The Development of Drug Metabolism Research as Exp

The Development of Drug Metabolism Research as Expressed

in the Publications of ASPET:Part 3,1984–2008

Patrick J.Murphy

College of Pharmacy and Health Sciences,Butler University,Indianapolis,Indiana

Received July 2,2008;accepted July 14,2008

ABSTRACT:

The dramatic changes in drug metabolism research in the last 25years are well documented in the publications of the American Society for Pharmacology and Experimental Therapeutics (ASPET).New analytical tools combined with modern molecular biological techniques have provided unprecedented access to the workings of the cell.A field that concentrated on only a handful of primary

enzymes now has a list of hundreds in its purview.Genetic varia-tion,environmental impact,and molecular diversity have all come under study in attempts to follow the fate of drugs and chemicals.Examples from ASPET journals will be used to illustrate the dra-matic advancements in the field.

The world of drug metabolism underwent a major metamorphosis in the last 25years,evolving from a field devoted to defining molec-ular transformations into a broad-ranging research effort directed to the delineation of all of the chemical,biochemical,and biological factors involved in the disposition of novel chemicals.These included the specific enzymes and receptors that had affinity for the drug,the mechanisms whereby the enzymes acted and the receptors reacted,the genetic makeup that gave rise to individuality of response,and finally the adaptive response elements that alter the components involved in response to new agents.Overlaying interpretations of these actions was the fact that the dynamic of the responding organism was con-tinually being altered by external and internal factors.

The number of articles published in the American Society for Phar-macology and Experimental Therapeutics (ASPET)journals grew from 763in 1984to 1246in 2007.Drug disposition-related articles went from 198to 380in the same period.In 1984,66%of these articles were in Drug Metabolism and Disposition (DMD),whereas,in 2007,80%were found in DMD.The changing face of metabolism and pharmacology was recognized by ASPET when it launched its newest journal,Molecular In(ter)ventions ,in 2001.This richly illustrated journal was aimed at a broad target audience and was meant “to reflect the entire range of pharmacological approaches,from reductionist genetic approaches to an integrated understanding of the impact of molecules on the whole organ-ism”(Duckles,2001).Articles on P450(Guengerich,2003;Frye,2004),molecular genetics (Weinshilboum,2003),and warfarin therapy (Rettie and Tai,2006)reflect the journal’s broad approach to drug metabolism and pharmacology and their applications.

Analytical Technology

In 1984,ASPET elected its first female president,Marjorie Horn-ing.This election was quite fitting for one of the major contributors to drug metabolism and drug analysis.Marjorie and her husband,Evan,were among the early innovators in GC/MS technology in the 1970s.The Hornings published many articles on metabolic intermediates and metabolites using GC/MS technology,including studies on formation of methylthio metabolites of indene (Bartels et al.,1986)and bromo-benzene (Lertratanangkoon and Horning,1987).

Although the use of GC/MS was a powerful analytical tool,the development of HPLC/MS in the 1980s provided the long-sought interface that revolutionized analytical laboratories.The discovery and development of the thermospray interface by Vestal (Vestal,1984)and the electrospray interface by Fenn 1(Fenn et al.,19)led to development and marketing of versatile LC/MS instruments using a variety of “soft ionization”techniques.The combination of the analytical power of MS and then MS/MS with the separation power of liquid chromatography made all molecules,even proteins,susceptible to rapid analyses.Subsequent development of LC/NMR instruments has expanded these analytical capabilities (Mutlib et al.,1995).

Numerous applications of this new technology combined with increasingly sophisticated biological model systems began to appear in the literature.Studies with acetaminophen illustrate these dramatic advances.Early papers focused on the chemical transformation of acetaminophen using the latest analytical methods.In 1984,the for-mation of the iminoquinone proposed by Mitchell in 1973(Mitchell et al.,1973)was confirmed using HPLC combined with electrochemical

1

John Fenn received the Nobel Prize in 2002.His lecture on the development of the electrospray interface is an excellent overview of this remarkable field (http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/fenn-lecture.html).

Article,publication date,and citation information can be found at http://dmd.aspetjournals.org.doi:10.1124/dmd.108.023226.

ABBREVIATIONS:P450,cytochrome P450;HPLC,high-performance liquid chromatography;UPLC,ultra-performance liquid chromatography;GC/MS,gas chromatograph/mass spectrometer;MS/MS,tandem mass spectrometers;LC/NMR,liquid chromatograph/nuclear magnetic resonance;LC/MS,liquid chromatograph/mass spectrometer;CAR,constitutive androstane receptor;PXR,pregnane X receptor.

