406 Chapter 7 Drug Metabolism 2.Glucuronic Acid Conjugation 456 3.Sulfate Conjugation. 460 4.Amino Acid Conjugation. 462 5.Glutathione Conjugation 464 6.Water Conjugation............... 466 7.Acetyl Conjugation........ 466 8.Fatty Acid and Cholesterol Conjugation. 468 9.Methyl Conjugation 469 D.Hard and Soft Drugs;Antedrugs 471 7.5 General References 473 7.5 Problems..… 475 7.7 References. 479 ☐7.1 Introduction When a foreign organism enters the body,the immune system produces antibodies to interact with and destroy it.Small molecules,however,do not stimulate antibody production.So how has the human body evolved to protect itself against low molecular weight environmental pollutants?The principal mechanism is the use of nonspecific enzymes that transform the foreign compounds(often highly nonpolar molecules)into polar molecules that are excreted by the normal bodily processes.Although this mechanism to rid the body of xenobiotics (molecules foreign to the organism)is highly desirable,especially when one considers all of the foreign materials to which we are exposed every day,it can cause problems when the foreign agent is a drug that we want to enter and be retained in the body sufficiently long to be effective. The enzymatic biotransformations of drugs is known as drug metabolism.Because many drugs have structures similar to those of endogenous compounds,drugs may get metabolized by specific enzymes for the related natural substrates as well as by nonspecific enzymes. The principal site of drug metabolism is the liver,but the kidneys,lungs,and GI tract also are important metabolic sites.When a drug is taken orally (the most common route of administration),it is usually absorbed through the mucous membrane of the small intestine or from the stomach.Once out of the GI tract it is carried by the bloodstream to the liver where it is usually first metabolized.Metabolism by liver enzymes prior to the drug reaching the sys- temic circulation is called the presystemic or first-pass effect.which may result in complete deactivation of the drug.If a large fraction of the drug is metabolized,then larger or multiple doses of the drug will be required to get the desired effect.Another undesirable effect of drug metabolism is that occasionally the metabolites of a drug may be toxic.even though the drug is not. The first-pass effect sometimes can be avoided by changing the route of drug admin- istration.The sublingual route (the drug is placed under the tongue)bypasses the liver. After absorption through the buccal cavity,the drug enters the systemie circulation.This is the route employed with nitroglycerin (7.1.Nitrostat),a drug used for the treatment of angina pectoris that is converted by mitochondrial aldehyde dehydrogenase to nitrite ion. which is then reduced to nitric oxide a second messenger molecule that dilates blood ves- sels in the heart.The recal roue in the form of a solid suppository or in solution as an
Section 7.1 Introduction enema.leads to absorption through the colon mucosa.Ergotamine (7.2.Ergomar).a drug 407 for migraine headaches,is administered this way (who would have guessed) (iv.)injection introduces the drug directly into the systemic circulation and is used when a rapid therapeutic response is desired.The effects are almost immediate when drugs are administered by this route,because the total blood circulation time in humans is 15-20 sec. Intramuscular (i.m.)injection is used when large volumes of drugs need to be administered. if slow absorption is desirable,or if the drug is unstable in the gastric acid of the stomach. A subcutaneous (s.c.)injection delivers the drug through the loose connective tissue of the subcutaneous layer of the skin.Another method of administration.particularly for gaseous or highly volatile drugs such as general anesthetics,is by pulmonary absorption through the respiratory tract.The asthma drug isoproterenol (7.3.Isuprel)is metabolized in the intestines and liver.but administration by aerosol inhalation is effective at getting the drug directly to the bronchi.Topical applicarion of the drug to the skin or a mucous membrane is used for local effects;few drugs readily penetrate the intact skin.Not all drugs can be administered by these alternate routes,so their structures may have to be altered to minimize the first-pass effect or to permit them to be administered by one of these alternate routes.These struc- tural modification approaches in drug design