Download Pharmacologically Active Drug Metabolites: Impact on Drug

1521-0081/65/2/578–640$25.00
PHARMACOLOGICAL REVIEWS
Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/pr.111.005439
Pharmacol Rev 65:578–640, April 2013
ASSOCIATE EDITOR: TIMOTHY A. ESBENSHADE
Pharmacologically Active Drug Metabolites: Impact
on Drug Discovery and Pharmacotherapy
R. Scott Obach
Pfizer Inc., Groton, Connecticut
Address correspondence to: Dr. R. Scott Obach, Pfizer Inc., Eastern Point Rd., Groton, CT 06340. E-mail: [email protected]
dx.doi.org/10.1124/pr.111.005439.
578
Downloaded from by guest on May 13, 2017
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
II. Absorption, Distribution, Metabolism, and Excretion Aspects of Active Metabolites . . . . . . . . . . 585
A. Metabolic Reactions That Can Yield Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
B. Pharmacokinetic Aspects of Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
C. Distributional Aspects of Active Metabolites in Relation to Contribution to
Pharmacological Effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
III. Active Metabolites in Drug Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
A. Research Tools in Metabolite Identification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
B. Identification of Active Metabolites in Preclinical Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
C. Identification of Active Metabolites in Clinical Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
D. Data Needed for Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
IV. Drugs with Active Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
A. Drugs with Active Metabolites That Dominate the Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
1. Albendazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
2. Allopurinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
3. Artesunate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
4. Astemizole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
5. Buspirone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
6. Chlordiazepoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
7. Clomiphene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
8. Codeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
9. Dihydroergotamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
10. Diphenoxylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
11. Dolasetron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
12. Ebastine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
13. Flutamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
14. Hydroxyzine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
15. Irinotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
16. Loratadine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
17. Losartan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
18. Oxcarbazepine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
19. Oxybutynin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
20. Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
21. Propoxyphene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
22. Remacemide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
23. Risperidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
24. Roflumilast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
25. Sibutramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
26. Tamoxifen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
27. Terfenadine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Pharmacologically Active Metabolites
579
28. Thiocolchicoside. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
29. Thioridazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
30. Tibolone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
31. Tolterodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
32. Tramadol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
33. Trimethadione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
34. Venlafaxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
B. Drugs with Active Metabolites That Contribute Comparably to the Parent . . . . . . . . . . . . . . . . 608
1. Acebutolol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
2. Acetohexamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
3. Amiodarone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
4. Aripiprazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
5. Carbamazepine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
6. Clarithromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
7. Clobazam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
8. Disopyramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
9. Fluoxetine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
10. Itraconazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
11. Ketamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
12. Levosimendan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
13. Metoprolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
14. Metronidazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
15. Morphine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
16. Pioglitazone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
17. Praziquantel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
18. Quazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
19. Quinidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
20. Saxagliptan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
21. Spironolactone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
22. Triamterene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
23. Verapamil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
24. Zolmitriptan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
C. Drugs with Metabolites That Possess Target Potency But Contribute Little
to In Vivo Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
1. Alprazolam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
2. Buprenorphine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
3. Carisoprodol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
4. Chloroquine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
5. Citalopram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
6. Cyclosporine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
7. Dabigatran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
8. Dasatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
9. Diazepam, Temazepam, and Oxazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
10. Diltiazem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
11. Donepezil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
12. Granisetron. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
13. Halofantrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
14. Imatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
15. Lidocaine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
16. Lumefantrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
17. Macitentan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
ABBREVIATIONS: CNS, central nervous system; COPD, chronic obstructive pulmonary disease; CSF, cerebrospinal fluid; EM, extensive
metabolizer; fu, fraction unbound in plasma; HMGCoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HPLC-MS, high pressure liquid
chromatography-mass spectrometry; 5-HT, serotonin; IC50, ligand concentration yielding 50% of the maximum response; Kp,uu, ratio of
unbound drug concentration within a tissue to unbound plasma concentration; mCPP, m-chlorophenyl piperazine; MEGX, monoethylglycine
xylidide; 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.