0090-9556/08/3610-1977–1982$20.00D RUG M ETABOLISM AND D ISPOSITION

Vol.36,No.10Copyright ©2008by The American Society for Pharmacology and Experimental Therapeutics 23226/3386118DMD 36:1977–1982,2008

Printed in U.S.A.

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analysis (Dahlin et al.,1984).In the same year,the formation of catechol metabolites was confirmed in the mouse (Forte et al.,1984).The enzymes involved in acetaminophen metabolism,both oxidative (Patten et al.,1993;Thummel et al.,1993;Chen et al.,1998;Dong et al.,2000)and conjugative (Court et al.,2001;Gamage et al.,2006),were elaborated.Binding of the reactive metabolite(s)of acetamino-phen to protein was demonstrated in vitro and in vivo using both chemical (Muldrew et al.,2002;Damsten et al.,2007)and immu-nologic methodology (Roberts et al.,1987;James et al.,2003).A transgenic humanized mouse model was used to show the participa-tion of CYP2E1in activation of acetaminophen (Cheung et al.,2005).Wolf and coworkers used knockout mice to show the participation of CYP3A and CYP2E1in hepatotoxicity caused by acetaminophen.The combination of knockout mice and advanced analytical techniques has shed further light on minor metabolites that may play a role in the toxicity profile of acetaminophen (Wolf et al.,2007).Three novel metabolites were identified when the metabolism of isotopically la-beled acetaminophen was compared in wild-type and CYP2E1-null mice using UPLC coupled to a time-of-flight mass spectrometer (Chen et al.,2008).Since the initial confirmation of the iminoquinone intermediate in 1984,there have been over 200articles published in society journals on acetaminophen metabolism and/or the toxicity caused by its metabolites.The advances in analytical methodology and biotechnology have been used to probe the actions of acetamin-ophen and its metabolites at the molecular level.

Reactive Intermediates

The area of reactive intermediates and active metabolites has rap-idly expanded with the help of the new technology.Of over 500references to metabolic activation published in the ASPET journals,95%of them have occurred in the last 25years.Examples of studies on drug activation include spironolactone (Sherry et al.,1986),di-sulfiram (Hu et al.,1997),diclofenac (Poon et al.,2001),losartan (Yasar et al.,2001),and troglitazone (He et al.,2004).Gram reviewed the role of reactive intermediates in pulmonary toxicity (Gram,1997),and Fred Guengerich gave an overview of bioactivation and detoxi-cation in his 1992Brodie Award lecture (Guengerich,1993).The role of glutathione-dependent activation of haloalkanes has been reviewed by Anders (2004).2Monitoring for reactive intermediates (Evans et al.,2004)or markers of cellular response to those intermediates (Takakusa et al.,2008)has become a focus of early drug development.The idea that drug metabolism is the process of “detoxication”must yield to the reality that many compounds are made active,more active,and/or toxic during biotransformation.

The P450s

The 1980s initiated a period of rapid expansion of our knowledge of P450s.Individual forms were isolated and characterized initially from animal livers and,subsequently,human liver samples.The first crystal structure of a P450came from the studies on the soluble P450found in Pseudomonas putida 3(Poulos and Howard,1986).The list of P450s was growing rapidly,and a need for a common nomenclature was widely acknowledged.Dan Nebert 4and collaborators proposed a

system related to the deduced amino acid sequence of the protein (Nebert et al.,1987).The P450s were assigned families,subfamilies,and a number relating to the order of discovery.Using detergent solubilization of the microsomes,individual P450s were isolated and identified.The major human P450s involved in drug metabolism were among the first of the multitude of P450s to be characterized.Human CYP1A1(Shimada et al.,1992),1A2(Distlerath et al.,1985),2A6(Yun et al.,1991),2B6(Mimura et al.,1993),2C9(Shimada et al.,1986),2C19(Wrighton et al.,1993),2D6(Distlerath et al.,1985;Birgersson et al.,1986;Gut et al.,1986),2E1(Wrighton et al.,1987),3A4(Watkins et al.,1985;Guengerich et al.,1986),3A5(Wrighton et al.,19),and 3A7(Wrighton and VandenBranden,19)were all characterized by initial isolation.The development of recombinant DNA technology allowed the cloning of human and animal p450s.The first successful cDNA-directed expression of a P450was per-formed using yeast (Oeda et al.,1985).Heterologous expression of a variety of human and animal p450s in mammalian,yeast,baculovirus,and bacterial systems followed.The status of the molecular biological approach to P450was summarized by Frank Gonzalez 5in his 1988review (Gonzalez,1988).Information on new and older drugs became incomplete if it did not include the determination of whether and which P450s were involved in the disposition of the molecule (Bjorns-son et al.,2003).The need to characterize the P450(s)involved in oxidizing a given drug has resulted in increasingly sophisticated methods for this evaluation (Lu et al.,2003).

