Pharmacologically Active Drug Metabolites: Impact on Drug … · 2017-11-06 · animals (Pluss et al., 1972) but the effect in humans would be due entirely to the generation of minoxidil

1521-0081/65/2/578–640$25.00 http://dx.doi.org/10.1124/pr.111.005439PHARMACOLOGICAL REVIEWS Pharmacol Rev 65:578–640, April 2013Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: TIMOTHY A. ESBENSHADE

Pharmacologically Active Drug Metabolites: Impacton Drug Discovery and Pharmacotherapy

R. Scott Obach

Pfizer Inc., Groton, Connecticut

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580II. Absorption, Distribution, Metabolism, and Excretion Aspects of Active Metabolites . . . . . . . . . . 585

A. Metabolic Reactions That Can Yield Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585B. Pharmacokinetic Aspects of Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588C. Distributional Aspects of Active Metabolites in Relation to Contribution to

Pharmacological Effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589III. Active Metabolites in Drug Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590

A. Research Tools in Metabolite Identification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591B. Identification of Active Metabolites in Preclinical Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591C. Identification of Active Metabolites in Clinical Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592D. Data Needed for Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

IV. Drugs with Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595A. Drugs with Active Metabolites That Dominate the Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596

1. Albendazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5962. Allopurinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5963. Artesunate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5974. Astemizole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5975. Buspirone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5986. Chlordiazepoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5987. Clomiphene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5988. Codeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5989. Dihydroergotamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59810. Diphenoxylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59911. Dolasetron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59912. Ebastine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59913. Flutamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59914. Hydroxyzine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60015. Irinotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60016. Loratadine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60117. Losartan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60118. Oxcarbazepine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60119. Oxybutynin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60220. Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60221. Propoxyphene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60322. Remacemide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60323. Risperidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60324. Roflumilast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60425. Sibutramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60426. Tamoxifen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60427. Terfenadine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605

Address correspondence to: Dr. R. Scott Obach, Pfizer Inc., Eastern Point Rd., Groton, CT 06340. E-mail: [email protected]/10.1124/pr.111.005439.

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28. Thiocolchicoside. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60529. Thioridazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60630. Tibolone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60631. Tolterodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60632. Tramadol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60733. Trimethadione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60734. Venlafaxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

B. Drugs with Active Metabolites That Contribute Comparably to the Parent . . . . . . . . . . . . . . . . 6081. Acebutolol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6082. Acetohexamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6093. Amiodarone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6104. Aripiprazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6105. Carbamazepine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6106. Clarithromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6107. Clobazam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6118. Disopyramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6119. Fluoxetine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61110. Itraconazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61111. Ketamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61112. Levosimendan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61213. Metoprolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61314. Metronidazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61415. Morphine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61416. Pioglitazone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61617. Praziquantel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61618. Quazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61619. Quinidine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61620. Saxagliptan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61621. Spironolactone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61722. Triamterene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61723. Verapamil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61724. Zolmitriptan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617

C. Drugs with Metabolites That Possess Target Potency But Contribute Littleto In Vivo Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6171. Alprazolam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6182. Buprenorphine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6183. Carisoprodol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6184. Chloroquine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6185. Citalopram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6196. Cyclosporine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6197. Dabigatran. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6198. Dasatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6199. Diazepam, Temazepam, and Oxazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61910. Diltiazem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62011. Donepezil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62012. Granisetron. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62013. Halofantrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62014. Imatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62015. Lidocaine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62116. Lumefantrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62117. Macitentan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621

ABBREVIATIONS: CNS, central nervous system; COPD, chronic obstructive pulmonary disease; CSF, cerebrospinal fluid; EM, extensivemetabolizer; fu, fraction unbound in plasma; HMGCoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HPLC-MS, high pressure liquidchromatography-mass spectrometry; 5-HT, serotonin; IC50, ligand concentration yielding 50% of the maximum response; Kp,uu, ratio ofunbound drug concentration within a tissue to unbound plasma concentration; mCPP, m-chlorophenyl piperazine; MEGX, monoethylglycinexylidide; MS, mass spectrometry; NaV, voltage-gated sodium channel; PDE, phosphodiesterase; PK/PD, pharmacokinetic-pharmacodynamic;PM, poor metabolizer; 1-PP, 1-(2-pyrimidyl)piperazine; PSA, polar surface area; SAR, structure-activity relationship.

Pharmacologically Active Metabolites 579

18. Mexiletine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62219. Mianserin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62220. Midazolam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62221. Mirtazapine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62322. Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62323. Propafenone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62324. Propranolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62425. Rifampin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62426. Rizatriptan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62427. Rosuvastatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62428. Sertraline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62429. Triazolam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62430. Valproic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62531. Zopiclone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625

D. Drugs with Metabolites Possessing Activity at Related Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . 6261. Amitriptyline and Nortriptyline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6262. Clomipramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6263. Clozapine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6274. Doxepin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6285. Imipramine and Desipramine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6286. Loxapine and Amoxapine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6297. Nefazodone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631

E. Drugs That Generate Active Metabolites but Assessment of In VivoContribution Is Ambiguous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6311. Atorvastatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6312. Bromhexine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6323. Bupropion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6324. Etretinate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6335. Ivabradine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6336. Mycophenolic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

Abstract——Metabolism represents the most preva-lent mechanism for drug clearance. Many drugs areconverted to metabolites that can retain the intrinsicaffinity of the parent drug for the pharmacologicaltarget. Drug metabolism redox reactions such asheteroatom dealkylations, hydroxylations, heteroatomoxygenations, reductions, and dehydrogenations canyield active metabolites, and in rare cases evenconjugation reactions can yield an active metabolite.To understand the contribution of an active metaboliteto efficacy relative to the contribution of the parent

drug, the target affinity, functional activity, plasmaprotein binding, membrane permeability, and phar-macokinetics of the active metabolite and parent drugmust be known. Underlying pharmacokinetic princi-ples and clearance concepts are used to describe thedispositional behavior of metabolites in vivo. Amethod to rapidly identify active metabolites in drugresearch is described. Finally, over 100 examples ofdrugs with active metabolites are discussed withregard to the importance of the metabolite(s) inefficacy and safety.

I. Introduction

Drugs are cleared from the human body by threemain mechanisms: renal excretion of unchanged drug,biliary excretion of unchanged drug followed byegestion in feces, and metabolism (there are alsoother rarer mechanisms of clearance). In the case ofmetabolic clearance, a drug is converted to newchemicals, i.e., metabolites. In the vast majority ofcases, metabolites have undergone enough chemicalchange from the parent drug that their capabilities to

bind to the target macromolecule are greatly di-minished or abolished altogether. However, in somecases a metabolite can retain enough intrinsic activityat the target receptor such that it can contribute tothe in vivo pharmacological effect(s) to a meaningfulextent. (The term “intrinsic” is used throughout thispaper to define the binding to the target in theabsence of any of the other factors that occur in a morecomplex system such as a whole organism, tissue, orin vitro cellular assay.) Since active metabolitescontribute to effect, they must be understood to the

580 Obach

same extent as the parent drug with regard to theirdispositional properties (pharmacokinetics, distribution,and clearance mechanisms). A metabolite that pos-sesses intrinsic activity at the target receptor in an invitro assay may or may not contribute to activity in vivo.The exposure of the target receptor to the metabolite,which is dictated by several factors, such as the rate andextent of its generation, rate of its subsequent clearance,target tissue penetration, and free fraction, must beunderstood for the metabolite as much as it is for theparent drug to ascertain whether the metabolite isimportant. This is critical in defining the proper doseneeded for efficacy and the causes of interindividualvariability in the dose-concentration-effect relationship.This is distinct from the case of prodrugs. In the

case of prodrugs, the parent drug is not pharmacolog-ically active itself, but it is converted into a drug,which essentially is an “active metabolite” whenadministered in this fashion. The metabolite pos-sesses all the pharmacological activity, the parentnone. Prodrugs are usually designed to addresssome dispositional flaw of an intrinsically activecompound (e.g., poor absorption, short half-life). Somedrugs that have active metabolites could be considered“accidental prodrugs.” Several older drugs that were

discovered through the use of animal models, and notthrough the use of biochemical screens, showed activityby virtue of a biochemically inert dosed compoundbeing converted into an active metabolite that exhibitedall the effect. Minoxidil could be considered an exampleof this, wherein its hypotensive effect was described in

Fig. 1. Schematic illustrating drug and metabolite binding to a target receptor. In case (A), the drug, represented as possessing a hydrophobic region,a hydrogen bonding region, and a solvent-exposed region, is shown binding to the receptor with points of interaction between atoms on each. (HA andHD refer to hydrogen bond acceptor and donors, respectively.) In case (B), the metabolic modification, represented by the triangle, does not affect theseinteractions, so the metabolite would be active. In case (C) the metabolic modification occurs on a position not involved in binding, so the metabolitewould be active. In (D), the metabolic modification is on a position that disrupts interaction, so the metabolite would be inactive. Such disruptions caninclude introduction of a polar or ionic substituent, or a substituent that provides steric bulk.

Fig. 2. Example illustrating formation rate–limiting and eliminationrate–limiting kinetics for metabolites. In this example, plasma concen-trations of the parent drug and two metabolites are measured followingintravenous administration of parent drug. Metabolite 1 demonstratesformation rate–limiting kinetics since its t1/2 is the same as the parentdrug. Metabolite 2 demonstrates elimination rate–limiting kineticsbecause its t1/2 is longer than that of the parent drug.


animals (Pluss et al., 1972) but the effect in humanswould be due entirely to the generation of minoxidilsulfate, a potent ligand of the ATP-dependent potassiumchannel (Buhl, et al., 1990; Messenger and Rundegren,2004). A discussion of prodrugs and their active metab-olites is beyond the scope of this article.

Over the past decade a great deal of attention hasbeen focused on the assessment of the safety of drugmetabolites (the so-called “MIST” or Metabolites inSafety Testing issue; Baillie et al., 2002). In this issue,the focus was on whether laboratory animals used insafety assessments of new drug candidates are

TABLE 1Comparison of plasma protein binding values for metabolites and their corresponding parent drugs

Parent Drug Metabolite fu(Parent Drug)

fu(Metabolite)

Acebutolol Diacetolol 0.88 0.92Albendazole Albendazole sulfoxide 0.10 0.35Amitriptyline Nortriptyline 0.05 0.08Amitriptyline E-10-Hydroxynortriptyline 0.05 0.31Artesunate Dihydroartimisinin 0.25 0.18Carbamazepine Carbamazepine-10,11-epoxide 0.14 0.33Carisoprodol Meprobamate 0.42 0.76Chlordiazepoxide Demoxepam 0.07 0.22Chloroquine Desethylchloroquine 0.42 0.60Citalopram Desmethylcitalopram 0.20 0.20Citalopram Didesmethylcitalopram 0.20 0.20Clobazam N-Desemethylclobazam 0.15 0.30Clozapine Norclozapine 0.06 0.10Codeine Morphine 0.93 0.74Dasatinib N-Dealkyldasatinib 0.04 0.07Desipramine 2-Hydroxydesipramine 0.10 0.20Desloratadine 3-Hydroxydesloratadine 0.15 0.13Diazepam Desmethyldiazepam 0.023 0.03Diazepam Temazepam 0.023 0.038Diazepam Oxazepam 0.023 0.04Diltiazem Desacetyldiltiazem 0.25 0.23Diltiazem N-Desmethyldiltiazem 0.25 0.32Disopyramide N-Desisopropyldisopyramide 0.35–0.95 0.90–0.95Doxepin Desmethyldoxepin 0.20 0.21Flutamide 2-Hydroxyflutamide 0.05 0.07Hydroxyzine Cetirizine 0.07 0.07Imatinib N-Desmethylimatinib 0.05 0.05Imipramine Desipramine 0.03 0.11Imipramine 2-Hydroxydesipramine 0.03 0.20Ketamine Norketamine 0.40 0.50Levosimendan Or-1896 0.02 0.60Lidocaine Monoethyl glycine xylidide (MEGX) 0.45 0.86Lidocaine Glycine xylidide (GX) 0.45 0.95Loratadine Desloratadine 0.03 0.15Losartan Exp3174 0.013 0.002Loxapine Amoxapine NA 0.10Meperidine Normeperidine 0.56 0.66Midazolam 19-Hydroxymidazolam 0.02 0.11Midazolam 19-Hydroxymidazolam glucuronide 0.02 0.57Morphine Morphine-6-glucuronide 0.74 0.89Nortriptyline 10-Hydroxynortriptyline 0.08 0.31Oxcarbazepine 10-Hydroxycarbamazepine 0.41 0.61Oxybutynin Desethyloxybutynin 0.0034 0.0018Pioglitazone Ketopioglitazone 0.016 0.02Pioglitazone Hydroxypioglitazone 0.016 0.02Primidone Phenobarbital 0.70 0.72Procainamide N-Acetylprocainamide 0.84 0.90Propafenone 5-Hydroxypropafenone 0.08 0.12Propoxyphene Norpropoxyphene 0.24 0.26Quinidine 3-Hydroxyquinidine 0.13 0.40Risperidone Paliperidone 0.10 0.23Roflumilast Roflumilast N-oxide 0.01 0.03Sibutramine Desmethylsibutramine 0.03 0.06Sibutramine Didesmethylsibutramine 0.03 0.06Spironolactone Canrenone 0.06 0.02Terfenadine Fexofenadine 0.05 0.35Thioridazine Sulforidazine 0.0016 0.017Thioridazine Mesoridazine 0.0016 0.017–0.09Tolterodine Desfesoterodine 0.037 0.36Triamterene 4-Hydroxytriamterene sulfate 0.39 0.10Trimethadione Dimethadione 0.91 0.94Valproic acid 2-Propyl-2-Pentenoic acid 0.10 0.007Venlafaxine Desvenlafaxine 0.73 0.70Verapamil Norverapamil 0.10 0.13

582 Obach

adequately exposed to metabolites to which humansare exposed, irrespective of known target receptoractivity. Following this, regulatory guidance wasissued stating that metabolites of potential interestneed to be present in humans at a predefined thresholdeither relative to the total drug-related material orrelative to the parent drug [Food and Drug Administra-tion, 2012 (http://www.fda.gov/downloads/Drugs/Gui-danceComplianceRegulatoryInformation/Guidan-ces/UCM079266.pdf); International Conference onHarmonization, 2012 (http://www.ich.org/products/guide-lines/multidisciplinary/article/multidisciplinary-guide-lines.html)]. If such a threshold is exceeded, then itmust be demonstrated that the species used in toxicol-ogy tests were also exposed to the same metabolites toan extent greater than humans. The focus of theguidance is on stable human metabolites present incirculation, presumably because all organs would beexposed to such metabolites via circulation. (This is inspite of the fact that many metabolites that areresponsible for toxicity are chemically reactive and arenot necessarily stable enough to be present in circula-tion.) Off-target pharmacological activity that yieldssafety concerns is also beyond the scope of this article,with the exception of those metabolites that can con-tribute to safety issues arising from exacerbated targetpharmacology. For example, while the right level ofactivity at opioid receptors yields the beneficial effect ofpain reduction, excessive activity can cause respiratorydepression. Thus, if there is a metabolite active at thisreceptor, it must be considered when relating theexposure to the safety effect.

In this review, pharmacologically active metaboliteswill be discussed with regard to drug research anddevelopment. Some pharmacokinetic concepts will bereviewed as they relate to the special case of metab-olites (Houston, 1981; Pang, 1985). The types ofbiotransformation reactions that can give rise topharmacologically active metabolites will be describedin the context of the impact that various chemicalmodifications can have on receptor/enzyme binding.Experimental approaches that can be applied atdifferent stages of new drug research to detect activemetabolites and understand their impact will bediscussed. Finally, these principles will be illustratedusing examples, along with a description of drugs withwell-established active metabolites in humans. Thedrugs/metabolites described were identified in a Sci-Finder search on the term “active metabolite,” andfollowing exclusion of prodrugs and drugs of abuse, theliterature for the remaining set was scrutinized for

Fig. 3. Method for finding active metabolites. In this scheme, anevaluation of the potential for active metabolites is made. An in vitrometabolism sample was generated and an extract prepared. The extractis injected onto HPLC-UV-MS and spectral data are collected. The eluentis split to a fraction collector. The solvent in the fractions is evaporated,and reconstituted in the buffer used in the pharmacological target assay.The samples are analyzed for target binding. The peaks in the UV andMS chromatograms representing metabolites are denoted with arrowswith the letter M, and the parent drug with the letter P. The UV dataoffers a crude estimate of relative abundances of the metabolites andparent drug. The MS data are used for proposal of the structures of themetabolites. A peak of target binding activity is detected in the fractionswhere the parent drug elutes (;35 minutes), which is expected and servesas an internal control that the target activity assay is functioningproperly. A second peak of target activity is also observed at;27 minutes,indicating the presence of an active metabolite. A second metabolite with

some activity is observed at ;25 minutes. Structures for thesemetabolites can be proposed from the MS data. Authentic standards ofthe active metabolites can then by prepared by synthetic or biosyntheticmethods for determination of potency and other pharmacologicalactivities.


http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM079266.pdf



http://www.ich.org/products/guidelines/multidisciplinary/article/multidisciplinary-guidelines.html



Fig. 4. Application of the activity-gram approach to finding active metabolites in humans. In this example, pooled human plasma samples from anearly phase 1 clinical study were extracted and analyzed by HPLC-UV-MS. Fractions were collected and tested for binding in an in vitropharmacological binding assay. In addition to the binding activity expected at the retention time for the parent drug (;25–26 minutes), there wasstrong binding activity at 21–23 minutes and some activity at 19 minutes too. Thus, in human plasma, target activity is caused by not only the parentdrug, but metabolites as well. The structure of the major metabolite at 21 minutes was proposed from the mass spectral data, and an authenticstandard prepared for determination of intrinsic potency. Potency was comparable to the parent drug.

584 Obach

specific information on the putative active metabolites.It should be noted that our level of thoroughness andsophistication with regard to the identification andcharacterization of active metabolites is much greaterthan it was in the past. Thus, the depth of ourknowledge regarding active metabolites for older drugsmay not be as developed as for more recently in-troduced drugs. For example, in a review on this topicfrom over 30 years ago (that had a special emphasis onthe increased exposure and effect of active metabolitesin renal insufficiency), a list of over 50 drugs withactive metabolites was included (Drayer, 1976), al-though some were prodrugs. Considerable knowledgehas been gained since that time. In fact, activemetabolites have been proposed to be potential newmaterial for new drug discovery and development(Kang et al., 2010). Certainly the presence of drugsused clinically that were once metabolites (e.g.,fexofenadine) or are prodrugs of these metabolites(fesoterodine) is a testament to this notion (Table 2).