580
Obach
18. Mexiletine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
19. Mianserin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
20. Midazolam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
21. Mirtazapine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
22. Primidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
23. Propafenone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
24. Propranolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
25. Rifampin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
26. Rizatriptan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
27. Rosuvastatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
28. Sertraline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
29. Triazolam. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
30. Valproic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
31. Zopiclone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
D. Drugs with Metabolites Possessing Activity at Related Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
1. Amitriptyline and Nortriptyline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
2. Clomipramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
3. Clozapine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
4. Doxepin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
5. Imipramine and Desipramine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
6. Loxapine and Amoxapine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
7. Nefazodone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
E. Drugs That Generate Active Metabolites but Assessment of In Vivo
Contribution Is Ambiguous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
1. Atorvastatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
2. Bromhexine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
3. Bupropion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
4. Etretinate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
5. Ivabradine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
6. Mycophenolic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
Abstract——Metabolism represents the most prevalent mechanism for drug clearance. Many drugs are
converted to metabolites that can retain the intrinsic
affinity of the parent drug for the pharmacological
target. Drug metabolism redox reactions such as
heteroatom dealkylations, hydroxylations, heteroatom
oxygenations, reductions, and dehydrogenations can
yield active metabolites, and in rare cases even
conjugation reactions can yield an active metabolite.
To understand the contribution of an active metabolite
to efficacy relative to the contribution of the parent
drug, the target affinity, functional activity, plasma
protein binding, membrane permeability, and pharmacokinetics of the active metabolite and parent drug
must be known. Underlying pharmacokinetic principles and clearance concepts are used to describe the
dispositional behavior of metabolites in vivo. A
method to rapidly identify active metabolites in drug
research is described. Finally, over 100 examples of
drugs with active metabolites are discussed with
regard to the importance of the metabolite(s) in
efficacy and safety.
I. Introduction
bind to the target macromolecule are greatly diminished or abolished altogether. However, in some
cases a metabolite can retain enough intrinsic activity
at the target receptor such that it can contribute to
the in vivo pharmacological effect(s) to a meaningful
extent. (The term “intrinsic” is used throughout this
paper to define the binding to the target in the
absence of any of the other factors that occur in a more
complex system such as a whole organism, tissue, or
in vitro cellular assay.) Since active metabolites
contribute to effect, they must be understood to the
Drugs are cleared from the human body by three
main mechanisms: renal excretion of unchanged drug,
biliary excretion of unchanged drug followed by
egestion in feces, and metabolism (there are also
other rarer mechanisms of clearance). In the case of
metabolic clearance, a drug is converted to new
chemicals, i.e., metabolites. In the vast majority of
cases, metabolites have undergone enough chemical
change from the parent drug that their capabilities to
Pharmacologically Active Metabolites
581
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 and
HD refer to hydrogen bond acceptor and donors, respectively.) In case (B), the metabolic modification, represented by the triangle, does not affect these
interactions, so the metabolite would be active. In case (C) the metabolic modification occurs on a position not involved in binding, so the metabolite
would be active. In (D), the metabolic modification is on a position that disrupts interaction, so the metabolite would be inactive. Such disruptions can
include introduction of a polar or ionic substituent, or a substituent that provides steric bulk.
same extent as the parent drug with regard to their
dispositional properties (pharmacokinetics, distribution,
and clearance mechanisms). A metabolite that possesses intrinsic activity at the target receptor in an in
vitro 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 and
extent of its generation, rate of its subsequent clearance,
target tissue penetration, and free fraction, must be
understood for the metabolite as much as it is for the
parent drug to ascertain whether the metabolite is
important. This is critical in defining the proper dose
needed for efficacy and the causes of interindividual
variability in the dose-concentration-effect relationship.