Individual variation in P450s is a hallmark of the genetic influence on drug metabolism.In a classic paper on variation of P450s,Shimada and coworkers determined the wide range of expression of P450s in microsomes from 60human livers (Shimada et al.,1994).As the studies on P450variations between individuals expanded,it became clear that genetic variation was a significant factor in racial,environ-mental,and population variability.Many of the allelic variants are a result of single nucleotide polymorphisms.The field of drug metab-olism became prominent in defining the area of pharmacogenomics.Altered nucleotides leading to single amino acid changes can result in proteins with modified catalytic activity.Alteration of nucleotide sequence can also cause partial deletions,frameshifts,and/or aberrant production of RNA.CYP2D6alone has over 70allelic variants.A nomenclature system has been established and is managed online by the Human Cytochrome P450(CYP )Allele Nomenclature Committee (http://www.cypalleles.ki.se).A major source of P450information is found on David Nelson’s Cytochrome P450Homepage (http://drnelson.utmem.edu/CytochromeP450.html).

The impact of enzyme variation due to genetics was exemplified in the P450family with the discovery of a segment of the population deficient in the ability to metabolize debrisoquine (Mahgoub et al.,1977)or sparteine (Eichelbaum et al.,1978).Deficiencies,or in some cases overexpression,of individual P450s can have a variety of effects depending on whether a drug is being inactivated,activated,or made toxic via the enzyme in question.The status of pharmacogenetics in drug metabolism and potential needs in clinical practice are summa-rized in the review of Gardiner and Begg (2006).

Molecular Biology

The biochemistry of drug metabolism advanced on many fronts.With recombinant technology in hand,it became possible to deter-2

Drag Anders was awarded the 1999Brodie Award in recognition of his contributions to the field over a 35-year period starting with studies on SKF 525-A inhibition of drug metabolism (Anders and Mannering,1966).3

Tom Poulos was the recipient of the 2004Brodie Award.His Brodie lecture provides an excellent history of the crystal structure of P450and a detailed comparison to nitric oxide synthase (Poulos,2005).4

Dan Nebert received the Brodie Award in 1986for his many contributions to the field.His award lecture details how an initial finding of a difference in induc-ibility between mouse strains led to the discovery of the Ah receptor and shaped his future career (Nebert,1988).5

Frank Gonzalez,in accepting the 2006Brodie Award,discussed the progress in our understanding of CYP2E1and particularly the value of the 2E1knockout mouse in elaborating the physiological functions of the enzyme (Gonzalez,2007).

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mine structures of P450s before function was understood.The number of P450s identified in nature grew to the thousands.The second crystal structure of a P450was that of BM3,a unique enzyme that contained the reductase as well as the P450heme in a single protein (Ravichandran et al.,1993).The ability to genetically engineer new proteins with altered amino acid sequence allowed the exploration of the substrate binding sites and other structural features of a given protein.Comparison of soluble P450s to membrane-bound structures pointed to the amino terminal portion of the mammalian P450s as the main membrane-binding sequence.Clipping this sequence off the protein maintained catalytic activity but led to soluble proteins that could be crystallized.The first mammalian P450crystal structure,CYP2C5,was published in 2000(Williams et al.,2000).6The crystal structures of all of the main human drug metabolizing P450s were soon to be determined (Rowland et al.,2006).Even this torrid pace of discovery was to be eclipsed with the elucidation of the human genome sequence.Analysis of the genome suddenly revealed that there were 57genes coding for human P450s.The physiological role of many of these P450s remains to be determined.The structures of new allelic variants present a continuing parade of minor and major variants that speak to human diversity at the molecular level.

It has become apparent that,to understand an individual response to a drug or multiple drugs,we must understand the makeup of the individual at an increasingly intimate level.New man-made biological tools have been developed to help decipher the milieu.Cloned en-zymes expressed in stable systems allow the direct analysis of indi-vidual enzymes on a given substrate.The use of knockout animals permits an assessment of the potential critical roles of the eliminated enzyme(s)in the physiological function of that animal.Replacement of animal enzymes with their human counterpart allows a new level of animal modeling of human metabolism.The “humanized”animals may allow new systems of drug discovery and development.

