and liquid/liquid extraction2 have been automated to speed up the process.On-line solid-phase extraction25]or direct plasma injection into the HPLC/MSI261 are other high-throughput methods of isolation. 7.3.B Separation The three most important techniques for resolving mixtures of metabolites are HPLC.cap- illary gas chromatography (GC)2 and capillary electrophoresis (CE)HPLC is more versatile than GC because the metabolites can be charged or uncharged,they can be ther- mally unstable,and derivatization is unnecessary.Normal phase columns(silica gel)can be used for uncharged metabolites,and reversed phase columns(silica gel to which C4 to C18 alkyl chains are attached to give a hydrophobic environment)can be used for charged metabolites.For GC separation the metabolites must be volatilized.This often requires prior derivatizatin nore for the metabolites to volatilize at lower temperatures.Carboxylic acids can be converted into the corresponding methyl esters with diazomethane:hydroxyl groups can be trimethylsilylated with bis-trimethylsilylacetamide or trimethylsilylimidazoe in pyridine.Ketone carbonyls can be converted intoO-substituted oximes.With radiolabeled compounds,the radioactivity can be monitored directly from the HPLC column using an in-line radioactivity detector
7.3 Analytical Methods in Drug Metabolism 7.3.C Identification 413 The two principal methods of metabolite structre idetionre masspeetromeryand nuclear magnetic resonance spectrometry.It is preferable to link the separation and identifi- cation steps by running tandem LC-MS,tandem GC-MS,or tandem CEMS.These methods are sufficiently sensitive to identify subnanogram amouns of material.The most popular methodology is tandem LC-electrospray ioniztion mass spectromery by which ametabolie extract (or rine directy)can be injected into the HPLC and each peakrun drecy the mass spectrometer Similarly,andem CE-electrospraypermry has become a very valuable tool for separation of biomolecules and drug metabolites In liquid chromatography/tandem mass spectrometry/mass spectrometry(LC/MS/MS).the HPLC is connected to a mass spectrometer for parent ion data,and this is connected to a sec- ond mass spectrometer for fragmentation of the parent ion.This technique can provide both mass data and fragmentation data for each metabolite rapidly.2)Ultra-fast gradient HPLC. tandem mass spectrometry can produce run times of less than 5 minutes.331 In this way there is less chance for metabolite degradation or loss,and workup procedures for mass spec- trometry sample preparation are eliminated.Mass spectrometric properties are determined using different ionization techniques.Common vacuum ionization sources include electron impact(ED).chemical ionization(CI),matrix-assisted laser desorption/ionization(MALDD). fast atom bombardment(FAB),and secondary-ion mass spectrometry (SIMS).The devel- opment of HPLC coupled to atmospheric pressure ionization sources.namely,electrospray ionization and atmospheric pressure chemical ionization mass spectrometry,have transformed the role of drug metabolism from its former minor role in drug discovery to its current impor- tant role in drug discovery and drug development.These latter LC/MS/MS methods are used not only for drug metabolism studies but also to investigate drug pharmacokinetics (absorp- tion,bioavailability,and clearance).As indicated in Chapter 2.Section 2.2.F,p.51,about three-quarters of drug candidates do not make it to clinical trials because of problems with pharmacokinetics in animals,4 and about 40%of the molecules that fail in clinical trials do so because of pharmacokinetic problems,such as poor oral bioavailability or short plasma half-lives.35]The trend in the pharmaceutical industry now is to initiate pharmacokinetic and metabolism studies as early as possible in the drug discovery process to aid in the selection of compounds that have the most drug-likeness and best chance for survival to avoid late attrition of drug candidates.36]With these HPLC/atmospheric pressure ionization mass spectrometric techniques,assessment of in vivo plasma half-lives and metabolic degradation can be made rapidly on a large number of drug candidates. A brief description of each of these mass spectrometric techniques follows.Electron impact mass spectrometry(EI-MS)involves the bombardment of the vaporized metabolite by high-energy electrons (0-100eV),producing a molecular radical cation (M)having a mass equivalent to the molecular weight of the compound.The electron bombardment causes bond fission and the positively charged fragments produced are detected.The mass spectrum isa plot of the percentage of relative abundance of each ion produced versus the mass-to-charge Chemical ionization mass spectrometry is important when compounds do not give spectra ratio (m/z). containing a molecular ion,generally because the molecular ion decomposes to give fragment ions.With CI-MS a reagent gas such as ammonia,isobutane,or methane is ionized in the mass spectrometer and then ion-molecule reactions such as protonation occur instead of electron- molecule reactions.This soft ionizarion process results in little fragmentation.Fragment ions