This is distinct from the case of prodrugs. In the
case of prodrugs, the parent drug is not pharmacologically active itself, but it is converted into a drug,
which essentially is an “active metabolite” when
administered in this fashion. The metabolite possesses all the pharmacological activity, the parent
none. Prodrugs are usually designed to address
some dispositional flaw of an intrinsically active
compound (e.g., poor absorption, short half-life). Some
drugs that have active metabolites could be considered
“accidental prodrugs.” Several older drugs that were
discovered through the use of animal models, and not
through the use of biochemical screens, showed activity
by virtue of a biochemically inert dosed compound
being converted into an active metabolite that exhibited
all the effect. Minoxidil could be considered an example
of this, wherein its hypotensive effect was described in
Fig. 2. Example illustrating formation rate–limiting and elimination
rate–limiting kinetics for metabolites. In this example, plasma concentrations of the parent drug and two metabolites are measured following
intravenous administration of parent drug. Metabolite 1 demonstrates
formation rate–limiting kinetics since its t1/2 is the same as the parent
drug. Metabolite 2 demonstrates elimination rate–limiting kinetics
because its t1/2 is longer than that of the parent drug.
582
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TABLE 1
Comparison of plasma protein binding values for metabolites and their corresponding parent drugs
Parent Drug
Metabolite
fu
(Parent Drug)
fu
(Metabolite)
Acebutolol
Albendazole
Amitriptyline
Amitriptyline
Artesunate
Carbamazepine
Carisoprodol
Chlordiazepoxide
Chloroquine
Citalopram
Citalopram
Clobazam
Clozapine
Codeine
Dasatinib
Desipramine
Desloratadine
Diazepam
Diazepam
Diazepam
Diltiazem
Diltiazem
Disopyramide
Doxepin
Flutamide
Hydroxyzine
Imatinib
Imipramine
Imipramine
Ketamine
Levosimendan
Lidocaine
Lidocaine
Loratadine
Losartan
Loxapine
Meperidine
Midazolam
Midazolam
Morphine
Nortriptyline
Oxcarbazepine
Oxybutynin
Pioglitazone
Pioglitazone
Primidone
Procainamide
Propafenone
Propoxyphene
Quinidine
Risperidone
Roflumilast
Sibutramine
Sibutramine
Spironolactone
Terfenadine
Thioridazine
Thioridazine
Tolterodine
Triamterene
Trimethadione
Valproic acid
Venlafaxine
Verapamil
Diacetolol
Albendazole sulfoxide
Nortriptyline
E-10-Hydroxynortriptyline
Dihydroartimisinin
Carbamazepine-10,11-epoxide
Meprobamate
Demoxepam
Desethylchloroquine
Desmethylcitalopram
Didesmethylcitalopram
N-Desemethylclobazam
Norclozapine
Morphine
N-Dealkyldasatinib
2-Hydroxydesipramine
3-Hydroxydesloratadine
Desmethyldiazepam
Temazepam
Oxazepam
Desacetyldiltiazem
N-Desmethyldiltiazem
N-Desisopropyldisopyramide
Desmethyldoxepin
2-Hydroxyflutamide
Cetirizine
N-Desmethylimatinib
Desipramine
2-Hydroxydesipramine
Norketamine
Or-1896
Monoethyl glycine xylidide (MEGX)
Glycine xylidide (GX)
Desloratadine
Exp3174
Amoxapine
Normeperidine
19-Hydroxymidazolam
19-Hydroxymidazolam glucuronide
Morphine-6-glucuronide
10-Hydroxynortriptyline
10-Hydroxycarbamazepine
Desethyloxybutynin
Ketopioglitazone
Hydroxypioglitazone
Phenobarbital
N-Acetylprocainamide
5-Hydroxypropafenone
Norpropoxyphene
3-Hydroxyquinidine
Paliperidone
Roflumilast N-oxide
Desmethylsibutramine
Didesmethylsibutramine
Canrenone
Fexofenadine
Sulforidazine
Mesoridazine
Desfesoterodine
4-Hydroxytriamterene sulfate
Dimethadione
2-Propyl-2-Pentenoic acid
Desvenlafaxine
Norverapamil
0.88
0.10
0.05
0.05
0.25
0.14
0.42
0.07
0.42
0.20
0.20
0.15
0.06
0.93
0.04
0.10
0.15
0.023
0.023
0.023
0.25
0.25
0.35–0.95
0.20
0.05
0.07
0.05
0.03
0.03
0.40
0.02
0.45
0.45
0.03
0.013
NA
0.56
0.02
0.02
0.74
0.08
0.41
0.0034
0.016
0.016
0.70
0.84
0.08