to avoid the first-pass effect are discussed in Chapter 8.Even if the first-pass effect is avoided,there are many enzymes in tissues other than the liver that are capable of catalyzing drug metabolism reactions.Once a drug has reached its site of action and elicited the desired response,it usually is desirable for the drug to be metabolized and eliminated at a reasonable rate.Otherwise,it may remain in the body and produce the effect longer than desired or it could accumulate and become toxie to the cells. CH HO NHI ONO2 ONO2 HO ONO2 HO H nitroglycerin ergotamine isoproterenol 7.1 7.2 7.3 Drug metabolism studies are essential for evaluating the potential safety and efficacy of drugs.Consequently,prior to approval of a drug for human use,an understanding of the metabolie pathways and disposition of the drug in humans and in preclinical animal species is required.The animal species used for metabolism studies are often those in which the toxicological evaluations are conducted.Additional toxicological studies have to be carried out on metabolites found in humans that were not observed in the animal metabolism studies. Metabolism studies also can be a useful lead modification approach.For example,after many years on the drug market,terfenadine(7.4.R=CH3:Seldane)was removed because it was found to cause life-threatening cardiac arrhythmias when coadministered with inhibitors of hepatic cytochrome P450.such as erythromycin and ketoconazole.2 The active metabolite
408 Chapter 7 Drug Metaboliam of terfenadine,fexofenadine (7.4.RCOOH:Allegra),however,produces no arhythmias. and it has replaced terfenadine on the market.31 CH HO OH terfenadine HCI(R=CH3) fexofenadine HCI(R COOH) 7.4 Once the metabolic products are known,it is possible to design a compound that is inactive when administered,but which utilizes the metabolic enzymes to convert it into the active form. These compounds are known as prodrugs,and are discussed in Chapter 8.In this chapter we consider the various reactions that are involved in the biotransformations of drugs.Because only very small quantities of drugs generally are required to elicit the appropriate response, it may be difficult to detect all of the metabolic products.To increase the sensitivity of the detection process,drug candidates are typically radioactively labeled.Radioactive compounds are useful for studying all aspects of absorption,distribution,metabolism,and excretion (ADME).4 Metabolite studies often can be done directly by tandem mass spectrometry/mass spectrometry techniques.51 In the next two sections we look briefly at how radiolabeling is carried out,how metabolites are detected,and how their structures are elucidated. 7，.2 Synthesis of Radioactive Compounds Because of the sensitivity of detection of particles of radioactive decay,a common approach used for detection,quantification,and profiling of metabolites in whole-animal studies is the incorporation of a radioactive label,typically a weak B-emitter such as 14Cl6]or3H.7 into the drug molecule.When this approach is used,it does not matter how few metabolites are produced or how small the quantities of metabolites,even in the presence of a large number of endogenous compounds.Only the radioactively labeled compounds are isolated from the urine and the feces of the animals,and the structures of these metabolites are elucidated(see Section 7.3).If one of the carbon atoms of a drug is metabolized to carbon dioxide.as is the case of erythromycin(7.5.Erythrocin),14C labeling of the carbon atom that becomes CO2(the NMe2 methyl groups of erythromyein are oxidized to CO2),makes it possible to measure the rate of metabolism of the compound by measuring the rate of exhaled To incorporate a radioactive label into a compound,a synthesis must be designed so that a commercially available,radioactively labeled compound or reagent can be used in one of the steps.It is highly preferably to incorporate the radioactive moiety in a step at or near the end of the synthesis because once the radioactivity is introduced.the scale of the reaction is generally diminished and special precautions and procedures regarding radiation safety and disposal of radioactive waste must be followed.Often the radioactive synthesis is quite different fromor longer than the synthesis of the unlabeled compound in order touse availableradioactive material.It is preferable to preparebeed hensrated in the drug.the site ofnrtion must be su that loss of therim by exchange with the medium does notoccurevenfer an early metabolic step