II. Absorption, Distribution, Metabolism,and Excretion Aspects of Active Metabolites

To appreciate the potential impact that pharma-cologically active metabolites can have on clinicalefficacy, some basic concepts regarding the disposi-tional aspects of metabolites must be understood. Inthis section, the metabolic reactions that can give riseto active metabolites are discussed followed by dis-cussions of distributional aspects and pharmacokineticphenomena.

A. Metabolic Reactions That Can YieldActive Metabolites

In order for a metabolite of a drug to retain bindingactivity to the same receptor to which the drug binds,the chemical modification must be as follows: 1) sominor as to not disrupt the atom-to-atom interactions

that occur between the drug and the receptor, or 2)occur on a position on the drug that is not orientedtoward the receptor when binding occurs. This isillustrated in the schematic in Fig. 1. Most metabolictransformations provide enough of a chemical changethat receptor potency is lost or greatly diminished.Furthermore, many metabolic reactions introduceenough hydrophilicity (by increasing the number ofhydrogen bonding substituents, charged entities, and/or size) and increased polar surface area that metab-olites are pharmacologically inactive by virtue of beingunable to penetrate the membranes of target tissues.However, in some cases, pharmacological activity canbe retained in a metabolite, while in other casesaffinity for the target can be diminished but pharma-cological activity retained due to high concentrations ofthe metabolite.

Among xenobiotic metabolizing enzymes, the cyto-chrome P450 family is the most important. Theseenzymes can catalyze a wide array of reaction types,and the specific biotransformation reactions that occur

Fig. 5. Metabolism of albendazole to the S-oxide metabolite.

Fig. 6. Metabolism of allopurinol to oxypurinol.

Fig. 7. Metabolism of artesunate to dihydroartimisinin.

Fig. 8. Metabolism of astemizole to desmethylastemizole.


for any given drug depend on the substituents presentin that drug (Guengerich, 2001). These include aliphaticand aromatic hydroxylations, heteroatom dealkylations,N- and S-oxygenations, dehydrogenations, and epox-idations. In the vast majority of examples, activemetabolites arise by one of these reactions. For ex-ample, among drugs active at neurotransmitter targetsthat contain a basic 2°- or 3°-aliphatic nitrogen, itis frequently the case that N-demethylated and

deethylated metabolites will also possess activity.The basicity of the amine, an important attribute forthese drugs, is retained or even somewhat increased.Thus, drugs such as fluoxetine, amitriptyline, imipra-mine, fenfluramine, citalpram, ketamine, mianserin,and many others have active N-dealkyl metabolites.Whether activity is exhibited in vivo depends on thepharmacokinetic and dispositional attributes of themetabolite.

Fig. 9. Metabolism of buspirone to hydroxyl and N-dealkyl metabolites.

Fig. 10. Metabolism of chlordiazepoxide to active metabolites.

586 Obach

In the case of hydroxylations, the likelihood thata metabolite is pharmacologically active is less pre-dictable. The addition of a single alcohol oxygen addsapproximately 20 Å2 in polar surface area (PSA) aswell as hydrogen-bond–donating potential to a positionwhere none existed in the parent structure. Such analteration can frequently yield an inactive metabolite ifthe target binding site cannot tolerate such a sub-stituent. However, in some cases the addition of –OHcan occur at a position that is not critical for targetbinding, or faces the solvent in the protein-ligandcomplex. In those cases, the metabolite can retainintrinsic activity, and its potential to contribute to invivo activity resides with other properties, such as invivo exposure and target tissue penetrability. It is notuncommon that alcohol and phenol metabolites are

rapidly cleared by conjugation reactions. It is alsofrequently the case that alcohol and phenol metabolitesdo not as readily penetrate target tissues, due toa decrease in membrane penetrability caused by the

Fig. 11. Metabolism of clomiphene to its hydoxylated and demethylated metabolites.

Fig. 12. Metabolism of codeine to morphine.


increased PSA. Nevertheless, there are several goodexamples of active hydroxyl metabolites and these aredescribed in Section IV (e.g., risperidone/paliperidone,saxagliptan/5-hydroxysaxagliptan, flutamide/2-hydrox-yflutamide, etc). Other reactions shown to yield activemetabolites include epoxidation (e.g., carbamazepine/carbamazepine-10,11-epoxide), reduction (dolasetron/hydrodolastron, buproprion/dihydrobupropion stereo-isomers, others), and heteroatom oxygenation (thiorid-azine/mesoridazine, roflumilast/roflumilast N-oxide).Other types of drug metabolism reactions usually do

not yield active metabolites, with some exceptions.Heteroatom dealkylations that result in loss of a largesubstituent usually inactivate a drug but there areexceptions [buspirone/1-(2-pyrimidyl)-piperazine (1-PP);nefazodone/m-chlorophenyl piperazine (mCPP)]. How-ever in such cases the pharmacological properties ofthe metabolite may be different from the parent(e.g., binds to a different member of the same receptorfamily). Conjugation reactions that add glucuronic acid,sulfuric acid, amino acid, and glutathione generallydo not yield active metabolites (Mulder, 1992a). Themost well known exception to this is the case ofmorphine-6-glucuronide, putatively an active metaboliteof morphine (Mulder, 1992b; Kilpatrick and Smith,2005). 4-Hydroxytriamterene sulfate represents anactive sulfate conjugate metabolite of triamterene. The

metabolite has nominally lower intrinsic potency anda lower free fraction; however, its plasma concentrationsare well in excess of parent and thus it contributes toeffect (Busch et al., 1996).

B. Pharmacokinetic Aspects of Active Metabolites

When compounds are administered directly, thedescription of pharmacokinetics is relatively straight-forward. However, when a compound arises fromwithin the organism, such as an active metabolite,the picture can be more complex (Pang, 1985; St. Pierreet al., 1988). This is primarily due to the fact that themetabolite represents an unknown fraction of the doseof the parent (i.e., dose is not truly known which isneeded for calculation of fundamental pharmacokineticparameters) and the rate of introduction into the bodyis not easily discerned.

From a pharmacokinetic standpoint, metabolites(active and inactive) are frequently referred to as tohaving formation rate–limiting kinetics or eliminationrate–limiting kinetics (Houston, 1981). A metabolitecannot have an elimination rate that exceeds theelimination rate of the parent drug from which it isformed. This is referred to as formation rate–limiting

Fig. 13. Metabolism of dihydroergotamine to 89-hydroxydihydroergotamine.

Fig. 14. Metabolism of diphenoxylate to difenoxin.

Fig. 15. Metabolism of dolasetron to hydrodolasetron.

Fig. 16. Metabolism of ebastine to carebastine.

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kinetics (Fig. 2). If an experiment is done wherein themetabolite is administered directly, then its t1/2 maybe shorter than when it is generated after adminis-tration of the parent drug. Many metabolites exhibitthis type of behavior by virtue of having higherclearances and/or lower volumes of distribution thanthe parent drug. In elimination rate–limiting kinetics,the t1/2 of the metabolite is longer than that of theparent drug (Fig. 2), and this value will be the sameirrespective of whether the metabolite arises afteradministration of the parent drug or whether it isadministered directly. If a metabolite exhibits elimi-nation rate–limiting kinetics, then with repeatedadministration of parent drug the metabolite has thepotential to accumulate to a greater extent than theparent drug at steady state.The factors that drive the total exposure to the

metabolite include the clearance rate of the parentdrug, the fraction of the dose of the parent drug that isconverted to the metabolite, and the subsequentclearance rate of the metabolite. The latter term canalso impact the amount of the metabolite formed thatescapes from the tissue from which it is formed andgets into the systemic circulation. In the vast majorityof cases, the focus can be on the liver as the main site ofmetabolite formation. When considering an activemetabolite, an important metric in understanding thepotential contribution of the metabolite to effectrelative to the parent drug is the area-under-the-curve (AUC) ratio. This can be defined as:

AUCmetabolite

AUCparent¼ fCL;m•Fm•CLparent

CLmetabolite

The terms are defined as follows: fCL,m is the fractionof the clearance of the parent drug that yields themetabolite, Fm is the portion of the total metabolitegenerated within an organ that is released into thesystemic circulation before it is either further metab-olized or released into bile (in the case of the liver) orurine (in the case of the kidney), CLparent is the totalclearance of the parent drug, and CLmetabolite is thetotal clearance of the metabolite. It is rarely the casethat these individual parameters are measured, espe-cially in humans, since they require the intravenousadministration of the metabolite directly. However,they offer conceptual insight as to the determinants ofmetabolite exposure. Prediction of metabolite exposureis a challenging undertaking when considering thenumber of contributing variables. Yet the importanceof active metabolites (as well as those that could causedeleterious off-target effects) in drug research andclinical practice makes research into methods that canpredict human metabolite exposures from in vitro and/or animal data an important endeavor (Lutz et al.,2010; Yeung et al., 2011; Lutz and Isoherranen, 2012;Smith and Dalvie, 2012).

C. Distributional Aspects of Active Metabolites inRelation to Contribution to Pharmacological Effect

The potential for the active metabolite to penetratethe target tissue relative to the parent drug is alsonecessary to understand whether the metabolite istruly an active metabolite. Even if the metabolitepossesses intrinsic potency that is equivalent to theparent, if the free concentration within the tissue islower than that of the parent, the metabolite will notcontribute as much as the parent. The main driversbehind the target-tissue free concentrations includeplasma protein binding, membrane penetrability,and the potential for active transporters to alterthe free tissue–to–free plasma concentration ratio(Kp,uu).

Distributional phenomena are largely driven by highcapacity, low affinity nonspecific binding interactionsbetween drugs and tissue macromolecules, and macro-molecular structures (e.g., phospholipid membranes).Such interactions are mostly a function of physico-chemical properties of the drug, especially lipophilicityand ionization state. Greater lipophilicity and greatercationic character are properties that tend to increasenonspecific binding to tissue macromolecular struc-tures. Since a majority of biotransformation reactionsresult in decreases in lipophilicity, metabolites tendnot to partition into tissues as well as their parentdrugs. An example of an exception to this is ketocona-zole, which undergoes deacetylation thereby convert-ing a neutral amide to a basic amine, and in this case itis the cationic metabolite that is better at binding tomembranes (Whitehouse et al., 1994).

Fig. 17. Metabolism of flutamide to 2-hydroxyflutamide.

Fig. 18. Metabolism of hydroxyzine to cetirizine.


Membrane penetrability is also frequently a functionof PSA, increases in which tend to debilitate penetra-bility. Since almost all biotransformation reactiontypes result in increases in PSA, metabolites will tendto be less membrane permeable than their respectiveparent drugs. If enough hydrophilicity is introduced,the metabolite could be completely membrane imper-meable. If a metabolite is still membrane permeable,but of lower permeability than the parent drug, thenthe result is that it will take longer for the metaboliteto achieve a steady-state exposure in the tissues thanthe parent drug. Increased hydrophilicity also rendersmetabolites better substrates for several of the drugefflux transport proteins, which also serves to decreasethe free tissue concentrations of metabolites relative toparent drugs.The effects of decreased tissue partitioning and

penetration can be offset by differences in plasmaprotein binding between parent drugs and theirmetabolites. Metabolites are frequently less protein-bound in plasma than their respective parent drugs,presumably due to their decreased hydrophobicity(Table 1). Thus, the potential decreases in membranepermeability and Kp,uu of metabolites relative to theirparent drugs can be offset by increases in free fraction.A potentially challenging situation for understandingthe relative importance of metabolites to pharmaco-logical effect occurs when the metabolite is generatedwithin the target tissue. For example, the hydroxylmetabolites of atorvastatin have potency at thepharmacological target. They are generated by CYP3Awithin the liver, which is the target tissue for

inhibition of cholesterol biosynthesis. Due to this, thefree concentrations within hepatocytes may not bereflected by systemic free plasma concentrations, andthus a true knowledge of the contribution of themetabolites to clinical effect would be unattainable.Estimates would need to be made from in vitro data ordata from laboratory animals.

III. Active Metabolites in Drug Research

For the purposes of this discussion, the process bywhich new drugs are created can be generally brokendown into two major portions: 1) the preclinical phase,in which medicinal chemists and pharmacologistscollaborate, along with other related discipline scien-tists, to identify a new molecular target and designligands that can bind and affect the activity of thattarget (termed “drug discovery”), and 2) the clinical

Fig. 19. Metabolism of irinotecan to SN-38.

Fig. 20. Metabolism of loratadine to desloratadine.

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phase, wherein the selected molecule is studied first inhealthy volunteers (phase 1) and then in patients forefficacy and safety (“drug development”). The impact ofpharmacologically active metabolites in these twophases is different. However, it is critical to accountfor activemetabolites in drug research as early as possible,as the insights that can be gleaned regarding targetstructure-activity relationship (SAR), pharmacokinetic-pharmacodynamic (PK/PD) relationships, and toxicityare invaluable (Fura, 2006)

A. Research Tools in Metabolite Identification

A lengthy discussion of how drug metabolites areidentified is beyond the scope of this review and theinterested reader is referred to other descriptions ofthis topic for greater depth (Prakash et al., 2007; Zhangand Comezoglu, 2007). However, to understand theissue of pharmacologically active metabolites, a verybrief description of how metabolites are identified isnecessary.In the early efforts (preclinical), the identification of

metabolites of new compounds is done using incubatesfrom in vitro liver-derived systems such as hepatocytesand microsomes, as well as various biologic fluids (e.g.,plasma, urine, bile) obtained from laboratory animalspecies following administration of the compound ofinterest. Identification of metabolites in the drugdiscovery phase is done for a variety of reasons,including 1) the identification of specific sites ofmodification on a rapidly metabolized molecule so that

drug design efforts can slow down metabolism, 2)identification of the main routes of metabolism andenzymes involved so that the overall clearance mech-anism in humans can be understood and predicted, 3)identification of the possibility of generation of chem-ically reactive metabolite types associated with toxic-ity, and, relevant to this article, 4) the identification ofpharmacologically active metabolites. In the earlystages of drug research, considerable effort is expendedthrough the use of animal models of disease tounderstand the biologic effects of manipulating theunderlying pharmacological target of interest. Develop-ing the relationship between the drug concentration andthe effect (i.e., the pharmacokinetic/pharmacodynamicrelationship) will offer insight into the duration anddegree to which the target receptor needs to be occupiedfor effect. The presence of an active metabolite willalter that relationship, and if an active metabolite ispresent but unaccounted for, the pharmacokinetic/pharmacodynamic relationship will be misinterpreted.

In the clinical stage, the same questions regardingestablishing the PK/PD relationship exist, albeit inhuman patients rather than animal disease models,and unlike the earlier work the efforts are focused onone or maybe two compounds for any given target. Inearly phase 1 studies, plasma and urine samples fromhuman study subjects can be used to seek metabolites.Later in clinical development, it is typical practice tocarry out a study wherein humans are administereda radiolabeled drug and excreta and plasma arethoroughly interrogated for a complete and compre-hensive picture of the metabolism of the new drug(Penner et al., 2012).

The main tool used by the drug metabolismscientist in metabolite identification is high-pressureliquid chromatography-mass spectrometry (HPLC-MS).HPLC-MS provides information on the molecularweight of the metabolite (so it can be compared withthe molecular weight of the parent compound) todiscern the type of metabolic modification, and thefragmentation of the metabolite in the MS offersinsight into the structure of the metabolite. While MSdata can sometimes yield enough information to assignthe chemical structure of a metabolite, it is frequentlythe case that the information is not enough to proposea precise structure. In those cases, additional datausing complementary methods is required, such asNMR spectroscopy, chemical derivatization, and/orsynthesis of an authentic standard of the metabolitefor comparison.

B. Identification of Active Metabolites inPreclinical Research

As stated above, knowledge of pharmacologicallyactive metabolites in animal models of disease isimportant. Furthermore, the prediction that humansmay also be exposed to pharmacologically active

Fig. 21. Metabolism of losartan to EXP-3174.

Fig. 22. Metabolism of oxcarbazepine to 10-hydroxycarbamazepine.


metabolites is important in predicting the efficaciousdose prior to human studies. In the preclinical stage,efforts are ongoing to develop knowledge of the SAR forreceptor binding potency, and the identification of anactive metabolite can offer the medicinal chemistgreater insight as to the types of substituents thatcan be tolerated in the ligand-receptor interaction.Finally, an active metabolite could be a better com-pound to develop than the drug itself (e.g., terfenadineversus fexofenadine).A sequential approach to identifying metabolites in

biologic matrices—proposing chemical structures ofthese metabolites, followed by organic synthesis andtesting of the metabolite at the target receptor—represents one strategy for identifying active metabo-lites in preclinical drug research. Some SAR insightmay already be known for a chemical series such thatproposed metabolite structures could be ruled out fromhaving pharmacological activity in the absence of data.For instance, N-demethylation of a tertiary amine drugto a secondary amine metabolite is frequently antici-pated to have minimal impact on target receptoractivity, thus it is prudent to establish this potentialactivity experimentally. However, N-deamination of anamine drug followed by oxidation to the carboxylic acidmetabolite is much less likely to yield an activemetabolite, due to the marked difference in structureand charge.An approach we have employed in our laboratories to

address the possibility of pharmacologically activemetabolites involves a close collaboration between thedrug metabolism scientist and pharmacologist (see Fig.3 and Fura et al., 2004). The drug metabolism scientistcarries out an analysis of a biologic sample (in vivo orin vitro) for metabolite identification on HPLC-MS as

usual. However, the HPLC eluent is split prior tointroduction into the MS. A small portion is introducedto the MS to yield spectral data. The remaining portionis diverted to a fraction collector. The fractions aredelivered to the pharmacologist for testing activity atthe target receptor. The HPLC-MS chromatogram iscompared with the receptor activity profile of thefractions to create an “activitygram.” If receptoractivity is detected in fractions corresponding to wherea metabolite eluted, then the metabolite is potentiallyan active metabolite. Since the amount of the metab-olite in the fraction is unknown, an absolute potencyvalue cannot be determined. This merely offers anearly signal regarding whether an active metabolite iseven possible, and if so, then synthesis of an authenticstandard of that metabolite for testing can be un-dertaken. If the metabolite poses a synthetic challenge,it can still be addressed by generating a biologicsample containing the metabolite, purifying it byHPLC, determining the concentration of the metaboliteusing proton NMR (Walker et al., 2011; Vishwanathanet al., 2009), and using that solution as a concentratedstock solution from which to dilute the metabolite intothe activity assay.