0.24
0.13
0.10
0.01
0.03
0.03
0.06
0.05
0.0016
0.0016
0.037
0.39
0.91
0.10
0.73
0.10
0.92
0.35
0.08
0.31
0.18
0.33
0.76
0.22
0.60
0.20
0.20
0.30
0.10
0.74
0.07
0.20
0.13
0.03
0.038
0.04
0.23
0.32
0.90–0.95
0.21
0.07
0.07
0.05
0.11
0.20
0.50
0.60
0.86
0.95
0.15
0.002
0.10
0.66
0.11
0.57
0.89
0.31
0.61
0.0018
0.02
0.02
0.72
0.90
0.12
0.26
0.40
0.23
0.03
0.06
0.06
0.02
0.35
0.017
0.017–0.09
0.36
0.10
0.94
0.007
0.70
0.13
animals (Pluss et al., 1972) but the effect in humans
would be due entirely to the generation of minoxidil
sulfate, a potent ligand of the ATP-dependent potassium
channel (Buhl, et al., 1990; Messenger and Rundegren,
2004). A discussion of prodrugs and their active metabolites is beyond the scope of this article.
Over the past decade a great deal of attention has
been focused on the assessment of the safety of drug
metabolites (the so-called “MIST” or Metabolites in
Safety Testing issue; Baillie et al., 2002). In this issue,
the focus was on whether laboratory animals used in
safety assessments of new drug candidates are
Pharmacologically Active Metabolites
Fig. 3. Method for finding active metabolites. In this scheme, an
evaluation of the potential for active metabolites is made. An in vitro
metabolism sample was generated and an extract prepared. The extract
is injected onto HPLC-UV-MS and spectral data are collected. The eluent
is 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 and
MS chromatograms representing metabolites are denoted with arrows
with the letter M, and the parent drug with the letter P. The UV data
offers a crude estimate of relative abundances of the metabolites and
parent drug. The MS data are used for proposal of the structures of the
metabolites. A peak of target binding activity is detected in the fractions
where the parent drug elutes (;35 minutes), which is expected and serves
as an internal control that the target activity assay is functioning
properly. A second peak of target activity is also observed at ;27 minutes,
indicating the presence of an active metabolite. A second metabolite with
583
adequately exposed to metabolites to which humans
are exposed, irrespective of known target receptor
activity. Following this, regulatory guidance was
issued stating that metabolites of potential interest
need to be present in humans at a predefined threshold
either relative to the total drug-related material or
relative to the parent drug [Food and Drug Administration, 2012 (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM079266.pdf); International Conference on
Harmonization, 2012 (http://www.ich.org/products/guidelines/multidisciplinary/article/multidisciplinary-guidelines.html)]. If such a threshold is exceeded, then it
must be demonstrated that the species used in toxicology tests were also exposed to the same metabolites to
an extent greater than humans. The focus of the
guidance is on stable human metabolites present in
circulation, presumably because all organs would be
exposed to such metabolites via circulation. (This is in
spite of the fact that many metabolites that are
responsible for toxicity are chemically reactive and are
not necessarily stable enough to be present in circulation.) Off-target pharmacological activity that yields
safety concerns is also beyond the scope of this article,
with the exception of those metabolites that can contribute to safety issues arising from exacerbated target
pharmacology. For example, while the right level of
activity at opioid receptors yields the beneficial effect of
pain reduction, excessive activity can cause respiratory
depression. Thus, if there is a metabolite active at this
receptor, it must be considered when relating the
exposure to the safety effect.