414 Chapter 7 Drug Metabollism in this case are almost always formed by loss of neutral molecules.and asa resumuch less structural information can be gleaned relative to EI-MS. A variety of mass spectral techniques for nonvolatile or higher mass compounds,including peptides and proteinsrenowavailaeMALDI isasootionechnique isimporant for analyzing biopolymers.3 It has the ability to produce gas-phaseoh little or no molecular fragmentation.FAB ionization involves the bombardment of a liquid film containing the nonvolatile sample with a beam of energized atoms of xenon or argon. This method also is useful for thermally unstable compounds.SIMS is similar to FABexcept that energetic ions(Xe+and Ar)instead of atoms are used in SIMS.(391 Two important atmospheric pressure ionization techniques arose out of the need for an ionization source that provided even softer ionization(less fragmentation of the molecular ion) and as a convenient interface with a liquid chromatograph.With electrospray ionization(ESI) ions are generated in solution phase,then the carrier solvent is evaporated,and a gas-phase ion is produced.4 In contrast to ESI,ammospheric pressure chemical ionization (APCI)is a gas-phase ionization process in which gas-phase molecules are isolated from the carrier solvent before ionization.4 In general,ESI is more applicable to high molecular weight. more polar compounds because it requires less heat and can produce multiple charged ions, whereas APCI is more useful for less polar molecules.Nonetheless,for most compounds with some acidic or basic characteristics and with relatively low molecular weight,either technique is applicable. In conjunction with HPLC profiling of metabolites,radioactively labeled drugs are useful to pinpoint retention times of metabolites for more focused mass spectrometric characteriza- tion.By incorporating a splitter into the HPLC sample stream that directs part of the effluent to a radioactivity detector and the rest to the mass spectrometer,simultaneous radioactivity and mass spectrometry monitoring can be carried out.421 In addition to tremendous advances in mass spectrometry,newer technologies in 2D and 3D nuclear magnetic resonance(NMR)spectrometry,particularly tandem LC-NMR(which became practical because of advances in solvent suppression techniques)have enhanced this analytical tool for studies in drug metabolism.431 This continuous-flow method is particularly valuable,allowing HandF spectra to be obtained with ony 5ngor less of metaboliteA mass spectrometer can be connected in tandem with the LC-NMR to give LC-NMR-MS spec- trometry,which enables high-quality NMR and mass spectra to be obtained simultaneously from a single HPLC injection of biological fluid.(451 7.3.D Quantification Quantification of drug metabolites is carried out by radioactive labeling,GC.HPLC,and mass spectrometry.The sensitivity and low volumes necessary for mass spectrometry reduce the assay development,sample preparation,and analysis time such that MS is ideally suited for the -well plate format of high-throughput metabolic screens In order for radioactive labeling techniques to be useful the various radiolabeled metabolitesare first separated by chromatography.Each is isolated and the rate of radioactive disintegration is determined by liquid scntllation countin methods.The amount of the metabolite isolaedn bed from the specific radioactivity of the drug(see Section 7.2). GC and HPLCboth require the construction of a calibration curve of known quantitisof reference compound usually of similar structure to that of the metabolite.From the integration
ectin74 Pathways for rug Deactivation and Ellminaion of the intea standard chromatography peak the amount of each metabolite oedbe 415 determined. Selecrednrin(SIM)isahighly selective method for detectond of small quantities of metabolites.SIM uses mass spectrometer as aselective detector of specific components in the effuent from a HPLC or gas chromatograph.Bysettin the spectrometer to detect characteristic fragmentionsat a singevalueothrm with the same retention times that do not produce those fragmentions will goundetected.When a full mass spectrum is recorded repetitively throughout a chromatogram and aseeed monitoring profile is reconstructed by computer,it is sometimes calledmass fragmenography. Subpicogram quantities of metabolites in a mixture can be detected by the SIM method. 07.4 Pathways for Drug Deactivation and Elimination 7.4.A Introduction The first mammalian drug metabolite that was isolated and characterized was hippuric acid (7.8)from benzoic acid in the early 19th century.1471 However,not until the late 1940s. when Mueller and Millerl48)demonstrated that the in vivo metabolism of 4-dimethyl- aminoazobenzene could be studied in vitro (see Section 7.4.B.1.p.418).was the disci- pline of drug metabolism established.As a result of the ready commercial availability of radioisotopes and sophisticated separation,detection,and identification techniques that were developed in the latter half of the 20th century (see Section 7.3).drug metabolism studies have burgeoned. COOH 7.8 The function of drug metabolism is to convert a molecule that can cross biological mem- branes into one that is cleared,generally in the urine:each progressive metabolic step usually reduces the lipophilicity of the compound.The lipophilicity of the drug molecule will deter- mine whether it undergoes direct renal clearance or is metabolically cleared.As the log D (see Chapter 2.Section 2.2.F.2.b,p.55)of the compound increases above zero,a marked decrease in direct renal clearance and a sharp increase in metabolic clearance occur(Fig- ure 7.1 shows the results for a series of chromone-2-carboxylic acid derivatives)indicating the contribution of lipophilicity to drug metabolism.Drug metabolism reactions have been divided into two general categories5 termed phase I and phase II reactionshase formations involve reactions that introduce or unmask a functional group.such as oxygenation or hydrolysis.Phase II transformations mostly generate highly polar derivatives (known as conjugates),such as glucuronides and sulfate esters,for exeretion in the urine. The rate and pathway of drug metabolism are affected by species.strain.sex,age.hor- mones,pregnancy,and liver diseases such as cirrhosis,hepatitis,porphyria.and hepatoma. Drug metabolism can have a variety of profound effects on drugs.It principally causes pharmacological deactivation of a drug by altering its structure so that it no longer inter acts appropriately with the target receptor and becomes more susceptible to excretion.Drug metabolism,however,also can convert a pharmacologically inactive prodrug intoan active