C. Identification of Active Metabolites inClinical Research

As humans are the target species, knowledge ofactive metabolites in human is essential to proper doseselection and understanding determinants of interpa-tient variability in drug response. By the time ofadministration to humans in phase 1 studies, thepresence (or not) of active metabolites in laboratory

Fig. 23. Metabolism of oxybutynin to desethyloxybutynin.

Fig. 24. Metabolism of procainamide to N-acetylprocainamide. Fig. 25. Metabolism of propoxyphene to norpropoxyphene.

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animals will have already been determined. In addi-tion, human in vitro systems will have been used topredict the metabolites that will be present in vivo.Thus, a good understanding of the potential for activemetabolites in humans should already have beenobtained. Nevertheless, it is still possible for an activemetabolite to be revealed for the first time in a clinicalstudy.From animal models, a prediction of the human

concentration-effect relationship can be made (eitherfor a biomarker or an effect on disease). If thatrelationship does not hold upon first examination inhuman (either the magnitude or duration of effect, orboth), then the possibility of pharmacologically activemetabolites must be entertained. The same type ofapproach described above can be performed usinghuman plasma samples. An example of this isillustrated in Fig. 4 (Hagen et al., 2008). In this

instance, the parent drug was targeting a subtype ofthe serotonin receptor family. During the phase 1 doseescalation study, suprapharmacological effects wereobserved at parent drug concentrations that were farlower than anticipated. Plasma samples that hadalready been used for quantitative analysis of the

Fig. 26. Metabolism of remacemide to desglycylremacemide.

Fig. 27. Metabolism of risperidone to paliperidone.

Fig. 28. Metabolism of roflumilast to roflumilast-N-oxide.

Fig. 29. Metabolism of sibutramine to its N-demethylated metabolites.


parent drug were subsequently used in the “activity-gram” approach, and the presence of two peaks of highreceptor binding activity was observed, with oneactivity peak corresponding to the retention time ofthe parent drug and an earlier eluting peak corre-sponding to where a hydroxylated metabolite eluted.This showed the likelihood of a circulating activemetabolite that could explain the clinical observations.Subsequent structure elucidation of the metabolite wasdone, and synthesis of an authentic standard showedthat the metabolite had intrinsic binding potencyequivalent to the parent drug.In addition to those instances wherein pharmacody-

namic observations trigger an aggressive search forpharmacologically active metabolites, the determinationof the metabolites present in human plasma and ex-creta is done for other purposes, such as gaining an

understanding of the important clearance pathways anddetermining whether human metabolites are alsoobserved in laboratory animal species that are used toexplore the toxicology of the drug. As those metabolitesare structurally identified, a judgment can be maderegarding whether they would potentially be active,based on the known structure-activity relationship forthe target receptor. Even if not contributing substan-tially to the efficacy of the drug, discovering a new activemetabolite in clinical samples can potentially offera new lead for a second-generation drug.

D. Data Needed for Active Metabolites

Once a metabolite is identified that has intrinsicbinding potency to the target receptor, the followinginformation needs to be gathered to properly charac-terize it:

Fig. 30. Metabolism of tamoxifen to its hydroxylated and N-demethylated metabolites.

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1. In addition to target binding potency, functionalactivity measurement is needed (antagonist,agonist, partial agonist, inhibitor, activator, etc.).

2. Plasma protein binding in humans and labora-tory animal species.

3. Penetration into the target tissue(s) and pre-diction of the free tissue–to–free plasma concen-tration ratio (i.e., Kp,uu).

4. Pharmacokinetics in humans and laboratoryanimal species, and the underlying clearancepathways for the active metabolite in humans.

Essentially, this set of information reflects the sameset that is needed to understand the pharmacology ofthe parent drug. For example, if an active metabolite isresponsible for the majority of the pharmacologicaleffect, knowledge of what can cause a change in theexposure to the metabolite (to either increase ordecrease it) must be known. Drug interactions thatalter the exposure to an active metabolite must beknown. For example, tamoxifen is converted to themore potent metabolite endoxifen by CYP2D6, so theconcomitant use of potent CYP2D6 inhibitors inpatients receiving tamoxifen should be avoided (Mann-heimer and Eliasson, 2010).

IV. Drugs with Active Metabolites

In this section, a description of numerous examplesof drugs with active metabolites is included. Themolecular targets, indications, and metabolic reactiontypes span a broad array, indicating that there is nosingle complement of circumstances that favor ordisfavor the possibility of an active metabolitephenomenon. As described in section III.D, the

information needed to truly assess the contribution ofan active metabolite to clinical effect is challenging andrequires knowledge of intrinsic receptor potency, freefraction, target tissue penetrability, and plasma phar-macokinetics. It is rare that all of these properties areknown for metabolites (especially plasma proteinbinding and target tissue penetrability), so in manycases inferences must be made and assumptionsaccepted when making such projections. Additionally,it is not uncommon that the reported intrinsic potencyvalues measured for active metabolites may have been

Fig. 31. Metabolism of terfenadine to fexofenadine.

Fig. 32. Metabolism of thiocolchicoside to its glucuronide.


made using nonhuman-derived reagents and tissues,so it must be assumed that relative potency valuesbetween parent and metabolite(s) are similar acrossspecies. From the assessments of these examples, it isclear that in many cases there is considerable datagathering that needs to be done to fully understand theaction of active metabolites. Many of the estimates ofcontributions of metabolites to efficacy relative to theparent drug described below were made using theproperties listed above that were obtained from thescientific literature.The examples have been divided into four categories:

1) active metabolites wherein the metabolite contrib-utes the majority of the activity, even though theparent drug has intrinsic target potency; 2) activemetabolites that contribute to target activity at a levelcomparable to the parent drug; 3) active metabolitesthat possess target affinity but would be estimated tocontribute little to in vivo effect, relative to the parent;and 4) metabolites that have activity at alternatepharmacological targets closely related to the intendedtarget that the parent drug interacts with, andputatively contribute to clinical effect via this alternatetarget. Section IV.E describes a few examples whereinthe delineation of metabolite and parent contributionsis highly ambiguous.

A. Drugs with Active Metabolites That Dominatethe Activity

In some instances, while the parent drug possessesaffinity for the intended target protein, a metabolitecan be present that actually dominates the in vivoeffect. Such cases are not truly prodrugs, since theparent drug has target activity, but their behavior canresemble those of prodrugs.1. Albendazole. Albendazole offers an interesting

example of site-dependent effects of parent drug versusits S-oxide metabolite (Fig. 5). Albendazole is ananthelminthic agent used in the treatment of variousparasitic diseases and its mechanism of action isthrough the binding of tubulin in nematodes. Theparent is about 6-fold more intrinsically potent attubulin binding than the S-oxide metabolite (Lubegaand Prichard, 1991). For systemic parasitic infection itis the metabolite that is believed to exhibit the effectbecause metabolite plasma concentrations achievedare in the range of 3 mM while the parent drug isbelow the limit of quantitation of plasma assays (Junget al., 1992; Medina et al., 1999; Mirfazaelian et al.,2003). It can be used in the treatment of neuro-cysticercosis because of the brain penetrability of theS-oxide metabolite. However, for parasitic infectionsof the gastrointestinal tract, it is likely the parentdrug has the effect since the S-oxide is generatedpostabsorption.2. Allopurinol. Allopurinol is oxidized to oxypurinol

by aldehyde oxidase (Fig. 6). Oxypurinol is approximately

10-times less potent an inhibitor of the target enzyme,xanthine oxidase (Tamta et al., 2006); however, thecirculating concentrations of oxypurinol in humansfollowing allopurinol administration are far greater andsustained for a longer time. Maximal concentrations ofoxypurinol are about 4- to 5-fold greater, and the half-lifeof parent and metabolite are 1 and 20 hours, respectively(Turnheim et al., 1999; Day et al., 2007). Thus, thecontribution of oxypurinol to xanthine oxidase inhibitionactivity dominates at later timepoints.

Fig. 33. Metabolism of thioridazine to its S-oxidized metabolites.

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3. Artesunate. Artesunate is an ester-containingderivative containing a hydroperoxide that is believedto form toxic adducts with heme as hemoglobin is beingdigested by the Plasmodium species that causesmalaria (Creek et al., 2008). It is more water-solublethan other artemisinin derivatives and thus offersmultiple dosing approaches. Upon oral administrationit is entirely cleaved to dihydroartimisinin, an activemetabolite (Fig. 7), but after intravenous administra-tion both parent drug and the active metabolite arepresent in circulation (Nealon et al., 2002; Morris et al.,2011). The intrinsic potency of the metabolite isapproximately 4-fold greater than the parent (Creeket al., 2008), and after correction for differences inplasma free fraction and plasma concentrations, it canbe estimated that the metabolite contributes about 4-fold greater activity than the parent.4. Astemizole. Astemizole is a histamine-1 receptor

antagonist that was withdrawn from clinical use dueto occurrence of cardiac arrhythmia caused by block-ade of the human ether-a-go-go-related gene potas-sium channel. It has been described to be convertedto O-desmethylastemizole (Fig. 8) which has a muchlonger t1/2 than astemizole (Heykants, 1984). Detailedinformation on the human H1 receptor potency of thismetabolite is not available in the literature, but ithas been described as being equipotent (Richardset al., 1984). It can thus be anticipated that themetabolite would contribute the majority of thepharmacological activity, especially upon accumula-tion at steady state. Other metabolites, such as 6-

hydroxydesmethylastemizole, have been shown tohave activity in guinea pig-derived models of antihis-tamine, but this may be due to alterations inhistamine release (Kamei et al., 1991).

Fig. 34. Metabolism of tibolone to hydroxylated and isomer metabolites.

Fig. 35. Metabolism of tolterodine to desfesoterodine.


5. Buspirone. Buspirone is an anxiolytic agentpurported to act via binding to the 5-HT1A (serotonin)receptor. It has long been known to undergo N-dealkylation to 1-(2-pyrimidyl)piperazine (1-PP), whichhas weak activity at the 5-HT1A receptor, and that thismetabolite could also play a role in the effect ofbuspirone (Fig. 9). However, in a clinical pharmacoki-netic study, it was shown that 6-hydroxybuspirone ispresent at much greater concentrations than buspironeand may contribute to activity (Dockens et al., 2006;Wong et al., 2007). If it is assumed that the freefraction of this metabolite is equivalent to buspironeitself, then it can be estimated that the metabolitecontributes seven times the activity of the parent.Reports in the literature on the process of making 6-hydroxybuspirone on a large scale suggest that there isinterest in this metabolite as a potential drug itself.Investigation of 1-PP as an active metabolite hasrevealed that it also could contribute considerably tothe action of buspirone by virtue of higher exposure. Inaddition, 1-PP has activity at the a2 receptor (Cacciaet al., 1986), the role of which is undefined.6. Chlordiazepoxide. Chlordiazepoxide generates

several active metabolites via sequential demethyla-tion, deamination, reduction, and hydroxylation reac-tions (Fig. 10). Which metabolite contributes thegreatest activity cannot be discerned, but it is clearthat metabolites, especially desmethyldiazepam, con-tribute to activity. The affinities of the entities with theN–O bond (chlordiazepoxide, norchlordiazepoxide, anddemoxapam) for the benzodiazepine receptor are 10- to20-fold lower than the desmethyldiazepam and oxaz-epam metabolites (Richelson et al., 1991). Concentra-tions of oxazapam after chlordiazepoxide were notavailable, but unbound concentrations of desmethyl-diazepam were comparable to its binding affinity

(Dixon et al., 1976; Boxenbaum et al., 1977; Greenblattet al., 1978; Ito et al., 1997).

7. Clomiphene. Similar to tamoxifen (section IV.A26.), clomiphene is metabolized by hydroxylation atthe para position of one of the phenyl rings by CYP2D6to yield a metabolite, 4-hydroxyclomiphene (Fig. 11),that has two orders of magnitude greater potency atbinding to the estrogen receptor (Ruenitz et al., 1983;Mürdter et al., 2012). Thus, CYP2D6 extensive-metabolizer (EM) and poor-metabolizer (PM) subjectsexhibit different responses, with the EM subjectsexpected to enjoy greater efficacy due to 7-fold greaterexposure (Mürdter et al., 2012). Clomiphene alsoundergoes N-deethylation, but N-desethylclomiphenehas similarly low activity to the parent drug. The 4-hydroxy metabolite of N-desethylclomiphene also hasactivity and would be estimated to contribute 50-foldmore than the parent drug. Furthermore, the picture iscomplicated by the fact that the drug is administeredas two geometric isomers, with the E-isomers possess-ing greater activity.

8. Codeine. Codeine is converted to the active O-demethylated metabolite morphine by CYP2D6 (Fig.12). After oral administration, the exposure to codeineis almost 40-fold greater than that of morphine(Quiding et al., 1986). However, the potency ofmorphine at the m-opioid receptor is 600-fold greater(Volpe et al., 2011), thus the pharmacological effects ofcodeine administration are almost exclusively attribut-able to the active metabolite morphine (and itssubsequent pharmacologically active glucuronidemetabolites; see Section IV.B.15.). In CYP2D6 poormetabolizers, morphine concentrations are very low(;50-fold lower than in extensive metabolizers; Kirch-heiner et al., 2007), and there is not enough generatedto elicit an effect. Codeine still has other clearancepathways besides CYP2D6, and thus exposures are notdifferent between EM and PM subjects. This supportsthe notion that in EM subjects, codeine itself does notcontribute to pharmacological effect.

9. Dihydroergotamine. Dihydroergotamine is a va-sodilator effective in the treatment and prophylaxis ofmigraine, with the serotonin 1A receptor considered asthe target. The oral bioavailability of dihydroergota-mine itself is very low, yet the drug is effective at lowparent drug concentrations. Investigation led to theidentification of the metabolite 89-hydroxydihydroer-gotamine as the putative active agent (Fig. 13). Itsintrinsic potency is about thrice that of the parent(Hanoun et al., 2003), but more importantly, theplasma concentrations of the metabolite are 6-timesgreater (Wyss et al., 1991; Chen et al., 2002). It can beestimated that the metabolite possesses 20-fold greaterin vivo activity than the parent, assuming that freefractions and brain penetrability properties are simi-lar. Considering that the addition of one hydroxylgroup to a molecule with a molecular weight of nearly

Fig. 36. Metabolism of tramadol to O-desmethyltramadol.

Fig. 37. Metabolism of trimethadione to dimethadione.

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600 would alter the physicochemical properties to onlya very small extent, such a consideration is reasonable.10. Diphenoxylate. Diphenoxylate is an ethyl ester

that is hydrolyzed to difenoxine (Fig. 14). It is used asan antidiarrheal agent that acts by agonizing theopiate receptor. The carboxylic acid metabolite issomewhat more potent than the parent drug (asassessed using a rat brain homogenate binding assay),and systemic concentrations of the metabolite aregreater than for the parent (Karim et al., 1972). Thetarget of action is the gut, so it cannot be necessarilystated that the metabolite bears the greater role inactivity since the effect could be due to local exposuresto parent drug upon oral administration (Wüster andHerz, 1978). In a comparison, dosing the metabolitedirectly to humans showed that five-times less wasneeded to achieve a constipatory effect the same as theparent ester (Rubens et al., 1972).11. Dolasetron. Dolasetron is a 5-HT3 receptor

antagonist used in the treatment of nausea andvomiting caused by emetogenic anticancer agents(Anzemet). It undergoes reduction to hydrodolasetronand this metabolite dominates the contribution toactivity (Fig. 15). When used orally, dolasetron is notmeasurable, thus it has been concluded that theactivity is dominated by the alcohol metabolite (Keunget al., 1997). The metabolite molecule has 20- to 60-times greater affinity at the 5-HT3 receptor than theparent (Gregory and Ettinger, 1998). Even whendolasetron is intravenously administered, hydrodola-setron is generated rapidly and exceeds the parent inconcentration by over 10-fold. Thus, even thoughdolasetron possesses intrinsic affinity for the 5-HT3

receptor, it offers no contribution to efficacy in vivo; theactivity resides with the reduced metabolite.12. Ebastine. Ebastine is a second-generation anti-

histamine that contains a t-butyl substituent thatundergoes extensive conversion to a carboxylic acidmetabolite, carebastine (Fig. 16). After administrationof ebastine, carebastine concentrations are 30-foldgreater (Sastre, 2008; Shon et al., 2010). Furthermore,carebastine is intrinsically more potent at the H1

receptor (Anthes et al., 2002). The exact values forplasma protein binding have not been reported as both

parent and metabolite are highly bound and merelyreported as .95%. On the basis of these measure-ments, it can be estimated that the metabolitecontributes virtually all of the antihistamine activityfollowing oral administration of ebastine.

13. Flutamide. Flutamide is an antiandrogen thatis converted to the active metabolite 2-hydroxyfluta-mide (Fig. 17). The intrinsic potency and plasmaprotein binding values of the parent and metaboliteare not very different (Feau et al., 2009), but whatdrives estimation of a high contribution of themetabolite to efficacy are the 20- to 40-fold greater

Fig. 38. Metabolism of venlafaxine to O-desvenlafaxine.

Fig. 39. Metabolism of acebutolol to diacetolol.


plasma concentrations of the metabolite (Belangeret al., 1988).14. Hydroxyzine. Hydroxyzine is a first-generation

antihistamine. Its use in the treatment of allergy hasfallen out of favor because it causes sedation, unlikemore recently available antihistamines. However,because of this side effect, it has found use as a sedativeand potentially as an anxiolytic. Hydroxyzine containsa 1° alcohol that is metabolized by oxidation to thecarboxylic acid metabolite cetirizine (Fig. 18). Cetir-izine also has intrinsic H1 potency that is about 3-foldweaker than the parent drug (Chen, 2008), yet thispotency was enough to permit introduction of this

metabolite as a drug itself. Cetirizine is zwitterionicand therefore not necessarily as good at penetratingthe brain as the parent hydroxyzine, and this serves asan advantage by offering antihistimic activity periph-erally while causing much less sedation. With hydroxy-zine dosing, the contribution of cetirizine toantihistamine activity is likely high, due to higherconcentrations of the metabolite (Simons et al., 1989).