Induction

In 1984the field was just beginning to understand that the process of enzyme induction involved a novel group of receptors character-ized by the newly discovered Ah receptor (Poland et al.,1976).It was the inductive response to the administration of aromatic hydrocarbons that had led to the discovery of P448and the concept of multiple P450s.The finding that aryl hydrocarbon hydroxylase induction was mediated through a separate protein with its own binding character-istics set the stage for the discovery of other receptors involved in induction.An overview of the role of the Ah receptor in endogenous functions provides additional insight regarding this important regula-tor (McMillan and Bradfield,2007).Handschin and Meyer (2003)have reviewed the development of nuclear receptor research focusing on the CAR-and PXR-mediated induction of P450s and other me-tabolizing enzymes.The crystal structure of the human PXR ligand binding domain in the presence and absence of ligands has been reported (Watkins et al.,2001;Xue et al.,2007).

Three human peroxisome proliferator-activated receptors have major roles in endogenous lipid metabolism and energy homeosta-sis (Michalik et al.,2006).An overview of the 48nuclear hormone receptor family stresses the unique characteristics and interdepen-dencies of these important human gene regulators (Moore et al.,2006).The complexity of the inductive process and its effects on endogenous as well as exogenous substrates is a focus of ongoing research (Moreau et al.,2008).

Drug-Drug Interactions

As it became clear that metabolism of a given drug may only in-volve one or a few of the P450s or forms of other classes of enzymes,the interpretation of drug interactions became more specific and predictable.A few unique situations increased general awareness of this phenomenon.In the late 1970s,the popular H2antagonist cime-tidine was shown to be a modest P450inhibitor,and this was the explanation for some of the observed drug interactions (Reimann et al.,1981;Rendic et al.,1983).The marketing of ranitidine,a new H2antagonist,emphasized that this compound was not a significant P450inhibitor.Suddenly,the language of our arcane field became the fodder of marketing specialists.

A second instance occurred when Bailey and coworkers unraveled the fact that their grapefruit juice “vehicle”for a study on alcohol interaction was actually causing potent inhibition of the metabolism of the study drug,felodipine (Bailey et al.,19).Inhibition of metab-olism came to the general public in the form of grapefruit juice prohibitions on the package inserts.The cause of this inhibition was found to be mainly the presence of bergamottin in the grapefruit juice (Schmiedlin-Ren et al.,1997).Bergamottin was one of a number of suicide substrates that,in the process of metabolism,were converted to protein-binding inactivators.

A third example of drug interaction via enzyme inhibition came with the histamine antagonist terfenadine.This popular antihistamine drug was found to cause a rare heart problem when it was given to patients who were taking erythromycin or ketoconazole at the same time (Monahan et al.,1990).The interaction was traced to the fact that the CYP3A4metabolism of terfenadine was inhibited by the macro-lides,and toxic levels of parent terfenadine could result (Jurima-Romet et al.,1994).This eventually led to Food and Drug Admin-istration withdrawal of terfenadine from the market and to new guidelines regarding the disposition studies necessary for approval of new chemical entities (http://www.fda.gov/cder/guidance/clin3.pdf).

Non-P450Oxidases

Although P450has held center stage in the development of the drug metabolism story,there are many other enzymes and enzyme systems that may be equally or more important for the fate of a given com-pound.The flavin monooxygenases,important for S-oxidation of compounds such as cimetidine (Cashman et al.,1993)or N-oxidation of heterocyclic amines like tamoxifen (Parte and Kupfer,2005)and clozapine (Tugnait et al.,1997),exhibit genes for five forms in humans (Krueger and Williams,2005).The oxidative step in the activation of the prodrug famciclovir to penciclovir is catalyzed by aldehyde oxidase (Clarke et al.,1995;Rashidi et al.,1997),a molyb-denum containing enzymes capable of oxidizing nitrogen heterocycles and aldehydes (Garattini et al.,2003).

There are 17human genes encoding the aldehyde dehydrogenases that are important for the metabolism of both endogenous aldehydes and aldehydes generated in the course of metabolism of administered drugs (Vasiliou et al.,2004;Marchitti et al.,2007).There are 23distinct human forms of alcohol dehydrogenase (Agarwal and Goedde,1992).The reductase required for the activity of P450,cytochrome P450reductase,was crystallized in 1995and the x-ray structure determined (Djordjevic et al.,1995).7Thus,much work has demonstrated the enzymes,in addition to the P450s,need to be carefully evaluated during drug development.