In this review, pharmacologically active metabolites
will be discussed with regard to drug research and
development. Some pharmacokinetic concepts will be
reviewed as they relate to the special case of metabolites (Houston, 1981; Pang, 1985). The types of
biotransformation reactions that can give rise to
pharmacologically active metabolites will be described
in the context of the impact that various chemical
modifications can have on receptor/enzyme binding.
Experimental approaches that can be applied at
different stages of new drug research to detect active
metabolites and understand their impact will be
discussed. Finally, these principles will be illustrated
using examples, along with a description of drugs with
well-established active metabolites in humans. The
drugs/metabolites described were identified in a SciFinder search on the term “active metabolite,” and
following exclusion of prodrugs and drugs of abuse, the
literature for the remaining set was scrutinized for
some activity is observed at ;25 minutes. Structures for these
metabolites can be proposed from the MS data. Authentic standards of
the active metabolites can then by prepared by synthetic or biosynthetic
methods for determination of potency and other pharmacological
activities.
584
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Fig. 4. Application of the activity-gram approach to finding active metabolites in humans. In this example, pooled human plasma samples from an
early phase 1 clinical study were extracted and analyzed by HPLC-UV-MS. Fractions were collected and tested for binding in an in vitro
pharmacological binding assay. In addition to the binding activity expected at the retention time for the parent drug (;25–26 minutes), there was
strong 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 parent
drug, but metabolites as well. The structure of the major metabolite at 21 minutes was proposed from the mass spectral data, and an authentic
standard prepared for determination of intrinsic potency. Potency was comparable to the parent drug.
Pharmacologically Active Metabolites
585
Fig. 5. Metabolism of albendazole to the S-oxide metabolite.
specific information on the putative active metabolites.
It should be noted that our level of thoroughness and
sophistication with regard to the identification and
characterization of active metabolites is much greater
than it was in the past. Thus, the depth of our
knowledge regarding active metabolites for older drugs
may not be as developed as for more recently introduced drugs. For example, in a review on this topic
from over 30 years ago (that had a special emphasis on
the increased exposure and effect of active metabolites
in renal insufficiency), a list of over 50 drugs with
active metabolites was included (Drayer, 1976), although some were prodrugs. Considerable knowledge
has been gained since that time. In fact, active
metabolites have been proposed to be potential new
material for new drug discovery and development
(Kang et al., 2010). Certainly the presence of drugs
used 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 pharmacologically active metabolites can have on clinical
efficacy, some basic concepts regarding the dispositional aspects of metabolites must be understood. In
this section, the metabolic reactions that can give rise
to active metabolites are discussed followed by discussions of distributional aspects and pharmacokinetic
phenomena.
Fig. 7. Metabolism of artesunate to dihydroartimisinin.
that occur between the drug and the receptor, or 2)
occur on a position on the drug that is not oriented
toward the receptor when binding occurs. This is
illustrated in the schematic in Fig. 1. Most metabolic
transformations provide enough of a chemical change
that receptor potency is lost or greatly diminished.
Furthermore, many metabolic reactions introduce
enough hydrophilicity (by increasing the number of
hydrogen bonding substituents, charged entities, and/
or size) and increased polar surface area that metabolites are pharmacologically inactive by virtue of being
unable to penetrate the membranes of target tissues.
However, in some cases, pharmacological activity can
be retained in a metabolite, while in other cases
affinity for the target can be diminished but pharmacological activity retained due to high concentrations of
the metabolite.
Among xenobiotic metabolizing enzymes, the cytochrome P450 family is the most important. These
enzymes can catalyze a wide array of reaction types,
and the specific biotransformation reactions that occur
A. Metabolic Reactions That Can Yield
Active Metabolites
In order for a metabolite of a drug to retain binding
activity to the same receptor to which the drug binds,
the chemical modification must be as follows: 1) so
minor as to not disrupt the atom-to-atom interactions
Fig. 6. Metabolism of allopurinol to oxypurinol.