15. Irinotecan. Irinotecan is a topoisomerase 1inhibitor structurally derived from natural productsfrom Camptotheca. It undergoes hydrolysis of thecarbamate bond to the active metabolite SN-38 (Fig.19), which is over 1000-times more potent at inhibition

TABLE 2Examples of drugs for which active metabolites are now used as drugs themselves

Original Drug Metabolite That Is Used as a Drug Mechanism/Indication Rationale for the Usefulness of the Metabolite as a Drug

Amitriptyline Nortriptyline Antidepressant Differential effect at neurotransmitter transportproteins

Bromhexine Ambroxol Mucokinetic agentand topical anesthetic

Diazepam Temazepam AnxiolysisDiazepam Oxazepam AnxiolysisEtretinate Acitretin Antipsoriatic Improved pharmacokineticsHydroxyzine Cetirizine Antihistamine Lower brain penetration, less sedationImipramine Desipramine Antidepressant Differential effect at neurotransmitter transport

proteinsLoratadine Desloratadine Antihistamine Lower brain penetration, less sedationLoxapine Amoxapine Antidepressant Differential effect at neurotransmitter transport

proteinsPrimidone Phenobarbital AntiseizureProcainamide Acecainide Antiarrythmic Molecular mechanism resulting in different

electrophysiological effectRisperidone Paliperidone Antipsychotic Decreased impact of CYP2D6 clearance, lower

intersubject variabilitySpironolactone Canrenone DiureticTerfenadine Fexofenadine Antihistamine Safety; reduced potassiumr channel activityThioridazine Mesoridazine AntipsychoticTolterodine Fesoterodine Urinary incontinence Decreased impact of CYP2D6 clearance, lower

intersubject variabilityVenlafaxine Desvenlafaxine Antidepressant Decreased impact of CYP2D6 clearance, lower

intersubject variability

Fig. 40. Metabolism of acetohexamide to hydroxyhexamide and structures of analogs.

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of the enzyme (Kawato et al., 1991). While SN-38 ispresent in circulation, it is only there at about a 40-foldlower concentration than the parent drug, and its freefraction is about 10-times lower (Wiseman andMarkham,1996; Iyer and Ratain, 1998; Mathijssen et al., 2001;Camptosar, 2012). Combining the inhibition potencyand free plasma concentrations, it would be estimatedthat SN-38 contributes 10-fold more than the parent.However, it is likely that irinotecan is cleaved in-side the tumor cell and bioactivated there, and SN-38would then make a much larger contribution (Kawatoet al., 1991).16. Loratadine. Loratadine was one of the first

nonsedating antihistamines and as such was a popularalternative to the older generation antihistamines thatreadily penetrated the blood-brain barrier and causedsedation. It is extensively metabolized to desloratadinevia hydrolysis of the carbamate moiety (Fig. 20). Theintrinsic potency of desloratadine far exceeds that ofthe parent (Anthes et al., 2002), the free fraction ishigher by 5-fold, and the circulating concentrations arealso higher (Ramanathan et al., 2007). When thesethree factors are combined, it suggests that loratadine

itself is responsible for none of the in vivo antihista-minic activity. Desloratadine is now used as a drugitself. The picture is further complicated by the factthat 3-hydroxydesloratadine is also listed as an activemetabolite; however, the intrinsic potency value is notavailable in the scientific literature. 3-Hydroxydeslor-atadine has a comparable free fraction and circulatesat about half the concentration of desloratadine.

17. Losartan. Losartan is an antihypertensiveagent that acts by binding to the angiotensin IIreceptor. It is a primary alcohol that is oxidized intwo steps to a carboxylic acid (EXP3174) by CYP3A andCYP2C9 (Fig. 21). An integrated examination of therelative potency (Le et al., 2003), free fraction inplasma (Cozaar, 2011), and plasma exposures (Loet al., 1995) yields the projection that the carboxylicacid metabolite would contribute approximately 14-times the activity than the parent drug. This isprimarily driven by the approximately 25-fold greaterpotency of the metabolite. The relevance of themetabolite to efficacy is reinforced by clinical observa-tions. As CYP2C9 has a major role in the generation ofEXP3174, subjects that are genotyped as containingCYP2C9*3 have shown lower metabolite concentra-tions (Yasar et al., 2002). Such subjects have also beenshown to have a lower therapeutic response to losartan(Lajer et al., 2007; Joy et al., 2009).

18. Oxcarbazepine. Oxcarbazepine is reduced to 10-hydroxycarbamazepine (Fig. 22) and it is the metabo-lite that has been claimed to carry the majority, if notall, of the antiepileptic activity. Exposure to the activemetabolite is considerably greater than the parentdrug and it is somewhat less protein-bound (Dickinson

Fig. 41. Metabolism of amiodarone to desethylamiodarone.

Fig. 42. Metabolism of aripiprazole to dehydroaripiprazole. Fig. 43. Metabolism of carbamazepine to the epoxide metabolite.


et al., 1989; Patsalos et al., 1990). Although themechanism of action may not be definitively proven,both compounds block sodium channels in vitro withequivalent potency (Schmutz et al., 1994). Con-sidering the relative potency, plasma exposure, andfree fraction values, and assuming equivalent brainpenetrability, it can be estimated that the me-tabolite carries 17-times the in vivo activity of theparent drug.19. Oxybutynin. Oxybutynin is an antimuscarinic

agent used in the treatment of urinary incontinence. Itundergoes an N-deethylation reaction to yield a majorcirculating metabolite present at 8-fold greater con-centrations (Fig. 23) (Reiz et al., 2007). Interestingly,this metabolite has shown a higher protein binding(Mizushima et al., 2007), which is counter to most caseswherein secondary amine metabolites have higher freefractions than their corresponding tertiary amineparent drugs. Both parent and metabolite are equallyactive in vitro (Waldeck et al., 1997). Thus, it can beestimated from the combination of these data thatdesethyloxybutynin has about 4-fold the in vivoactivity as the parent drug. It should be noted thatthese estimates are based on data for the racemate,and somewhat different estimates could be possible byconsidering the stereoisomers independently.

20. Procainamide. Procainamide is a class 1 anti-arrythmic agent that blocks the sodium channel. Itundergoes acetylation to the active metabolite N-acetylprocainamide (Fig. 24), which also has beenshown to bind to this channel (Sheldon et al., 1994b).Combining the circulating concentrations, plasma pro-tein binding (which is low for both), and the sodiumchannel affinity leads to the conclusion that theacetylated metabolite contributes twice as much tothe receptor activity as the parent drug. Despite thisestimation, there are other data to suggest that N-acetylprocainamide does not make this contributionand actually works as a class III antiarrythmic viapotassium channel block (Harron and Brogden, 1990).Direct intravenous administration showed that N-acetylprocainamide concentrations needed to begreater than what would be expected from findingswhen the metabolite was measured after administra-tion of procainamide and that the electrophysiologicalprofile qualitatively differed from the parent drug(Roden et al., 1980; Dangman and Hoffman, 1981;Jaillon et al., 1981). To further complicate this picture,acetylation is catalyzed by the polymorphic N-acetyl-transferase 2 enzyme, thus the ratio of parent tometabolite in vivo differs between extensive and poormetabolizers (Reidenberg et al., 1975).

Fig. 44. Metabolism of clarithromycin to 14-hydroxyclarithromycin.

Fig. 45. Metabolism of clobazam to N-desmethylclobazam.

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21. Propoxyphene. Propoxyphene is a m-opioidagonist that was recently removed from clinical usein the United States due to cardiac toxicity. It under-goes N-demethylation to norpropoxyphene (Fig. 25),which has nearly equivalent intrinsic potency andplasma binding (Giacomini et al., 1978; Neil andTerenius, 1981). However, it is present in nearly 10-fold excess to parent after steady-state dosing, withnonstationary clearance for both parent and metaboliteresulting in high accumulation (Inturrisi et al., 1982).This would suggest that norpropxyphene carries thebulk of the target activity. However, in rats it wasshown that the norpropoxyphene metabolite does notpartition into brain as readily as the parent drug (Wayand Schou, 1979) and that propoxyphene itself is aP-glycoprotein substrate in mouse (Doran et al., 2005).Thus, the relative central effects of the two agents areunclear. The cardiotoxicity may be largely due to theactive metabolite (Way and Schou, 1979; Ulens et al.,1999).22. Remacemide. Remacemide is an antiepileptic

agent that undergoes loss of a glycine to an activemetabolite (Fig. 26). While a complete examination ofall input parameters necessary for assessing therelative contributions to efficacy of parent drug andactive metabolite are not available, it has been shownthat the metabolite is over 100-fold more potent at thetarget ion channel and achieves greater free exposurein the brain than the parent drug (Palmer et al., 1992;Schachter and Tarsy, 2000). Assuming equivalentplasma protein binding and target tissue penetrationleads to the estimate that the desglycine metaboliteactually possesses nearly all of the pharmacologicaleffects, essentially making the parent a prodrug.23. Risperidone. Risperidone is an effective anti-

psychotic agent active at several human neurotrans-mitter receptors, particularly the serotonin-2A and

dopamine-2 types (Schotte et al., 1996). It is exten-sively metabolized to the active metabolite 9-hydrox-yrisperidone (paliperidone) by CYP2D6 (Fig. 27)(Huang et al., 1993; Fang et al., 1999). The metabolitepossesses intrinsic potency similar to the parent.Deconvolution of the relative contributions of each toefficacy is challenging because of potential differencesin brain penetrability caused by P-glycoprotein, forwhich both parent and metabolite are substrates(Wang et al., 2004; Ejsing et al., 2005). In mouse,risperidone and 9-hydroxyrisperidone brain penetra-tion appear to be hampered 10- and 20-fold, respec-tively, as shown by comparison of brain/plasma ratiosin multidrug resistance knockout mice. Since CYP2D6is polymorphically expressed, risperidone demon-strates different exposure levels in EM and PMsubjects; however, this difference does not appear tomanifest itself in different levels of efficacy and this isattributed to the fact that 9-hydroxyrisperidone is alsoan active antipsychotic agent. In EM subjects, it can beprojected that the metabolite carries the large share of

Fig. 46. Metabolism of disopyramide to its desisopropyl metabolite.

Fig. 47. Metabolism of fluoxetine to norfluoxetine.

Fig. 48. Metabolism of itraconazole to hydroxyitraconazole.

Fig. 49. Metabolism of ketamine to norketamine.


receptor occupancy (maybe as much as 10-fold), evenwith a potentially greater P-glycoprotein-catalyzedefflux. The efficacy of 9-hydroxyrisperidone has beenleveraged in that this compound is now used as a drugitself.24. Roflumilast. The phosphodiesterase (PDE) 4

inhibitor roflumilast is used for chronic obstructivepulmonary disease (COPD) and it possesses an activeN-oxide metabolite (Fig. 28). A thorough analysis of thepharmacokinetics of these two and their variability, asthey relate to contribution to activity, has been done(Lahu et al., 2010). In this analysis the followingequation was used:

tPDE4i ¼ AUCp•fu;pIC50;p•t

þ AUCm•fu;mIC50;m•t

in which tPDE4i refers to a unitless value for in-hibition of the target enzyme, which is related to thesum of a parent term and a metabolite term, each ofwhich contain values for the total exposure, freefraction, potency, and dosing interval. The contributionof the N-oxide metabolite was modeled to be greaterthan the parent, and interestingly, based on compar-isons of healthy volunteers and COPD patients, thiscontribution was modeled to be even greater in thepatients. Using exposure values from Böhmer et al.,(2009) during the control phase of a drug interaction

study done at steady-state roflumilast dosing, alongwith values of 0.8 and 2.0 nM for PDE4 inhibition invitro (Sanz et al., 2007), with free fractions yieldsvalues for the two terms of:

roflumilast contribution :AUCp•fu;pIC50;p•t

¼ 35:2 mg � hr=l•0:010:8 nM•24h

•1000 nmol

mmol•mmol400mg

¼ 0:046

roflumilast N-oxide contribution :AUCm•fu;mIC50;m•t

¼ 417 mg � hr=l•0:032:0 nM•24h

•1000 nmol

mmol•mmol416mg

¼ 0:62

thus projecting that the N-oxide metabolite contrib-utes 93% of the activity, provided it can penetrate thetarget tissue equivalently to the parent.

25. Sibutramine. Sibutramine offers an interestingexample of two active metabolites and an instancewherein the parent drug probably contributes littleeffect. It was used as a weight-loss agent, althoughsafety problems led to its removal from clinicalpractice. Its mechanism of action is purported to bevia action on serotonin, norepinenephrine, and possiblydopamine transporters as well, although it does notwork in models of depression in which other drugspossessing these activities work. The most potentbinding is to the norepinephrine transporter, but thedesmethyl and didesmethyl metabolites (Fig. 29)possess 100- and 60-times greater intrinsic potency(Cheetham et al., 1996). Furthermore, the metabolitesare present in circulation at concentrations in the samerange as the parent drug (Kim et al., 2009), and theparent drug is more bound in plasma (fu = 0.03 versus0.06 for both metabolites) (Meridia, 2010). Thus, themetabolites are really driving the in vivo activity.Understanding the relationship among the activeentities can be further confounded by the fact thatsibutramine is administered as a racemate and thatthe metabolism can show quantitative differencesbetween the enantiomers.

26. Tamoxifen. Tamoxifen offers a particularly in-teresting example of active metabolites in that it hadbeen used clinically for many years before it wasuncovered that a secondary metabolite, endoxifen, maycontribute the majority of the antiestrogenic activity.

Fig. 50. Metabolism of levosimendan to OR-1896.

Fig. 51. Metabolism of metoprolol to a-hydroxymetoprolol.

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Endoxifen arises via sequential hydroxylation andN-demethylation reactions that are catalyzed byCYP2D6 and CYP3A, respectively (Fig. 30). CYP2D6generates 4-hydroxytamoxifen, which is also pharma-cologically active. In CYP2D6 poor metabolizers orpatients taking CYP2D6 inhibitors, like paroxetine,the levels of endoxifen will be lower and may reduceefficacy in these patients (Goetz et al., 2005; Jin et al.,2005), although this may not be that clear cut (Nowellet al., 2005; Regan et al., 2012). An examination of theintrinsic potency of the metabolites and circulatingconcentrations would suggest that endoxifen is impor-tant in efficacy. While a side-by-side comparison oftamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen,

and endoxifen has not been reported, assembling thedata from several reports on these compounds suggeststhat 4-hydroxytamoxifen and endoxifen are two ordersof magnitude more potent than their respective non-hydroxylated counterparts (Furr and Jordan, 1984;Johnson et al., 1989, 2004). Endoxifen is present incirculation at approximately 1/10th that of tamoxifen,whereas 4-hydroxytamoxifen is present at about 1/100th that of tamoxifen. Summing these up, andassuming similar free fractions (Bourassa et al., 2011),since affinities for albumin are nearly equivalent, it canbe projected that endoxifen contributes approximately10 times the receptor activity as tamoxifen, whereas thecontributions of 4-hydroxytamoxifen and tamoxifen areabout equal. N-Desmethyltamoxifen, which quantita-tively is the major metabolite present in circulation atconcentrations in the same range as tamoxifen and in20- to 100-fold greater levels than endoxifen and 4-hydroxytamoxifen, would have a contribution similar totamoxifen itself. One final interesting point regardingtamoxifen active metabolites is that a sophisticatedanalytical method was developed whereby an estrogenreceptor binding assay was placed in-line with an HPLCfor the facile detection of active estrogen receptorcompounds, and it can be used to detect activemetabolites (Oosterkamp et al., 1996; Kool et al., 2006).

27. Terfenadine. Terfenadine was the first of thenonsedating antihistamines; however, it was with-drawn from clinical use due to serious cardiac toxicity,particularly when coadministered with CYP3A inhib-itors such as azole antifungals. Terfenadine undergoesa very high first-pass extraction that results in muchhigher exposures to its major carboxylic acid metabo-lite, fexofenadine (Fig. 31), while terfenadine itself isbarely measurable (Lalonde et al., 1996). Fexofenadinehas about 4-fold lower intrinsic potency at the H1

receptor (Anthes et al., 2002); however, due to its veryhigh relative exposure, it contributes extensively to invivo efficacy. Essentially, terfenadine was a prodrug.When terfenadine was removed from clinical use, itwas replaced with fexofenadine. The exposure valuesfor fexofenadine that are efficacious after directadministration are similar to those achieved followingadministration of efficacious doses of terfenadine,lending support to the notion that terfenadineexhibited its effect primarily via the active metabolite.

28. Thiocolchicoside. Thiocolchicoside (Fig. 32) isan unusual derivative of a natural product that hasmuscle-relaxant activity believed to be mediated bybinding to GABA receptors. It possesses a glucosesubstituent that undergoes deglycosylation to thephenol intermediate, which is subsequently glucuroni-dated. After an oral dose that shows efficacy, theglucuronide is present but the glucose-containingparent drug is undetectable (Trellu et al., 2004).Administration of the metabolite directly to ratsyielded the same effect on polysynaptic reflex as the

Fig. 52. Metabolism of metronidazole to hydroxymethylmetronidazole.

Fig. 53. Metabolism of morphine to morphine-6-glucuronide.


same dose of parent drug. Data comparing the in vitroactivity of parent and metabolite was not found, so theimportance of the metabolite to effect in humans mustbe inferred.29. Thioridazine. Thioridazine is an old antipsy-

chotic agent that is still used, but not as much as themore modern antipsychotic agents. It undergoes S-oxidation reactions on the thiomethyl side chain toform mesoridazine (sulfoxide) and sulforidazine (sul-fone) (Fig. 33). Mesoridazine has also been used asa drug itself. Mesoridazine and thioridazine possessaffinity for the dopamine D2, D3, and D4 receptors(Richtand et al., 2007), with mesoridazine showingsomewhat greater affinity. After administration ofthioridazine, total exposure to thioridazine is about6-fold greater than the mesoridazine; however, it hasbeen shown that the parent drug is vastly moreprotein-bound than the metabolite. Values for fu havebeen reported to be 0.0016 and 0.09 for parent andmesoridazine, respectively, in one report (Freedberget al., 1979) and 0.0015 and 0.017 in a second report

(Nyberg et al., 1978); the reasons for the differencereported for mesoridazine are not apparent. However,in either case, it can be estimated that mesoridazinecontributes virtually all of the receptor occupancyrelative to parent. Additionally, the second S-oxidationto mesoridazine yields the other active metabolite,sulforidazine. This metabolite has similar potency andfree fraction as mesoridazine, but it circulates at abouta third the concentration. Nevertheless, it is estimatedthat this metabolite also plays a more important role inefficacy than thioridazine.