6

Eric Johnson,in his acceptance of the 2002Brodie Award,detailed the wealth of information gained from the crystal structure of CYP2C5(Johnson,2003).

7

Bettie Sue Siler Masters,who was involved in many of the pioneering studies on this reductase,received the 2000Brodie Award.A retrospective of her studies of P450reductase and nitric oxide synthase has been published (Masters,2005).

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Phase II Enzymes

The multiplicity of enzymes involved in phase I metabolism is also reflected in phase II disposition.The number and structures of most of the human conjugating enzymes have now been elucidated.The sulfotransferases are found in two families with a total of 13enzymes (Allali-Hassani et al.,2007).Similarly,glucuronosyltransferases are found in two families with a total of 16forms (Tukey and Strass-burg,2000).There are multiple N-,O-,and S-methyl transferases,2N-acetyl transferases (Wu et al.,2007),and 24glutathione trans-ferases (Hayes et al.,2005).With the multiplicity of enzymes,unique compound specificities,and potential for inhibition and induction,the development of any new drug candidate demands a detailed overview of these phase II enzymes.For example,the major form(s)responsible for conjugation of gemfibrozil (2B7)(Mano et al.,2007),tamoxifen active metabolites (1A10,2B7,1A8)(Falany et al.,2006;Sun et al.,2007),and troglitazone (liver 1A1,intestine 1A8,1A10)(Watanabe et al.,2002)illustrate selectivity within the glucuronosyltransferase family.Tissue-and gender-specific expression of glucuronosyltrans-ferases has recently been explored in mice (Buckley and Klaassen,2007).8High affinity substrates for specific sulfotransferases include raloxifene (SULT2A1)and 4-hydroxytamoxifen (SULT1E1)(Falany et al.,2006;Sun et al.,2007),ethinylestradiol (SULT1E1)(Schrag et al.,2004),and troglitazone (SULT1A3)(Honma et al.,2002).

Transporters

The role of transporters in drug disposition has blossomed since the discovery of P-glycoprotein in 1976(Juliano and Ling,1976).Early efforts were aimed at understanding why patients became resistant to cancer chemotherapy after continued dosing.P-glycoprotein was found to actively pump anticancer drugs out of the cell.Subsequently,this protein was found to play a major role in all tissues,especially the intestine,liver,and brain.For example,fexofenadine,a drug that is essentially unmetabolized,is a substrate for P-glycoprotein,and this protein,along with another transporter,OATP,controls the rate of absorption and elimination of the compound (Cvetkovic et al.,1999).9P-glycoprotein was found to control the penetration of many drugs into the brain (Schinkel et al.,1994).Umbenhauer and coworkers showed that a genetic deficiency in P-glycoprotein was correlated with the penetration of avermectin into the brain in mice (Umben-hauer et al.,1997;Kwei et al.,1999).

The ATP-binding cassette transporter family,which contains P-glycoprotein,consists of 47members.The analysis of the human genome revealed a total of 770transporters.The latest information and structural details on transporters can be found in the transporter protein analysis database (Ren et al.,2007)

Conclusion

The progress of drug metabolism research in the last 25years has been truly astounding.Any attempt to cover all of the important findings in contemporary research is bound to fall short.Many of the critical findings will only become appreciated with the passage of time.When Axelrod presented his findings on microsomal metabo-lism of drugs at a Federation of Societies for Experimental Biology meeting in Atlantic City,New Jersey,in 1954,only a handful of

people were in the audience on that Friday afternoon (Axelrod,1954).Who would have realized that the paper was one of the seminal discoveries in our field?It is a certainty that many papers of the same quality lie unmentioned in this historical summary.Only the future can reveal the riches of the past.What seems sure is that ASPET will continue to provide the publishing and presentation venues necessary for the rapid communication of sound scientific studies on the fore-front of science.With a rich 100year history at its back,the society is poised for the challenges of the future.

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Curt Klaassen was awarded the 2008Brodie Award in honor of his many outstanding contributions to our understanding of the role of metabolism in toxicity.9

Grant Wilkinson,one of the authors of the fexofenadine study,was a pioneer in pharmacokinetics who passed away in 2006.Grant was an active member of ASPET,and his many contributions to the field are memorialized in an article in Clin Pharmacol Ther (Wood,2006).

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Address correspondence to:Dr.Patrick J.Murphy,35Brumley Mews,Carmel,Indiana 46033.E-mail :pjmurphy12@aol.com

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