Fig. 8. Metabolism of astemizole to desmethylastemizole.
586
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Fig. 9. Metabolism of buspirone to hydroxyl and N-dealkyl metabolites.
for any given drug depend on the substituents present
in that drug (Guengerich, 2001). These include aliphatic
and aromatic hydroxylations, heteroatom dealkylations,
N- and S-oxygenations, dehydrogenations, and epoxidations. In the vast majority of examples, active
metabolites arise by one of these reactions. For example, among drugs active at neurotransmitter targets
that contain a basic 2°- or 3°-aliphatic nitrogen, it
is frequently the case that N-demethylated and
deethylated metabolites will also possess activity.
The basicity of the amine, an important attribute for
these drugs, is retained or even somewhat increased.
Thus, drugs such as fluoxetine, amitriptyline, imipramine, fenfluramine, citalpram, ketamine, mianserin,
and many others have active N-dealkyl metabolites.
Whether activity is exhibited in vivo depends on the
pharmacokinetic and dispositional attributes of the
metabolite.
Fig. 10. Metabolism of chlordiazepoxide to active metabolites.
Pharmacologically Active Metabolites
587
Fig. 11. Metabolism of clomiphene to its hydoxylated and demethylated metabolites.
In the case of hydroxylations, the likelihood that
a metabolite is pharmacologically active is less predictable. The addition of a single alcohol oxygen adds
approximately 20 Å2 in polar surface area (PSA) as
well as hydrogen-bond–donating potential to a position
where none existed in the parent structure. Such an
alteration can frequently yield an inactive metabolite if
the target binding site cannot tolerate such a substituent. However, in some cases the addition of –OH
can occur at a position that is not critical for target
binding, or faces the solvent in the protein-ligand
complex. In those cases, the metabolite can retain
intrinsic activity, and its potential to contribute to in
vivo activity resides with other properties, such as in
vivo exposure and target tissue penetrability. It is not
uncommon that alcohol and phenol metabolites are
rapidly cleared by conjugation reactions. It is also
frequently the case that alcohol and phenol metabolites
do not as readily penetrate target tissues, due to
a decrease in membrane penetrability caused by the
Fig. 12. Metabolism of codeine to morphine.
588
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Fig. 15. Metabolism of dolasetron to hydrodolasetron.
metabolite has nominally lower intrinsic potency and
a lower free fraction; however, its plasma concentrations
are well in excess of parent and thus it contributes to
effect (Busch et al., 1996).
B. Pharmacokinetic Aspects of Active Metabolites
Fig. 13. Metabolism of dihydroergotamine to 89-hydroxydihydroergotamine.
increased PSA. Nevertheless, there are several good
examples of active hydroxyl metabolites and these are
described in Section IV (e.g., risperidone/paliperidone,
saxagliptan/5-hydroxysaxagliptan, flutamide/2-hydroxyflutamide, etc). Other reactions shown to yield active
metabolites include epoxidation (e.g., carbamazepine/
carbamazepine-10,11-epoxide), reduction (dolasetron/
hydrodolastron, buproprion/dihydrobupropion stereoisomers, others), and heteroatom oxygenation (thioridazine/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 large
substituent usually inactivate a drug but there are
exceptions [buspirone/1-(2-pyrimidyl)-piperazine (1-PP);
nefazodone/m-chlorophenyl piperazine (mCPP)]. However in such cases the pharmacological properties of
the metabolite may be different from the parent
(e.g., binds to a different member of the same receptor
family). Conjugation reactions that add glucuronic acid,
sulfuric acid, amino acid, and glutathione generally
do not yield active metabolites (Mulder, 1992a). The
most well known exception to this is the case of
morphine-6-glucuronide, putatively an active metabolite
of morphine (Mulder, 1992b; Kilpatrick and Smith,
2005). 4-Hydroxytriamterene sulfate represents an
active sulfate conjugate metabolite of triamterene. The
Fig. 14. Metabolism of diphenoxylate to difenoxin.