Secon 7.2 Synthesis of Radioactive Compounds Generally,only one radioactive label is incorporated in a drug because drug metabolism 409 typicalyadstoastrucurewthiamiooemoeyobk farenough inasynthesis,however.it is possible to synthesizeadrug with several om radioactively labeled.Radioactive labeling at multipeiofaoceoudethe identification of more fragments of the drug and consequently the elucidation of metabolite structures and the fate of the molecule invivo. Me Me HO OH OH Me Me O.HOZ OMe Me Me Me -OH Me erythromycin 7.5 Industrially,the radioactive drug is synthesized with high specific radioactivity (a measure of the amount of radioactivity per mole of compound),often>57 mCi/mmol of14C(the theo- retical maximum is 64 mCi/mmol).When needed,the specific radioactivity is diluted with non-[14C]-labeled drug for use in metabolism studies.Typically,commercially available radio- active compounds have relatively low specific radioactivities.This means that may be only one in 106 or fewer molecules actually contains the radioactive tag:the remainder of the molecules are unlabeled and are carriers of the relatively few radioactive molecules.In the case of 4C there will be no difference in the reactivity of the labeled and unlabeled molecules. so the statistical amount of radioactivity in the products formed is the same as that in the starting materials.The specific radioactivity of the metabolites formed during metabolism, then,should be identical to the specific radioactivity of the drug.In the case of tritiated drugs. however,if a carbon-hydrogen bond is broken,the radiolabel will be lost as tritiated water, and satisfactory recovery of total radioactivity in animal studies cannot occur.Also,a kineric isorope effect will occur on those molecules that are tritiated.This will lead to metabolite formation with a lower specific radioactivity than that of the drug.As a result,quantitation of the various metabolic pathways,where some involve C-H bond cleavage and others do not,may require knowledge of the tritium isotope effect.This,then,is another reason why it is preferable to use [4C]-labeling of a drug for metabolism studies rather than tritium labeling. If the drug is a natural product or derivative of a natural product,the easiest procedure for incorporation of a radioactive label could be a biosynthetie approach.namely,to grow the organism that produces the natural product in the presence of a radioactive precursor.and let Nature incorporate the radioactivity into the molecule.Because of the volume of media generally involved and,therefore,the large amount of radioactive precursor required,this could be a very expensive approach:however,generally the expense is compensated by the ease of the method and the attractive yield of product obtained. An example ofadrug class that could use this approach is the penicillins.which are biosyn- thesized by Penicillium fungi from valine,cysteine,and various carboxylic acids(Scheme7.1). Valine is commercially available with a c label at the carboxylate carbon or it may be
Chapter 7 Drug Metabolism 410 +HS P所 COOH NH Penicillim +COOH Scheme 7.1Biosynthesis of penicillins (iPr)2EtN H NO 98% Cbz-Cl NaHCO 91% 00 85% 1.MsCI/EN Ac2O 2.NaNs pyridine 3.H2/Pd-C 68% CH 93% linezolid 7.7 Scheme 7.2Chemical synthesis of linezolid obtained uniformly labeled:that is,all of the carbon atoms are labeled to some small extent with 4C(albeit very few molecules would contain all of the carbon atoms labeled in the same molecule).It also can be purchased with a tritium label at the 2-and 3-positions or at the 3-and 4-positions.Cysteine is available uniformly labeled in 14C or with a35s label. Penicillin G could be produced if phenylacetic acid(available with a 4C label at either the 1-or 2-position)were inoculated into the Penicillium growth medium. If the drug is not a natural product (the more common case)a chemical synthesis must be carried out.For example,the synthesis of the first in a new class of antibacterial drugs. (7.Zyvx)sshowninScheme72The last step in the synthesis.acetytion of heprmymne can becedouth Clacetic anhydride toinraedoacive labethe acetyl group of 7.7.The oxoido earbonyl carbon lso coul have been abenCC-Clomke.6 but radioactive Cbz-Clsommly availablesoit would have had to be synthesized fromChosgene and benzyl alcohol
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