30. Tibolone. Tibolone is a steroidal agent that hasactivity at the a-estrogen receptor and the androgenreceptor and is used in osteoperoisis and breast cancerprevention. It undergoes reduction of the 3-keto groupto two stereoisomeric alcohols and isomerization of thedouble bond to the 4-position (Vos et al., 2002; Fig. 34).The alcohols are more potent than parent at theestrogen receptor, while the alkene isomer is morepotent at the androgen receptor (Escande et al., 2009).The 3a-hydroxy isomer is present at more than 9-foldgreater concentrations than tibolone (Timmer et al.,2002) and thus can be estimated to have a 20-foldgreater contribution than tibolone itself, assumingother distributional aspects are similar between parentand metabolite. The 3b isomer is also present atgreater concentrations, but not as much as the otherisomer. The alkene isomer metabolite is estimated tocontribute about twice as much androgen receptorbinding as the parent.

31. Tolterodine. Tolterodine, a muscarinic antago-nist used to treat urinary incontinence and overactivebladder, offers an interesting example whereina marked difference in plasma protein binding proba-bly contributes to the metabolite contributing a greater

Fig. 54. Metabolism of pioglitazone to its hydroxy and ketone metabolites.

Fig. 55. Metabolism of praziquantel to trans-4-hydroxypraziquantel.

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share to the efficacy. Tolterodine is metabolized to 5-hydroxymethyltolterodine (desfesoterodine; Fig. 35) byCYP2D6, as demonstrated by marked differences inexposure to both the parent and the metabolitebetween CYP2D6 EM and PM subjects. CYP2D6 EMsubjects have high metabolite and low parent; vice-versa for CYP2D6 PM subjects (Brynne et al., 1998).Both parent and metabolite bind to the target withsimilar potency (Yono et al., 1999) and have similarexposures in EM subjects; however, the metabolite is10-fold less protein-bound (Pahlman and Gozzi, 1999;Olsson et al., 2001). Thus, it can be extrapolated that inEM subjects, the metabolite contributes the lion’sshare of the activity, while in PM subjects the parentdrug would be the major contributor. Furthermore, itwas shown in animal studies that 5-hydroxymethyl-tolterodine has a lower capability to penetrate thebrain (as assessed using Kp,uu values; Callegari et al.,2011), thus it could be proposed that the metabolitewould have lower propensity to cause side effectscaused by antimuscarinic activity in the brain, such asmemory impairment. The lack of an impact of CYP2D6on the metabolism of 5-hydroxymethyltolterodineserved as a rationale for the development of fesoter-odine, an ester prodrug of the metabolite (Malhotraet al., 2009).32. Tramadol. Tramodol is an opiate used in pain

treatment. It is dosed as a racemate and undergoes O-demethylation to an active metabolite (Fig. 36). The (+)enantiomer of the active metabolite possesses m-opioidreceptor binding that is over 100-fold greater than theparent [as a racemate or the (+)enantiomer] (Lai et al.,

1996). This biotransformation reaction is catalyzed byCYP2D6, thus poor metabolizers do not form appre-ciable amounts of the metabolite and as a consequencedo not experience the same antinociceptive effect(Paulsen et al., 1996). This also suggests that themetabolite may be entirely responsible for action,essentially making tramadol itself a prodrug.

33. Trimethadione. Trimethadione is an old oxazo-lidinedione antiepileptic agent that undergoes N-demethylation to dimethadione (Fig. 37). It offers aninteresting example in that N-demethylation results inthe generation of an acidic 2° imide which substan-tially alters the physicochemical properties of themolecule. At the neuromuscular junction the effect oftrimethadione and dimethadione differ (Alderdice andMcMillan, 1982). By use of frog tissue, the parent drugwas shown to alter the postjunctional sensitivity,whereas the metabolite acts by altering acetylcholinerelease. The concentrations at which these effects aredemonstrated are relevant to those measured in vivofor the metabolite; however, the potency of the parentis not as great and the antiepileptic effects may be duemostly to the metabolite.

34. Venlafaxine. Venlafaxine is an inhibitor of theserotonin reuptake transporter that has been used asan antidepressant. It has an active metabolite, O-desmethylvenlafaxine (Fig. 38) that is also now used asa drug itself (Deecher et al., 2006). Venlafaxine ismetabolized to the metabolite by CYP2D6, thusexposure values differ between CYP2D6 EM and PMsubjects with the former having higher metabolite

Fig. 56. Metabolism of quazepam to 2-oxoquazepam.

Fig. 57. Metabolism of quinidine to 3-hydroxyquinidine. Fig. 58. Metabolism of saxagliptin to 5-hydroxysaxagliptin.


concentrations and the latter having higher parentdrug concentrations (Fukuda et al., 1999). Thus, thecontribution of parent versus metabolite to in vivoefficacy will differ between the two populations.Considering the free plasma levels and in vitroaffinities (Owens et al., 1997), in EM subjects themetabolite can be calculated to contribute almost 4-times the activity relative to parent, whereas for PMsubjects the parent can be estimated to contributeabout twice the activity relative to the metabolite. Bothparent and metabolite have been shown to haveequivalent brain partitioning and impact of P-glycoprotein (in mouse; Karlsson et al., 2010), thus itis not anticipated that different metabolite-versus-parent ratios would alter this relationship. The affinityof both for the norepinephrine transporter is consider-ably less and a low occupancy of this protein would bepredicted from pharmacokinetic data.

B. Drugs with Active Metabolites That ContributeComparably to the Parent

1. Acebutolol. Acebutolol is an antagonist of theb-adrenergic receptor used in the treatment of

hypertension and cardiac arrythmias. It is metabolizedby hydrolysis of a butyl amide group followed byacetylation to diacetolol (Fig. 39), a pharmacologicallyactive metabolite with a longer half-life than theparent (and similarly low plasma protein binding)which permits once-per-day dosing (Coombs et al.,1980; DeBono et al., 1985; Piquette-Miller et al., 1991).Diacetolol has been shown to have activity by directadministration to humans (Ohashi et al., 1981). Whiledata on the in vitro potency at the human receptor arenot reported, considerable exploration of the relativeeffects of acebutolol and diacetolol has been done inanimals and using animal tissues. In a guinea pigcardiac preparation, the metabolite was shown to haveabout 3-fold lower potency (Basil and Jordan, 1982).At early timepoints following oral administration,acebutolol and diacetolol plasma concentrations are

Fig. 59. Metabolism of spironolactone to canrenone.

Fig. 60. Metabolism of triamterene to 4-hydroxtriamterene sulfate.

Fig. 61. Metabolism of verapamil to norverapamil.

Fig. 62. Metabolism of zolmitriptan to N-desmethylzolmitriptan.

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about equal, but at later timepoints diacetolol ispresent at much greater concentrations. Thus, it islikely that the parent contributes more activity at earlytimepoints (up to Tmax ;4 hours) and the metabolitecontributes at the later timepoints.2. Acetohexamide. Acetohexamide is reduced to

hydroxyhexamide (Fig. 40), which is also active atstimulating insulin secretion and lowering bloodglucose. The glucose-lowering effect is better correlatedto the pharmacokinetics of the metabolite, which hasa longer average half-life (4.6 versus 1.3 hours) andlater Tmax (4 hours versus 1) (Smith et al., 1965). Theimportance of knowing that an active metabolite can be

responsible for effect is illustrated by the observationthat coadministration of phenylbutazone results in anenhancement of the effect of acetohexamide, despite nochange in acetohexamide exposure (Field et al., 1967).Hydroxyhexamide is excreted in urine, and phenylbu-tazone is known to affect renal clearance of anionicdrugs; thus it can be proposed that the phenylbutazone-acetohexamide interaction is due to an increase inhydroxyhexamide exposure caused by inhibition of therenal clearance of the metabolite. The activity ofhydroxyhexamide can be rationalized by comparisonwith other members of the sulfonylurea class of drugs.Tolbutamide, chlorpropamide, and tolazemide possess

Fig. 63. Metabolism of alprazolam to 19-hydroxyalprazolam.

Fig. 64. Metabolism of buprenorphine to N-dealkyl and glucuronide metabolites.


similar structures, with a site of structural variationbeing on the 4-position of the central phenyl ring, whichis the site of reduction of acetohexamide to hydroxyhex-amide (Gopalakrishnan et al., 2000) (Fig. 40).3. Amiodarone. Amiodarone is an unusual antiar-

rhythmic agent that putatively has multiple mecha-nisms of action, thus delineating the relative roles ofthe parent drug and its active desethyl metabolite (Fig.41) is challenging. Multiple cation channels areaffected by both parent and metabolite as well asinhibition of the conversion of thyroxine to triiodothy-ronine (Kodama et al., 1997). Plasma concentrations ofamiodarone and desethylamiodarone associated withefficacy are about 2 and 1.5 mM, respectively, and dueto high lipophilicity the plasma protein binding is high(Stäubli et al., 1985; Ujhelyi et al., 1996). In vivostudies suggest that desethylamiodarone has greatereffects in animal models at equivalent total concen-trations and that these effects may be due to a higherfree fraction (Nattel and Talajic, 1988).4. Aripiprazole. Aripiprazole is a dopamine-receptor

partial agonist used in the treatment of variouspsychiatric disorders. It undergoes dehydrogenation ofa dihydroquinolone ring to generate quinolone (Fig. 42).

The metabolite has intrinsic affinity and functionalactivity at the dopamine receptors equivalent to theparent drug (Tadori et al., 2011) and steady-stateconcentrations in patients have been shown to becomparable, with somewhat greater concentrations ofthe metabolite (Molden et al., 2006). Protein binding isvery high for both (Abilify, 2012), making a preciseestimate challenging, but if assumed to be equal to theparent (which is reasonable considering the verymoderate difference in physicochemical properties be-tween the two), it could be estimated that the dehydrometabolite contributes slightly more than the parent.Both are substrates of P-glycoprotein and this may alterestimates of the relative contributions of parent andactive metabolite to efficacy in vivo (Kirschbaum et al.,2010).

5. Carbamazepine. Carbamazepine is used in thetreatment of epilepsy, as well as some other centralnervous system (CNS) disorders. It is oxidized bycytochrome P450 enzymes to the active 10,11-epoxidemetabolite (Fig. 43), which subsequently undergoeshydrolysis to the inactive diol. The mechanism ofaction is believed to arise via blockage of voltage-dependent sodium channels in the brain, and the invitro activity of carbamazepine and the epoxide aresimilar (McLean and Macdonald, 1986). Circulatingconcentrations of the parent exceed those of themetabolite by about 5-fold, while the metabolite hasa free fraction about twice that of the parent.Cerebrospinal fluid (CSF) concentrations have beenmeasured in humans for both, and consistent with thetotal plasma concentrations and free fractions, themetabolite is present at about one-third of the parent(Johannessen et al., 1976), suggesting equilibration offree plasma and CSF levels. Thus, it can be estimatedthat the epoxide metabolite will contribute abouta third of the activity in vivo. Interestingly, in a studyin neuralgia patients, blinded replacement of effectivecarbamazepine therapy with carbamazpine-10,11-epoxide dosing resulted in no alteration in reportedeffect on pain (Bertilsson and Tomson, 1984).

6. Clarithromycin. Clarithromycin is metabolizedby CYP3A to 14-hydroxyclarithromycin, which also hasantibacterial activity (Fig. 44). The plasma concentra-tion of the metabolite is about half of that of the parent

Fig. 65. Metabolism of carisoprodol to meprobamate.

Fig. 66. Metabolism of chloroquine to desethylchloroquine.

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drug (Rodvold, 1999), but its in vitro minimallyinhibitable concentration for Haemophilus influenzaeis also about half that of parent (Hardy et al., 1990).Thus, it can be estimated that the parent andmetabolite contribute equally, depending on the po-tential for differential tissue penetration.7. Clobazam. Clobazam is a benzodiazepine drug

that is used as an anxiolytic and anticonvulsant. Itundergoes N-demethylation (Fig. 45) to a metabolitewith one-fifth of the potency (Onfi Product Label). Themetabolite has a longer t1/2 and accumulates to greaterexposures than the parent (Ochs et al., 1984; Bunet al., 1986). Taking into consideration a 2-fold

difference in free fraction, it can be predicted that themetabolite and parent drug would have approximatelyequal contributions to efficacy.

8. Disopyramide. Disopyramide is a class I anti-arrythmic agent that blocks the cardiac sodiumchannel. It possesses two isopropyl groups on an amineand undergoes N-dealkylation of one of these to theactive metabolite N-desisopropyldisopyramide (Fig.46). The metabolite possesses comparable potency forthe channel, but the free plasma concentrations of themetabolite are about a third of those of the parent (Hillet al., 1988; Sheldon et al., 1994b). [The parent drughas been shown to be subject to saturable plasmaprotein binding (Hinderling et al., 1974), so thecontribution of the metabolite may increase as theparent drug concentration decreases.] An estimate canbe made from these data that the metabolite contrib-utes about a third of the activity of the parent drug(Hinderling and Garrett, 1976).

9. Fluoxetine. Fluoxetine has a major active me-tabolite norfluoxetine (Fig. 47). The half-life of themetabolite is about a week, as compared with theparent drug, which has a half-life of about 2 days(Aronoff et al., 1984). CYP2D6 has been shown to havea role in the conversion of fluoxetine to norfluoxetine,and poor metabolizers have less of the metabolite incirculation (Llerena et al., 2004; Scordo et al., 2005).Also, after repeated dosing, CYP2D6 in EM subjectsbecomes inhibited so that CYP2C9, another enzymeinvolved in fluoxetine metabolism, plays an increasingrole in fluoxetine clearance as shown by comparison ofpharmacokinetics in subjects containing a defectiveCYP2C9 allele (Scordo et al., 2005). Affinity for thehuman serotonin uptake transporter is almost thesame between the parent and metabolite, and concen-trations of the two are also similar (Owens et al., 1997;Scordo et al., 2005). Initially, fluoxetine was approvedas a once-per-day drug—recognition that the norfluox-etine metabolite possesses the same activity whilehaving a longer half-life led to the introduction ofa once-per-week formulation. Brain permeation forboth parent drug and metabolite was high and theresidence time was long with a half-life the same as inplasma (as assessed with 19F NMR for combinedparent and metabolite; Bolo et al., 2000).

10. Itraconazole. The antifungal agent itraconazoleundergoes metabolism to hydroxyitraconazole (Fig.48). In vitro the metabolite possesses similar antifun-gal activity (Mikami et al., 1994; Odds and Bossche,2000). It also circulates in plasma at levels similar toparent (Barone et al., 1998), so it can be estimated thatthe metabolite and parent drug will have a similarcontribution to antifungal activity.

11. Ketamine. Ketamine is an N-methyl-D-alanine–receptor antagonist used as an intravenous anesthetic.It is converted to an N-demethyl metabolite that alsopossesses N-methyl-D-alanine–receptor binding activity

Fig. 67. Metabolism of citalopram to demethyl metabolites.


(Fig. 49) (Ebert et al., 1997). After oral administration,norketamine exposure values are greater than ket-amine values and it could be estimated that themetabolite may contribute almost equally to effect(Hijazi and Boulieu, 2002). However after intravenousadministration, exposures are about equal, and thecontribution to activity would be mostly due to theparent compound (Clements et al., 1982). Ketamine isthus illustrative of the potential impact the route ofadministration may have on metabolite exposures andhence contribution of a metabolite to pharmacologicaleffect. For drugs that undergo high first pass metabo-lism (hepatic and/or intestinal), there can be a largeamount of active metabolite generated following oraladministration. Metabolite/parent ratios can differ, andthis is illustrated by the example of ketamine.12. Levosimendan. Levosimendan is an intrave-

nously administered inotropic agent for heart failurepatients, believed to cause its effect via sensitization oftroponin-C to Ca2+ binding, although the mechanism is

not definitively proven. It possesses a propanedinitrile-substituted hydrazinylidene substituent that is re-moved via reduction of the N–N bond by gut microflorato yield an intermediate aniline metabolite that sub-sequently undergoes N-acetylation to the active me-tabolite OR-1896 (Fig. 50). While the half-life oflevosimendan is short (;1 hour), the active metabolitehas a half-life of a few days, and the beneficial effect isobserved for considerably longer than what the parenthalf-life would indicate, suggesting an important rolefor OR-1896 in the extended duration of activity(Antoniades et al., 2007). In contrast to this, noapparent difference in efficacy has been observedbetween poor and extensive acetylators despite a dif-ference in exposure to OR-1986 between the twogroups (Antila et al., 2004; Kivikko et al., 2011).Delineating the relative contributions of OR-1896 tolevosimendan is not straightforward; the pharmacoki-netics are such that during the intravenous infusion,the parent drug concentrations are far greater than the

Fig. 68. Metabolism of cyclosporine to active hydroxylated metabolites.

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metabolite and cardiovascular effect is observed(Kivikko et al., 2002). Upon cessation of the infusion,levosimendan concentrations drop rapidly. OR-1896concentrations slowly increase to levels about 10% ofthose that were observed for the parent drug duringthe infusion phase, presumably due to biliary secretionof parent, reduction in the gut by microflora, absorp-tion of the reduced intermediate, and acetylation toyield OR-1896. OR-1896 concentrations are measure-able for days, while levosimendan levels are undetect-able. Thus, it may be the case that levosimendanparent is responsible for the hemodynamic effectsduring infusion and the metabolite takes overpostinfusion.13. Metoprolol. Metoprolol is a b-blocker that is

subject to a pharmacokinetic difference in CYP2D6 EMand PM subjects. It undergoes extensive metabolism tomostly inactive metabolites with the exception of thehydroxylation to a-hydroxymetoprolol, an active me-tabolite by CYP2D6 (Kaila and Iisalo, 1993) (Fig. 51).Delineation of the temporal relationship between thepharmacokinetics of metoprolol (short t/1/2, 3–4 hours),a-hydroxymetoprolol (t/1/2, 7–10 hours), and the effecton tremor measured over time in humans suggeststhat the metabolite can contribute between 50–100% of

the activity relative to parent drug (Quarterman et al.,1981; Gengo et al., 1984). It has also been observedthat the pharmacodynamic responses in EM versus PM

Fig. 69. Metabolism of dabigatran to its acyl glucuronide.

Fig. 70. Metabolism of dasatinib to dealkyldasatinib.


subjects do not differ to the extent that the pharma-cokinetic differences in parent drug exposure betweenthese two groups would suggest; i.e., the PK/PDrelationship for metoprolol differs between EM andPM subjects in that EM subjects have lower metoprololconcentrations but the efficacy is not diminished(Jonkers et al., 1991). This also supports the presenceand importance of an active metabolite.14. Metronidazole. The nitroimidazole drug metro-

nidazole is an antibacterial that is metabolized byhydroxylation of the methyl substituent and thismetabolite also has bacteriocidal activity (Fig. 52).The relative activities depend on the species andstrain. For Gardnerella vaginalis, the metabolite hasbeen reported to be 8-fold more efficacious (as de-termined by comparison of minimum inhibitoryconcentration; Ralph and Amatnieks, 1980), whereasfor Bacteroides fragilis the parent is more potent thanmetabolite (Ralph and Amatnieks, 1980; Pendlandet al., 1994). The parent is present at greatercirculating concentrations and neither entity hasappreciable plasma protein binding (Easmon et al.,1982). For G. vaginalis, an estimate that the metab-olite and parent contribute nearly equally can be

made, whereas for B. fragilis it is the parent drug thatshould dominate the efficacy. Fractional inhibitoryconcentration values were calculated using in vitroactivity and in vivo exposure data and were about 0.7for the effect of the metabolite for B. fragilis (Pend-land et al., 1994).

15. Morphine. Morphine represents a fascinatingexample of the potential for an active metabolite tocontribute to the pharmacological activity of the parentdrug because it has been well studied, and the readerinterested in a greater level of detail is directed toa thorough review by Kilpatrick and Smith (2005).Morphine is metabolized by glucuronidation to the 3-and 6-glucuronides (Fig. 53). The 3-glucuronide is themajor circulating metabolite in humans but does notpossess activity at the m-opioid receptor. Morphine-6-glucuronide possesses nearly equivalent in vitro re-ceptor activity (binding and agonism) as the parentdrug. Morphine-6-glucuronide is present in humancirculation at about the same concentrations asmorphine after intravenous administration of thelatter, and it has been estimated that about 10% ofmorphine is cleared by the 6-glucuronidation route(Lotsch et al., 1996). The analgesic effects of morphine-

Fig. 71. Metabolism of diazepam and its metabolites.

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6-glucuronide have been studied directly in humansfollowing intravenous administration of the metaboliteand such data offer the best approach to delineatewhether a metabolite can contribute to activity in vivo.(This represents a rather unique situation as it is rarethat a metabolite is administered directly to humans,unless the metabolite is being tested as a drug itself.)Morphine-6-glucuronide showed good efficacy at stan-dard endpoints of opioid activity, albeit the centraleffects occurred later (6–8 hours), relative to morphineitself (2–3 hours) (Lotsch et al., 2001; Murthy et al.,2002; Skarke et al., 2003). This could be due to eithera slow uptake into human brain or a delayed egress ofthe metabolite from the brain. Based on physicochem-ical attributes, high penetration of glucuronide

metabolites into the brain would be unexpected;however in the case of morphine-6-glucuronide it hasbeen suggested that a folded conformation may beadopted that reduces hydration and increases lip-ophilicity (Gaillard et al., 1994). In later work, thepartitioning of unbound drug into brain (Kp,uu), a metricused to assess the potential for free-plasma and free-brain (hence efficacious) concentrations, showed a valuefor morphine-6-glucuronide that was far lower thanthat for morphine (in rat) (Friden et al., 2009). Animalstudies suggest that drug transport may be involved inmorphine-6-glucuronide brain disposition, but data inhumans are lacking or inconclusive. Furthermore,interpretation can be confounded by the possibilitythat morphine is glucuronidated within the brain, as it

Fig. 72. Metabolism of diltiazem to desmethyl and desacetyl metabolites.

Fig. 73. Metabolism of donepezil to 6-O-desmethyldonepezil.


has been shown in vitro that human brain tissue iscapable of this (Yamada et al., 2003).16. Pioglitazone. Pioglitazone is a peroxisome

proliferator-activated receptor–g agonist useful in thetreatment of diabetes. It is metabolized on the twobenzylic positions adjacent to the pyridine to hydroxyland ketone metabolites (Fig. 54) (Krieter et al., 1994;Actos, 2011). The potencies of these two metaboliteshave been measured in a cellular lipogenesis assay andmouse glucose lowering assay (Sohda et al., 1995;Tanis et al., 1996; Young et al., 1998) and shown to beequivalent to the activity of pioglitazone itself. Proteinbinding is comparable and the metabolites are presentin circulation at concentrations of 10–50% of parent(Kalliokoski et al., 2008). As such, it can be estimatedthat these two metabolites combined can makea considerable contribution to the activity of pioglita-zone, in the range of 40% of the total activity.17. Praziquantel. Praziquantel, a drug to treat

schistosomiasis, is converted to a hydroxyl metabolite(Fig. 55) that could contribute some effect. The in vitropotency of the metabolite at killing the parasite is 60-fold less than parent (Ronketti et al., 2007); however, itis present at over 20-fold greater exposure (Westhoffand Blaschke, 1992; Cioli et al., 1995). If free fractionsare assumed to be nearly equal, it can be estimatedthat the metabolite may contribute about a third of theeffect of the parent drug.18. Quazepam. Quazepam is a benzodiazepine an-

xiolytic with an unusual thioamide structure. Metab-olism results in the replacement of the =S to =O, the 2-oxoquazepam metabolite (Fig. 56). This metabolite has

2- to 3-fold greater potency than the parent (Sieghart,1983) and is present in circulation at about two-thirdsof the parent (Chung et al., 1984). As such, it can beestimated that 2-oxoquazepam contributes substan-tially to the total activity.

19. Quinidine. Quinidine has been used for deca-des in the management of cardiac arrhythmias.Despite this length of use, its molecular mechanismis largely unknown, although it is known to interactwith several ion channels (Na+ and K+). It gives rise tothe active 3-hydroxyquinidine metabolite (Fig. 57).Following oral quinidine administration, concentra-tions of the metabolite are not as great as quinidine;however, it has lower plasma protein binding andthus the free concentrations of quinidine and 3-hydroxyquinidine are about the same (Wooding-Scottet al., 1988). 3-Hydroxyquinidine has been adminis-tered to humans directly, with measurement ofcardiac pharmacodynamic endpoints (Vozeh et al.,1985), which is an excellent way to gather data thatcan be used in estimating the contribution of themetabolite to the parent drug action. When adminis-tered directly, plasma concentrations of 1.5 mM wereshown to yield similar effects to those observed whenquinidine was administered, and 1.5 mM concentra-tions of metabolite are similar to those observed afterquinidine administration. Thus, it can be concludedthat 3-hydroxyquinidine contributes equivalently toquinidine in effect.

20. Saxagliptan. Saxagliptan is an antihyperglyce-mic dipeptidyl peptidase-4 inhibitor for the treatmentof diabetes. It possesses a hydroxyl adamantyl sub-stituent, and metabolic introduction of a second hy-droxyl group yields 5-hydroxysaxagliptan (Fig. 58),which retains about half of the intrinsic potency (Furaet al., 2009). In consideration of the SAR for the parentdrug (Augeri et al., 2005), it is not surprising that theaddition of a second hydroxyl to this substituent doesnot abolish activity. The metabolite circulates atconcentrations about twice that of the parent andneither compound is bound to plasma proteins(Onglyza, 2011), thus the 2-fold difference in potencyand 2-fold difference in exposure essentially canceleach other out and lead to the conclusion that parent

Fig. 74. Metabolism of granisetron to 7-hydroxygranisetron.

Fig. 75. Metabolism of halofantrine to desbutylhalofantrine.

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and metabolite could contribute to in vivo efficacyabout equally, provided that free target tissue concen-trations are similar to free plasma concentrations.21. Spironolactone. Spironolactone is an aldoste-

rone inhibitor used as a diuretic in hypertension. Itpossesses a thioacetate group that is converted to analkene bond in the metabolite canrenone (Fig. 59)(Abshagen et al., 1976). Canrenone itself is used asa drug, as well as an analog in which the lactone ring isopen and dosed as the potassium salt (canrenoate). It isnot entirely clear regarding all the entities’ contribu-tions to activity and whether activity is better relatedto plasma or urinary concentrations; however, canre-none has been estimated to contribute to 73% of theactivity (Ramsay et al., 1977). While spironolactoneand potassium canrenoate yield similar levels ofcanrenone, spironolactone is more potent, suggestingthat other spironolactone metabolites may contributeto activity (Kojima et al., 1985; Peile, 1985). Spirono-lactone had been shown to generate metabolites thatlost the acetyl group but retained the sulfur (Abshagenet al., 1976). A comparison of the concentrations ofspironolactone and canrenone (Dong et al., 2006) showsthat the metabolite is present at about 3-fold excess atmaximum plasma concentration but vastly greaterconcentrations at later timepoints. When correcting forrelative free fractions (Chien et al., 1976) and intrinsicpotencies (Funder et al., 1976), it would suggest that

canrenone contributes a quarter of the activity ofparent. At later timepoints, this contribution wouldincrease.

22. Triamterene. Triamterene is used in the treat-ment of hypertension as a potassium-sparing antihy-pertensive acting at sodium channels. It is frequentlyco-dosed with antihypertensives that cause loss ofpotassium. A major circulating metabolite in humansarises by sequential hydroxylation and sulfationreactions, 4-hydroxytriamterene sulfate (Fig. 60), withconcentrations in excess of 10-times those of the parentdrug (Sörgel et al., 1985). The metabolic reactions arecatalyzed by CYP1A2 and platelet phenol sulfotrans-ferase. Accounting for differences in free fraction andestimates of intrinsic potency (Busch et al., 1996), itcan be estimated that the metabolite contributesapproximately half of the activity of the parent drug.The metabolite has been shown to be active in animalmodels (Voelger, 1991).

23. Verapamil. Verapamil undergoes extensive me-tabolism, including a major N-demethylation pathwaythat yields the active circulating metabolite norverapa-mil (Fig. 61). The metabolite has been shown to be ac-tive in vivo and an estimate of its intrinsic potency at theCa2+ channel places it approximately 5-fold less active(Ferry et al., 1985; Johnson et al., 1991). It circulates ata greater concentration than parent and is slightly lessprotein-bound in plasma (Yong et al., 1980; Powell et al.,1988), thus it can be estimated that it contributes abouta third of the activity of the parent drug.

24. Zolmitriptan. Zolmitriptan is a serotonin re-ceptor agonist used in the treatment of migraine. Itundergoes N-demethylation to yield a secondary aminemetabolite that is claimed to be twice as intrinsicallypotent (Fig. 62) (Jandu et al., 2001). Following oraladministration, the metabolite circulates at about halfthe level of the parent drug (Dixon et al., 1997; Pecket al., 1998), thus it can be projected that thecontributions of zolmitriptan and the N-desmethylmetabolite are about equal.

C. Drugs with Metabolites That Possess TargetPotency But Contribute Little to In Vivo Effect

In some cases, metabolites are identified in labora-tory animals or in vitro systems and are then shownto have some affinity to the receptor targeted by the

Fig. 76. Metabolism of imatinib to N-desmethylimatinib.

Fig. 77. Metabolism of lidocaine to its deethylated metabolites.


parent drug. However, when examined in humans invivo, it is shown that the metabolite is estimated toactually contribute little effect. This can be due to lowin vivo concentrations, higher plasma protein binding,or a lower ability to enter the target tissue.1. Alprazolam. Alprazolam is a highly used anxio-

lytic agent that is cleared by oxidative metabolism andrenal excretion. Metabolism is on the methyl group onthe triazole (19-hydroxyalprazolam) and on the diaze-pine (4-hydroxyalprazolam), with the 19-hydroxyalpra-zolam possessing intrinsic binding potency that isabout 3-fold lower than the parent drug (Richelsonet al., 1991; Fig. 63). The metabolite is present at farlower concentrations in plasma (Smith and Kroboth,1987; Garzone and Kroboth, 1989) and it is thusunlikely to contribute significantly to the pharmaco-logical effects of alprazolam.2. Buprenorphine. Buprenorphine is an opioid

agent that acts as a partial agonist at the m receptorand also binds the k subtype as well. Data regardingthe contribution to activity of the metabolites norbu-prenorphine and buprenorphine 3-glucuronide (Fig.64) have only recently emerged (Brown et al., 2011),

and thus the full picture of data needed to assess therelative contributions of parent and metabolites to invivo effect in humans has not emerged. The drug istypically administered intravenously, sublingually, ortransdermally, and plasma exposure to the metabolitesis less than that for the parent drug (Polettini andHuestis, 2001). Protein binding values have not beenreported for the metabolites. In postmortem brainsamples, it has been shown that buprenorphine ispresent in the brain, but the desmethyl and glucuro-nide metabolites were undetectable (Elkader andSproule, 2005). Furthermore, it has been shown thatnorbuprenorphine is a substrate of P-glycoprotein,which may diminish brain exposure (Tournier et al.,2010). Thus, while buprenorphine metabolites mayshow intrinsic receptor activity, in vivo data suggestthat only the parent drug contributes to the effect.

3. Carisoprodol. Carisoprodol is a sedative andmuscle relaxant that yields an active metabolitemeprobamate, which itself was a drug used as ananxiolytic in the 1960s (Fig. 65). The mechanism ofaction is not entirely clear, although some activity maybe mediated through the GABAA receptor (Gonzalezet al., 2009). The conversion of carisprodol to meprob-mate is partially catalyzed by CYP2C19 (Dalen et al.,1996), an enzyme subject to genetic polymorphism, andthus concentrations of parent drug and metabolitediffer between extensive and poor metabolizers (Olsenet al., 1994). If GABAA is the receptor responsible forthe activity, it can be estimated that meprobamatecontributes about 10% of the activity; less in CYP2C19poor metabolizers.

4. Chloroquine. The antimalarial agent chloroquineis converted to an active N-desethyl metabolite (Fig.66). The metabolite is present in human circulation atabout a third of the concentration of the parent drugfollowing a single dose. The mean residence time forthe metabolite is longer than for the parent drug, soafter repeated administration the metabolite concen-trations will increase more than the parent. Incorpo-rating the differences in free fraction and intrinsicpotency, it can be estimated that the desethyl

Fig. 78. Metabolism of lumefantrine to desbutyllumefantrine.

Fig. 79. Metabolism of macitentan to ACT-132577.

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metabolite contributes about 20% to activity (Ofori-Adjei et al., 1986; Augustijns and Verbeke, 1993;Vippagunta et al., 1999).5. Citalopram. Citalopram is a serotonin selective

reuptake inhibitor widely used as an antidepressant inboth its racemic forms and as the S-isomer alone(escitalopram). It is metabolized via sequential N-demethylation reactions to yield N-desmethylcitalo-pram and didesmethylcitalopram (Fig. 67). The metab-olites have lower affinity for the reuptake transporterthan the parent (6- to 8-fold) (Owens et al., 1997;Deupree et al., 2007) and circulate at lower steady-state concentrations than the parent (Sidhu et al.,1997). Thus, the metabolites contribute very little tothe antidepressant effect of citalopram.6. Cyclosporine. The immunosuppressant cyclo-

sporine undergoes complex metabolism, and consider-able attention has been paid to whether it is importantto measure metabolites during therapeutic monitoringof this drug (Santori et al., 1997; Ozbay et al., 2007).Among the metabolites identified, two hydroxylatedmetabolites possess measureable in vitro activity

(Copeland et al., 1990; Fig. 68). Combining bloodconcentrations (Christians et al., 1991) and in vitropotencies, and assuming similar tissue penetrability ofthe metabolites and cyclosporine [which, consideringthe very minor alteration introduced by metabolisminto such a large molecule, along with little differencebetween plasma binding (Kodobayashi et al., 1995), islikely a reasonable assumption], it can be estimatedthat the two metabolites probably only contribute5–15% to efficacy.

7. Dabigatran. Dabigatran is a thrombin inhibitorintended to prevent deep vein thrombosis. It is orallyadministered as a carbamate ester of an amidine thatyields dabigatran, which is the major entity incirculation. Dabigatran undergoes glucuronidation ofits carboxylic acid group and the resulting 1-O-acylglucuronide (Fig. 69) as well as its rearrangementproducts were all shown to cause prolongation ofthromboplastin time. It was hypothesized that theCOOH group is remote from the amidine group that isessential for target binding, thus alteration of theCOOH, even with a change as large as esterification ofglucuronic acid, would not affect activity (Ebner et al.,2010). However, in humans the circulating concen-trations of glucuronides are less than a tenth of that fordabigatran itself (Blech et al., 2008; Stangier et al.,2008), thus while this represents an interestingexample of a glucuronide conjugate that retains invitro potency, its relevance to in vivo effect is minimal.

8. Dasatinib. Dasatinib is a second-generationkinase inhibitor affecting the Bcr-Abl Src family ofenzymes and is used in the treatment of leukemia. Itundergoes extensive metabolism (including an N-deal-kylation of an ethanol side chain) to yield a metabolitewith equivalent intrinsic potency (Christopher et al.,2008) (Fig. 70). The free fraction of the metabolite isalmost twice as high (Sprycel, 2011); however, thecirculating concentrations of the metabolite are farlower than the parent (Furlong et al., 2012), thus themetabolite likely contributes less than 10% to theactivity.

9. Diazepam, Temazepam, and Oxazepam. Diaze-pam is metabolized to several metabolites via oxidationreactions, and three of these metabolites havebeen used as drugs themselves. Diazepam under-goes N-demethylation and this metabolite is the majorcirculating metabolite, relative to temazepam (ahydroxyl metabolite) and oxazepam (the correspondinghydroxyl metabolite of desmethyldiazepam) (Ghabrialet al., 1986; Rouini et al., 2008) (Fig. 71). All threemetabolites have lower affinity for the benzodiazepinereceptor (4- to 7-fold; Richelson et al., 1991) andcirculate at concentrations much lower than the parentdrug (8- to 50-fold lower). With only small differencesin free fraction, it is unlikely that any of thesemetabolites contribute substantially to the effects ofdiazepam; even when summed the contributions of the

Fig. 80. Metabolism of mexiletine to hydroxylated metabolites.

Fig. 81. Metabolism of mianserin to N-desmethylmianserin.


active metabolites are less than 10% of that of parentdrug. The half-lives for temazepam and oxazepam areshorter than diazepam, indicating formation-rate limi-tation, while the half-life for N-desmethyldiazepam issimilar to that of diazepam.10. Diltiazem. Diltiazem represents an interesting

example of active metabolites. The N-desmethyl anddesacetyl metabolites have been a focus as contributingto activity (Fig. 72). These circulate at concentrationsthat are lower than the parent (Montamat andAbernethy, 1987) and are comparably protein-bound(Boyd et al., 1989). The in vitro potency values of themetabolites relative to the parent drug are nominallylower (Li et al., 1992), thus overall the contribution ofmetabolites to the efficacy of diltiazem is probablyquite low. It is interesting to note that in a single-dosestudy a clockwise hysteresis was observed for diltiazemconcentrations (i.e., decreased effect at the sameconcentrations at a later time), and the authorsproposed the possibility of metabolites that antagonizethe P-R interval–prolongation effect of diltiazem,among other possibilities (Boyd et al., 1989). Such aneffect of the desmethyl and desacetyl metaboliteswould not be consistent with their diminished activityat the Ca2+ channel but would need to be due to analternate target.

11. Donepezil. Donepezil, an acetylcholinesteraseinhibitor used in the treatment of dementia, possessesan active metabolite that arises via O-demethylation ofthe 6-methoxy substituent on the indanone ring (Fig.73). The metabolite is reported as possessing equiva-lent intrinsic potency (Sugimoto et al., 1995); however,when plasma concentrations have been measured, theyare far below those measured for the parent compound(at least 150-fold), thus it is unlikely that 6-O-desmethyldonepezil actually contributes to in vivoefficacy (Okereke et al., 2004; Patel et al., 2008).

12. Granisetron. Granisetron is a serotonin-3 re-ceptor antagonist used as an antiemetic in cancerpatients undergoing treatment with emetogenic che-motherapeutics (Fig. 74). It undergoes hydroxylationon the indazole ring at the 7-position (Clarke et al.,1994) to yield a metabolite with slightly greater targetpotency than the parent drug (Vernekar et al., 2010).After both oral and intravenous administration, themetabolite is present at about 10% of that of the parent(Clarke et al., 1994; Boppana, 1995). Assuming similarfree fractions, it can be estimated that the metabolitewould contribute about 20% of the activity of theparent.

13. Halofantrine. Halofantrine is an antimalarialagent that is not used very much due to QT-intervalprolongation. However, it has a major circulatingmetabolite (Milton et al., 1989; Veenendaal et al.,1991) that possesses some in vitro activity as well,desbutylhalofantrine, in which one of the two butylsubstituents are removed (Fig. 75) (Bhattacharjee andKarle, 1998). While protein binding of desbutylhalo-fantrine has not been reported, if potency and totalconcentrations are compared, the metabolite couldhave about a quarter of the activity.

14. Imatinib. Imitinib is a protein–tyrosine kinaseinhibitor selective for the v-ABL type as well aspotency for the platelet-derived growth factor kinase.

Fig. 82. Metabolism of midazolam to 19-hydroxymidazolam and its glucuronide.

Fig. 83. Metabolism of mirtazepine to N-desmethylmirtazepine.

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It undergoes N-demethylation by CYP3A4 (Fig. 76),and the metabolite circulates at approximately 10–15%of levels of the parent drug. Other attributes (freefraction, intrinsic potency) are the same between theparent and metabolite, thus it can be projected that theparent will dominate the contribution to activity byvirtue of its greater exposure (Kretz et al., 2004; LeCoutre et al., 2004; Gschwind et al., 2005; Peng et al.,2005; Treiber et al., 2008).15. Lidocaine. Lidocaine is an antiarrhythmic

agent that undergoes sequential N-deethylation reac-tions to yield putatively active metabolites monoethyl-glycine xylidide (MEGX) and glycine xylidide (Fig. 77).The metabolites are not as active at sodium channelblock as the parent drug using rat in vitro data(Sheldon et al., 1991); however, after comparison to

free circulating concentrations it can be estimated thatMEGX contributes about a fifth of the activity whileglycine xylidide contributes very little. MEGX is verypoorly bound to plasma proteins so free concentrationsare almost equal to parent and the potency is aboutone-fourth that of parent. Given earlier animal data, ithad been concluded that MEGX might contribute moreto the activity (Drayer et al., 1983).

16. Lumefantrine. The example of lumefantrine isvery similar to that of halofantrine. Similar tohalofantrine, it undergoes N-dealkyation to desbutyl-lumefantrine (Fig. 78). The metabolite has 6-foldgreater in vitro potency at killing Plasmodium falci-parum but is present in circulation of patients un-dergoing successful treatment of malaria at about 1/20th the concentration of the parent compound (Wonget al., 2011). Plasma binding has not been reported, butif assumed to be equal, it can be predicted that themetabolite carries about 30% of the activity of theparent.

17. Macitentan. Macitentan is an endothelin an-tagonist being developed to treat pulmonary arterialhypertension. It has an N-propylsulfonylurea thatundergoes N-depropylation to yield an active metabo-lite that possesses about an eighth of the intrinsicactivity of the parent (Fig. 79) (Iglarz et al., 2008). Inhumans, the metabolite is present at about 50%

Fig. 84. Metabolism of primidone to phenobarbital.

Fig. 85. Metabolism of propafenone to hydroxylated and N-dealkylated metabolites.


greater plasma concentrations (Sidharta et al., 2011).The free fraction is unreported, so making an estimateof the contribution of the metabolite relative to parentsuggests that the metabolite contributes to about 20%of the endothelin inhibition.18. Mexiletine. Mexiletine is a sodium channel

blocker used as an antiarrythmic agent. In humans it isconverted to several hydroxyl metabolites (Paczkowskiet al., 1992), two of which have been shown to have invitro activity (p-hydroxymexiletine and hydroxymethyl-mexiletine) (Fig. 80) (Catalano et al., 2004; DeBelliset al., 2006). While the free fraction for the parent isknown, there is no report of the free fraction for themetabolites, so the comparison on contributions toactivity in vivo must be made based on total concen-trations. Neither metabolite is projected to contributemore than 10% of activity, and even if they are as-sumed to be entirely free, their contributions would notexceed 20% of the parent drug.19. Mianserin. Mianserin is a tetracyclic antide-

pressant that is metabolized to its N-demethyl analog(Fig. 81). It binds many receptors, the a2-adrenergicbeing one of the most important. Upon demethylation,the intrinsic affinity for a2 decreases by about 3-fold(Nickolson et al., 1982). Circulating concentrations ofthe metabolite are about 60% of those of parent (Reiset al., 2009). The free fraction of the parent is available(Kristensen et al., 1985), but for the metabolite it is notreported, so estimation of relative contributions to

activity can only be made assuming that proteinbinding and central penetration are equivalent. Underthese assumptions, and also assuming that the a2-adrenergic receptor is the most relevant for effect, it isestimated that the metabolite contributes about 25% ofthe efficacy of the parent.

20. Midazolam. Midazolam is used as an anestheticagent frequently by the intravenous route. It is initiallymetabolized to 19-hydroxy and 4-hydroxy metabolites(Fig. 82), with the former possessing intrinsic bindingactivity to the benzodiazepine receptor similar to thatof the parent drug (Richelson et al., 1991). Further-more, the glucuronide conjugate of 19-hydroxymidazo-lam may also contribute to activity, based on theobservation of prolonged activity in renal insufficiencypatients who are less able to renally clear thismetabolite (Bauer et al., 1995). Plasma protein bindingof the glucuronide metabolite is considerably lowerthan midazolam; however, the ability of the glucuro-nide metabolite to penetrate the brain relative to theparent is unknown, thus precluding a true assessmentof the relative contributions of parent and metabolite toeffect. The 19-hydroxymidazolam metabolite has beenshown to have activity in humans following its directadministration, which is a highly valuable approach tounderstanding the activity of a metabolite. However, itis infrequently carried out (Mandema et al., 1992). Thisactivity was comparable to midazolam itself. However,the clearance of 19-hydroxymidazolam is high and thus

Fig. 86. Metabolism of propranolol to 4-hydroxypropranolol.

Fig. 87. Metabolism of rifampin to 23-deacetylrifampin.

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exposure to this metabolite after administration ofmidazolam is a lot lower than exposure to midazolamitself. Thus, the contribution of 19-hydroxymidazolamto the sedative/anesthetic properties of midazolam islikely to be low.21. Mirtazapine. Mirtazepine, a close analog of

mianserin described above, also undergoes N-deme-thylation to an active metabolite with reported 3- to 4-fold lower intrinsic potency than the parent drug (Fig.83) (Stimmel et al., 1997). Mirtazepine acts byantagonizing the a2-adrenergic receptor, which resultsin downstream effects on serotonin and norepinephrinerelease (de Boer et al., 1988). It also binds to someserotonin receptors and the H1 receptor. The metabo-lite is present at about half the exposure of the parentdrug (Shams et al., 2004), but the role that themetabolite can play in all of these activities has notbeen described in the literature. For the a2-adrenergicreceptor activity, if it is assumed that the free fraction

does not differ between parent and metabolite andbrain penetration is equal, it would be anticipated thatthe metabolite would contribute less than 20% of theactivity. A more accurate assessment would requirefurther investigation.

22. Primidone. Primidone is a relatively old drugthat is used in the treatment of epilepsy. It is oxidizedto phenobarbital (Fig. 84), a drug itself, but a metabo-lite when primidone is the parent drug. Affinity for theGABA receptor is approximately 2.5-fold greater forprimidone (Ticku and Olsen, 1978), and phenobarbitalconcentrations following oral primidone are lower thanthe parent drug (Martines et al., 1990). Plasma proteinbinding is comparable between the two compounds, soif brain penetration is equivalent it can be estimatedthat phenobarbital contributes about a quarter of theactivity. A second metabolite, phenylethylmalonamideis present in circulation but does not contribute to invivo activity.

23. Propafenone. Propafenone is a sodium channelblocker used in the treatment of cardiac arrhythmias.It is metabolized by the polymorphic enzyme CYP2D6to 5-hydroxypropafenone (Fig. 85), which is an activemetabolite (Zoble et al., 1989). Propafenone is subjectto a supraproportional exposure-dose relationship inCYP2D6 extensive metabolizers that is not shown inpoor metabolizers, consistent with saturation ofCYP2D6-catalyzed first-pass metabolism (Siddoway

Fig. 88. Metabolism of rizatriptan to monodesmethylrizatriptan.

Fig. 89. Metabolism of rosuvastatin to N-desmethylrosuvastatin. Fig. 90. Metabolism of sertraline to N-desmethylsertraline.


et al., 1987). At the higher doses, EM and PMdifferences are about 2-fold and no dose adjustmentis made between the two populations. 5-Hydroxypro-pafenone, which is also subject to nonlinear clearance(Vozeh et al., 1990), has about half the intrinsicpotency as the parent and is present at about 4-foldlower unbound exposure. Thus, it does not contributemuch to the efficacy of propafenone. The N-despropylmetabolite is also weakly active in vitro (Oti-Amoakoet al., 1990) but is present at even lower concentrationsin vivo and therefore would not be expected tocontribute to in vivo effect.24. Propranolol. Propranolol is oxidized to 4-

hydroxypropranolol (Fig. 86) and this metabolite ispresent in human circulation following administrationof propranolol but at 25- to 150-times lower exposures(depending on CYP2D6 genotype) (Raghuram et al.,1984). The metabolite is less potent at the b-adrenergicreceptor (compared using turkey as the source speciesfor receptor; Bilezikian et al., 1978), so it is unlikelythat the metabolite has any substantive contribution toactivity.25. Rifampin. Rifampin (rifampicin) is an antibiotic

drug that has been used for decades to treat tubercu-losis. It undergoes hydrolysis of an acetyl group at the23-position to yield a metabolite that has similarefficacy against Mycobacterium (Fig. 87; Tenconiet al., 1970) and increased potency against many otherbacteria (Nakazawa et al., 1970). After oral adminis-tration, 23-desacetylrifampicin is present in circulationat about 1/12th the concentrations (Loos et al., 1985;Tenconi et al., 1970). Plasma protein binding for the

metabolite is not reported, but if assuming a similarvalue (i.e., 81% bound; Kochansky et al., 2008), then itcan be assumed that the metabolite contributes lessthan 10% to the antibacterial activity in vivo.

26. Rizatriptan. Rizatriptan is an antimigrainedrug that acts by binding serotonergic receptors (5-HT1B and 5-HT1D) with high affinity (Hargreaves,2000). The mono N-desmethyl metabolite (Fig. 88) hasintrinsic potency that is about 3-fold lower. Itscontribution to in vivo efficacy is probably very low ornegligible, since circulating concentrations are aboutone-tenth those of the parent drug (Goldberg et al.,2000). Consideration of any potential free-fractiondifferences are not necessary to understanding theeffect of the metabolite since rizatriptan itself is mostlyfree in plasma (Maxalt, 2011).

27. Rosuvastatin. Rosuvastatin is a widely usedlipid-lowering agent that inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase,with an inhibitory potency greater than other clinicallyused compounds of this class (McTaggart et al., 2001).It undergoes N-demethylation of a sulfonamide nitro-gen to yield N-desmethylrosuvastatin, which isclaimed to have 2- to 6-fold less potency at inhibitingthe enzyme (Crestor, 2012; Fig. 89). It also is present incirculation at concentrations 7-fold less than theparent drug (Schneck et al., 2004). Thus it is projectedto have very little contribution to the activity ofrosuvastatin.

28. Sertraline. Sertraline is a serotonin reuptakeinhibitor widely used in the treatment of depression. Itundergoes N-demethylation to desmethylsertraline(Fig. 90), which has only about 1/50th the bindingaffinity of the parent drug (Owens et al., 1997). Itcirculates at about 1.5-fold greater exposure at steadystate (Ronfeld et al., 1997) but the free fraction isunknown. Because of the considerably lower potency, itis doubtful that desmethylsertraline contributes to invivo efficacy.

29. Triazolam. Similar to midazolam and alprazo-lam described above, triazolam is also metabolized onthe methyl group of the azole ring to the 19-hydroxy

Fig. 91. Metabolism of triazolam to 19-hydroxytriazolam.

Fig. 92. Metabolism of valproic acid to 2-propyl-2(E)-pentenoic acid.

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metabolite (Fig. 91), and also similar to the other twodrugs, this metabolite possesses intrinsic potency thatis approximately 3- to 5-fold lower than the parent(Richelson et al., 1991). However, less is known aboutthis metabolite regarding pharmacodynamics, and thehuman free fraction is not reported. Combining theplasma exposure relative to parent (Eberts et al., 1981;O’Connor-Semmes et al., 2001) and the relative potencyvalues, it can be estimated that 19-hydroxytriazolamwould only possess a minor contribution to the efficacyof triazolam. However, if the relationship between thefree fractions of triazolam and 19-hydroxytriazolamfollow those for midazolam and 19-hydroxymidazolam,then the contribution of 19-hydroxytriazolam to efficacycould be greater.30. Valproic Acid. Valproic acid is an antiepileptic

agent that has been used successfully in the control ofseizures for many years. While its molecular targetmay be ambiguous, studies in systems in vitro ofnerve cell firing suggest that its dehydrogenatedmetabolite trans-2-propyl-2-pentenoic acid (Fig. 92)also possesses this activity at about half of the potency(Löscher, 1981). However, valproic acid concentra-tions are extremely high (;0.1 mM) and considerablyexceed those of this metabolite (Addison et al., 2000).Furthermore, in a unique study wherein human brainsamples were able to be taken from patients on

valproic acid therapy who were also undergoing brainsurgery, it was shown that the brain concentrationswere a lot less than those of valproic acid itself andthat the free fraction for the metabolite was lowerthan that of valproic acid (Adkison et al., 1995). It canbe concluded from these data that it is unlikely thatvalproic acid metabolites actually contribute much tothe efficacy in vivo, despite the in vitro potency shownfor trans-2-propyl-2-pentenoic acid. This exampleillustrates the importance of gathering dispositionaldata for metabolites that are shown to have in vitroactivity.

31. Zopiclone. Zopiclone is a sedative agent activeat the benzodiazepine receptor family. In one paper itwas claimed that the N-oxide metabolite possessedactivity and the N-desmethyl did not (Fernandez et al.,1995). However in receptor binding studies (using rat),the N-oxide was at least two orders of magnitude lesspotent than parent (Trifiletti and Snyder, 1984), andthe desmethyl was one order of magnitude less potent(Fig. 93). Subsequently, a closer examination of the S-enantiomer of the N-desmethyl metabolite showed thatit possesses pharmacological activity by binding betterto GABAA receptors possessing the g2 subunit (Fleck,2002). Nevertheless, after administration of zopiclone,neither the N-oxide nor desmethyl metabolites aredetected in plasma (Fernandez et al., 1993), and

Fig. 93. Metabolism of zopiclone to desmethyl and N-oxide metabolites.


therefore, it is unlikely that any drug-related effect invivo can be attributed to metabolites.

D. Drugs with Metabolites Possessing Activity atRelated Targets

In some instances, changes in structure introducedby metabolism can cause a metabolite to have in-creased affinity at a receptor other than the onetargeted by the parent drug. It is typically the casethat the receptor or enzyme that the metabolite bindsis one among those in a family that the parent binds (e.g., alternate G-protein coupled receptors). This cancause subtle alterations in clinical effect. Examples ofthis are below.1. Amitriptyline and Nortriptyline. Amitriptyline,

a tricyclic antidepressant, is metabolized by N-deme-thylation to nortriptyline, which undergoes furthermetabolism to several hydroxyl metabolites includingE-10-hydroxynortriptyline (Fig. 94). The activity profile

contribution of these entities is complex. Amitriptylineis more potent at the serotonin transporter than thenorepinephrine transporter, whereas the reverse is truefor nortriptyline (Owens et al., 1997). Consideration ofthe affinity and free plasma concentrations associatedwith efficacy with amitriptyline dosing suggests that forthe serotonin transporter, amitriptyline would contrib-ute to about 50% occupancy and norytriptyline aboutanother 10%. For the norepinephrine transporter,nortriptyline free concentrations are about the sameas the potency value, while for amitriptyline the freeconcentrations are about a fifth of the reported affinitysuggesting a similar level of occupancy for the norepi-nephrine transporter with the metabolite contributingthe greater share (Breyer-Pfaff et al., 1982). This is allfurther complicated by the observation that 10-hydrox-ynortriptyline also can contribute to effect (Nordin andBertilsson, 1995). After amitriptyline administration,this metabolite has a concentration that is above its IC50

for the norepinephrine transporter (measured in wholehuman plasma). Relative to amitriptyline, it can beestimated that 10-hydroxynortriptyline contributes al-most twice as much to norepinephrine transport in-hibition than the parent drug, but still less thannortriptyline.

When nortriptyline is administered as the parentdrug the picture is somewhat simpler because theserotonin transporter is no longer of much importance(since that activity was driven by amitriptyline). Usingthe respective in vitro potency values of nortriptylineand the 10-hydroxy metabolite (which were generatedusing human plasma as the matrix) and the in vivoconcentrations after repeated administration (Dahlet al., 1996) suggests that the metabolite contributesto about a third of the activity. 10-Hydroxynortripty-line is generated by CYP2D6 and research on thepolymorphism in this enzyme has shown that there aredifferences among CYP2D6 extensive and poor metab-olizers, with lower concentrations of the metabolite inthe latter. Examination of CSF concentrations haveshown that there may be a partial barrier to brainpenetration of 10-hydroxynortriptyline, since concen-trations are lower than corresponding plasma ultrafil-trate concentrations (Nordin et al., 1985; Bertilssonet al., 1991). Finally, it has been suggested that 10-hydroxynortriptyline could itself be an antidepressantthat would have a better side-effect profile because ithas less activity at muscarinic receptors.

2. Clomipramine. Clomipramine is a tricyclic anti-depressant agent that is metabolized by N-demethyla-tion and hydroxylation to produce three putativelyactive metabolites: norclomipramine, 8-hydroxycli-mipramine, and 8-hydroxynorclomipramine (Fig. 95).Intrinsic potency comparisons have been made for theserotonin and norepeinephrine reuptake transporters,as well as the cholinergic receptors (which may beresponsible for side effects). N-Demethylation seems to

Fig. 94. Metabolism of amitriptyline to nortriptyline and 10-hydroxynortriptyline.

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favor increased norepinephrine transporter potencywhile retention of the N-methyl favors serotonintransporter potency (Thomas and Jones, 1977; Linnoilaet al., 1982; Nunez and Perel, 1995; Agnel et al., 1996;Tatsumi et al., 1997). Plasma concentrations amongthe four entities are relatively comparable (;50–300ng/ml), but unfortunately the lack of free fraction dataon these entities precludes reliable estimations of therelative contributions of each (Bertilsson et al., 1979).If free fractions and CNS penetration are consideredcomparable, it would suggest that norclomipramineand 8-hydroxyclomipramine contribute 10-times theactivity of parent drug at the norepinephrine trans-porter. For the serotonin transporter, norclomipramineis only projected to contribute less than 20% of theparent, while 8-hydroxyclomipramine would be ex-pected to contribute very substantially (about equallyto parent).3. Clozapine. Clozapine is N-demethylated to nor-

clozapine and while both entities possess activity at the5-HT2A and 5-HT2C receptors, they appear to possessdiffering activities at the various dopamine receptorswhich will help to describe the efficacy profile of thedrug as an antipsychotic agent (Fig. 96). Norclozapine

and clozapine circulate at about the same free plasmaconcentrations, albeit the total concentrations and freefractions differ somewhat (Schaber et al., 1998).Clozapine binds most tightly to the D4 receptor andis an antagonist, while it also binds with 10-fold loweraffinity to D2 and D3 receptors, where it is an inverseagonist (Burstein et al., 2005). Norclozapine binds withsimilar affinity to D2 and D3 as clozapine but isa partial agonist at these two receptors. It binds to D4

Fig. 95. Metabolism of clomipramine by N-demethylation and hydroxylation.

Fig. 96. Metabolism of clozapine to norclozapine.


as well but with ten-fold lower affinity and is anantagonist. This offers a truly confusing picture re-garding what may be happening in vivo. For the 5-HT2

activity, the contribution of the parent and metabolite

to occupancy and activity can be considered morestraightforward and it is estimated that the norcloza-pine metabolite contributes about two-thirds of theactivity (Kuoppamaki et al., 1993). But for thedopamine receptors, with the varying affinities andfunctional activities of the two circulating entities, it ismuch more difficult to claim what is actually occurring.

4. Doxepin. Doxepin is an unusual agent in that theparent and N-desmethyl metabolite appear to possessvery different pharmacological activities (Mundo et al.,1974). Doxepin is a potent antihistamine that readilypenetrates the brain and thus causes sedation and isindicated for this purpose at low doses (Silenor, 2010).N-Desmethyldoxepin (Fig. 97) binds to the norepineph-rine transporter and thus has antidepressant proper-ties like tricyclic antidepressants. High doses areindicated for antidepressant activity. An estimationof relative norepineprine transporter occupancies forparent and metabolite suggest that the metabolite hasnearly seven-times the activity.

5. Imipramine and Desipramine. Like the tricyclicantidepressant amitriptyline and its metabolites nor-triptyline and hydroxynortriptyline, a similar situationexists for imipramine and its metabolites desipramineand 2-hydroxydesipramine (Fig. 98). Imipramine hasactivities at both the serotonin and norepeinephrinetransporters with greater affinity for the serotonintransporter while desipramine and 2-hydroxydesipr-amine have greater affinities for the norepinephrinetransporter (Owens et al., 1997). Thus, a complexpicture emerges regarding the molecular activitiesoccurring over the time course of drug and metaboliteexposures as well as among different individuals(Caccia and Garattini, 1990). Following administrationof imipramine, it can be estimated that the norephi-nephrine transporter is almost completely occupied bydesipramine (free plasma concentration/IC50 ratio of~20) (Borga et al., 1969; Sutfin et al., 1988; Szymura-Oleksiak et al., 2001). 2-Hydroxydesipramine concen-trations are approximately half of those of the parentand is approximately 3-fold more potent at thenorepeinephrine transporter than imipramine suggest-ing that it also can contribute more to the occupancy ofthat protein (Javaid et al., 1979; Nelson et al., 1983).Imipramine would be predicted to drive the serotonintransport inhibition since its potency is far greater atthat target relative to the two metabolites; however,because of the higher free concentrations of desipra-mine it can also contribute appreciably to this activity.

When desipramine itself is dosed as the parent drug,the activity is projected to be mostly at the norepei-nephrine transporter, with contributions from desipra-mine and the hydroxyl metabolite. Based oncomparative in vitro potency values and free concen-tration values (Cooke et al., 1984), it can be estimatedthat 2-hydroxydesipramine has about 40% of the invivo potency. This must be interpreted with some

Fig. 97. Metabolism of doxepin to N-desmethyldoxepin.

Fig. 98. Metabolism of imipramine to desipramine and 2-hydroxydesipramine.

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caution since it is based on potency values measured inrat brain. Finally, since desipramine is converted to theactive 2-hydroxdesipramine metabolite by CYP2D6,there are differences between EM and PM subjects inexposure to desipramine and its metabolite (Shimodaet al., 2000).6. Loxapine and Amoxapine. The antipsychotic

agent loxapine, and its N-desmethyl metabolite amox-apine (which is also used as a drug itself) posesa complex picture of the contribution of parent drugand metabolites to pharmacological activity. Loxapineundergoes N-demethylation to amoxapine as well ashydroxylation to 7-hydroxyloxapine and 8-hydroxylox-apine. Amoxapine also undergoes these same hydrox-ylations to the 7- and 8-hydroxyamoxapine metabolites(Fig. 99). The drugs and their metabolites bind to 5-HT2A and D2 receptors with varying potencies, as well

as other CNS receptors to lesser extents (Ereshefsky,1999). Following administration of loxapine, concen-trations of parent drug, amoxapine, 7-hydroxyloxapine,and 8-hydroxyloxapine are approximately 51, 11, 32,and 180 nM, respectively (Cheung et al., 1991). Amongthe four entities, 7-hydroxyloxapine has the greatestpotency and can be projected to deliver 3-times the 5-HT2A receptor activity as loxapine and 11-times the D2

receptor activity. 8-Hydroxyloxapine is less potent, buthas greater concentrations and can be projected tohave 70% of the activity at 5-HT2A receptors and 1.5-fold the activity at D2 receptors. Amoxapine hassimilar potency as loxapine at these two receptors,and as it circulates at about one-third of the levels, itcan be estimated to have one-third the activity.

Administration of amoxapine yields the correspond-ing 7- and 8-hydroxy metabolites. 7-Hydroxyamoxapine

Fig. 99. Metabolism of loxapine and amoxpine.


is more potent than amoxapine at both the 5-HT2A andD2 receptors (Ereshefsky, 1999) and is present incirculation at about half the concentration (Takeuchiet al., 1993). From this, it can be estimated that 7-hydroxyamoxapine contributes to 5-HT2A occupancy atabout an equal amount as parent but is the dominant

contributor to D2 occupancy. 7-Hydroxyamoxapine hasbeen shown to readily partition into brain tissue (in rat),but CSF concentrations of it and parent drug are low(Wong et al., 2012). The 8-hydroxyamoxapine metabo-lite is present in greater abundance, but is 10- to 20-foldless potent at both receptors and therefore contributes

Fig. 100. Metabolism of nefazodone to hydroxynefazodone, mCPP, and its triazolodione metabolites.

Fig. 101. Metabolism of atorvastatin to hydroxy metabolites.

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about a third of the activity of parent. This contributionlikely increases with multiple dosing, since the t1/2 of 8-hydroxyamoxapine is much longer than that of amox-apine or 7-hydroxyamoxapine (Calvo et al., 1985).7. Nefazodone. The antidepressant nefazodone is

metabolized to three active metabolites, hydroxylationon the ethyl side chain (hydroxynefazodone), whichundergoes further oxidative metabolism to lose theethyl group altogether (triazolodione metabolite), andN-dealkylation to yield mCPP (Fig. 100). Nefazodoneand its hydroxyl metabolite have very similar receptorbinding profiles with the most potent binding occurringat the 5-HT2A receptor (Taylor et al., 1995; Owenset al., 1997). The triazolodione metabolite appears tohave far weaker activity. The mCPP metabolite hasa different receptor binding profile, is more potent at 5-HT1A and is an agonist. While plasma protein bindingis very high for nefazodone (fu = 0.009), data are notavailable for the metabolites, so estimations of contri-bution to activity relative to parent drug must be made

assuming that these are similar, which is reasonablefor the hydroxynefazodone metabolite, but likely notreasonable for mCPP. For hydroxynefazodone, it can beestimated that it has approximately ;25% of theactivity of nefazodone itself. Protein binding data formCPP is available in rat (fu = 0.7), and if the value inhuman is similar, it would suggest that mCPP couldhave profound activity in humans [maximum plasmaconcentration (Cmax) = 0.5 mM; Barbhaiya et al., 1996]including activity at serotonin and norepinephrinereuptake transporters (Owens et al., 1997).

E. Drugs That Generate Active Metabolites butAssessment of In Vivo Contribution Is Ambiguous

1. Atorvastatin. Atorvastatin is a very commonlyused lipid lowering agent for the prevention ofatherosclerotic disease. It is metabolized by CYP3Ato two metabolites claimed to be active, 2- and 4-hydroxyatorvastatin (Fig. 101); however actual inhibi-tion potencies for the HMGCoA reductase target arenot available in the scientific literature. In the productlabel for atorvastatin, it is claimed that 70% of theactivity is attributable to metabolites (Lipitor, 2012).However, examinations of the relationship betweenatorvastatin efficacy and various CYP3A polymor-phisms that could cause alterations in parent andmetabolite ratios have yielded mixed results (Kivistöet al., 2004; Gao et al., 2008; Li et al., 2011). It isimportant to note that since the target tissue forinhibition of HMGCoA reductase is the liver withinwhich the active metabolites are generated and that ithas been shown that atorvastatin and its metabolitesare substrates for hepatic uptake transporters, it maybe difficult and even inappropriate to relate systemic

Fig. 102. Metabolism of bromhexine to ambroxol.

Fig. 103. Metabolism of bupropion to hydroxybupropion and stereoisomeric dihydrobupropion metabolites.


circulating concentrations of atorvastatin and its activemetabolites to effect.2. Bromhexine. Bromhexine is a relatively old

agent that has been used as an expectorant, to helpmake mucus less viscous and more easily expectorated.Its active metabolite that arises from N-demethylationand hydroxylation, ambroxol, has also been used inthat manner (Fig. 102). These agents could find use inthe treatment of serious respiratory disorders such ascystic fibrosis or COPD (Malerba and Ragnoli, 2008).When bromhexine is administered orally, the systemicexposure to ambroxol is much lower (;8-fold; Liu et al.,2010), but since the true molecular action of theseagents is not entirely known, it cannot be ascertainedwhether bromhexine or ambroxol, or both, contributeto activity. Ambroxol has also been associated witha reduction in throat pain, which has triggeredinvestigation into the activity of it and bromhexine atsodium channels (such as NaV1.8 and the tetrodotoxinsensitive channel; Leffler et al., 2010).3. Bupropion. Bupropion (Fig. 103) represents an

interesting challenge when attempting to discernwhether the metabolites are relevant to pharmacolog-ical effect. In the product labels for Wellbutrin andZyban (two products containing bupropion), it is statedthat the hydroxyl, and the two reduced metabolites are

active (Wellbutrin, 2011). However, even for bupropionitself, it is not entirely clear as to the mechanism ofaction as an antidepressant or smoking-cessation aid.Bupropion does interact with dopamine and norepi-nephrine transporters, with somewhat greater affinityto the former (Damaj et al., 2004). The CSF concen-tration of bupropion is about 40% of that in plasma,which is slightly higher than what would be projectedwhen comparing to plasma protein binding (fu = 0.16)(Golden et al., 1988). The CSF concentration is,however, far lower than the affinity (by ~20-fold),casting some doubt on whether bupropion is the activeentity and whether the dopamine transporter is thetarget. The same goes for hydroxybupropion, althoughthe affinity is only 4-fold lower than CSF concentra-tions. Affinity for the reduced metabolites is notpublished, but as the concentration of threohydrobu-propion in CSF is nearly 1 mM and if its affinity for thedopamine transporter is similar to that of bupropion, itis possible that this metabolite carries the majority ofthe in vivo activity. However, direct administration toanimals has suggested that bupropion itself andperhaps the hydroxyl metabolite are actually moreactive than the reduced metabolites threo- and

Fig. 104. Metabolism of etretinate to acitretin.

Fig. 105. Metabolism ivabradine to N-desmethylivabradine.Fig. 106. Metabolism of mycophenolic acid to its glucuronidemetabolite.

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erythrohydrobupropion (Butz et al., 1982; Martin et al.,1990). Furthermore, plasma concentrations of themetabolites were shown to be higher in nonresponderpatients suffering from bipolar disorder (Golden et al.,1988).4. Etretinate. Etretinate is a drug no longer used in

the treatment of psoriasis that had an active metab-olite acitretin that arises via hydrolysis of the ethylester (Fig. 104). Etretinate had a very long half-life,due presumably to sequestration and slow release fromdeep tissue compartments. The teratogenic effect ofretinoids with this long half-life made etretinateundesirable for theapy. Acitretin shows a formationrate–limiting half-life when generated from etretinate,and a much shorter half-life when given directly. Theshorter half-life is more amenable for therapy, in lightof the teratogenic effect. The molecular mechanism(i.e., target binding protein) is unknown (Saurat, 1999),so delineating the relative contributions of etretinateand acitretin to activity when etretinate is adminis-tered is challenging. With etretinate dosing, acitretinachieves concentrations of ;150 ng/ml, whereas etre-tinate is present at 4- to 5-fold higher concentrations(Larsen et al., 1988). The free fraction of the ester isalso considerably greater (Urien et al., 1992; Soriatane,2011). When acitretin is dosed directly, higher concen-trations are observed (Pilkington and Brogden, 1992)than when generated from etretinate. This suggeststhat etretinate also likely had some contribution toactivity when administered directly. Finally, thegeneration of etretinate by esterification in vivo hasalso been observed following acitretin dosing, whicheven further complicates the understanding of relativecontributions to effect (Lambert et al., 1992).5. Ivabradine. Ivabradine is a drug used in the

treatment of angina which undergoes demethylation toan active metabolite (Fig. 105). The delineation of theparent drug and metabolite to effect was made usingpopulation PK/PD modeling after oral and intravenousadministration, because the pharmacological proper-ties of the metabolite are not published (Ragueneauet al., 1998). The metabolite circulates at much lowerconcentrations than parent. These investigators pro-posed an indirect PK/PD model wherein the metabolitecontributed to effect at the earlier times followingdosing and a sustained effect contributed by the parentdrug.6. Mycophenolic Acid. The immunosuppressive

agent mycophenolic acid is used to reduce rejection oftransplanted organs. It is metabolized by glucuronida-tion to a phenol glucuronide and an acyl glucuronide(Fig. 106). The latter had been tested in in vitro assaysthat model immunosuppression and shown to havepotency about half that of the parent drug (Shipkovaet al., 2001); however, such a determination ischallenged by the observation that the acyl glucuronideis converted partially to the parent drug under the

conditions used to measure intrinsic activity. In vivo,the acyl glucuronide circulates at a far lower level thanthe parent drug (Shipkova et al., 2002). To considerwhether this metabolite can contribute to in vivoactivity, the free fraction needs to be assessed forparent and metabolite. A protein binding value for theparent is known at approximately 98% bound. How-ever, the acyl glucuronide forms an irreversible co-valent bond with albumin, which makes measurementof the free fraction technically challenging to obtain. Itis thus not presently possible to ascribe relativeparticipation of the parent drug and acyl glucuronidein the pharmacological effect.

V. Conclusions

In drug research, it is clearly the case that thepotential for pharmacologically active metabolitesmust be considered. Knowledge of the properties ofactive metabolites, i.e., intrinsic potency, functionalactivity, pharmacokinetics, clearance mechanism andrate, free fraction, and membrane permeability arenecessary to understand concentration-effect relation-ships in humans as well as interpatient variability inresponse. The occurrence of active metabolites amongdrugs is frequent, consistent with the notion that smallalterations in chemical structure of a drug will notnecessarily alter its biologic properties. In the researchof new drug candidates, the early identification ofactive metabolites is of critical importance in de-veloping optimal clinical study designs and correctdata interpretation. Proactively hunting for activemetaboilites in ex vivo and in vitro samples is a soundstrategy in drug research. Finally, active metabolitesmay possess advantages as drugs themselves (i.e., greaterpotency/efficacy, superior dispositional properties, im-proved safety profile), and there are many examples ofdrugs used in clinical practice that were originallyobserved as metabolites of other drugs.

Acknowledgments

The author greatly appreciates Dr. Doug Spracklin for criticalevaluation of this manuscript.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Obach.

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