When compounds are administered directly, the
description of pharmacokinetics is relatively straightforward. However, when a compound arises from
within the organism, such as an active metabolite,
the picture can be more complex (Pang, 1985; St. Pierre
et al., 1988). This is primarily due to the fact that the
metabolite represents an unknown fraction of the dose
of the parent (i.e., dose is not truly known which is
needed for calculation of fundamental pharmacokinetic
parameters) and the rate of introduction into the body
is not easily discerned.
From a pharmacokinetic standpoint, metabolites
(active and inactive) are frequently referred to as to
having formation rate–limiting kinetics or elimination
rate–limiting kinetics (Houston, 1981). A metabolite
cannot have an elimination rate that exceeds the
elimination rate of the parent drug from which it is
formed. This is referred to as formation rate–limiting
Fig. 16. Metabolism of ebastine to carebastine.
Pharmacologically Active Metabolites
Fig. 17. Metabolism of flutamide to 2-hydroxyflutamide.
kinetics (Fig. 2). If an experiment is done wherein the
metabolite is administered directly, then its t1/2 may
be shorter than when it is generated after administration of the parent drug. Many metabolites exhibit
this type of behavior by virtue of having higher
clearances and/or lower volumes of distribution than
the parent drug. In elimination rate–limiting kinetics,
the t1/2 of the metabolite is longer than that of the
parent drug (Fig. 2), and this value will be the same
irrespective of whether the metabolite arises after
administration of the parent drug or whether it is
administered directly. If a metabolite exhibits elimination rate–limiting kinetics, then with repeated
administration of parent drug the metabolite has the
potential to accumulate to a greater extent than the
parent drug at steady state.
The factors that drive the total exposure to the
metabolite include the clearance rate of the parent
drug, the fraction of the dose of the parent drug that is
converted to the metabolite, and the subsequent
clearance rate of the metabolite. The latter term can
also impact the amount of the metabolite formed that
escapes from the tissue from which it is formed and
gets into the systemic circulation. In the vast majority
of cases, the focus can be on the liver as the main site of
metabolite formation. When considering an active
metabolite, an important metric in understanding the
potential contribution of the metabolite to effect
relative to the parent drug is the area-under-thecurve (AUC) ratio. This can be defined as:
AUCmetabolite fCL;m •Fm •CLparent
¼
AUCparent
CLmetabolite
Fig. 18. Metabolism of hydroxyzine to cetirizine.
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The terms are defined as follows: fCL,m is the fraction
of the clearance of the parent drug that yields the
metabolite, Fm is the portion of the total metabolite
generated within an organ that is released into the
systemic circulation before it is either further metabolized or released into bile (in the case of the liver) or
urine (in the case of the kidney), CLparent is the total
clearance of the parent drug, and CLmetabolite is the
total clearance of the metabolite. It is rarely the case
that these individual parameters are measured, especially in humans, since they require the intravenous
administration of the metabolite directly. However,
they offer conceptual insight as to the determinants of
metabolite exposure. Prediction of metabolite exposure
is a challenging undertaking when considering the
number of contributing variables. Yet the importance
of active metabolites (as well as those that could cause
deleterious off-target effects) in drug research and
clinical practice makes research into methods that can
predict 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 in
Relation to Contribution to Pharmacological Effect
The potential for the active metabolite to penetrate
the target tissue relative to the parent drug is also
necessary to understand whether the metabolite is
truly an active metabolite. Even if the metabolite
possesses intrinsic potency that is equivalent to the
parent, if the free concentration within the tissue is
lower than that of the parent, the metabolite will not
contribute as much as the parent. The main drivers
behind the target-tissue free concentrations include
plasma protein binding, membrane penetrability,
and the potential for active transporters to alter
the free tissue–to–free plasma concentration ratio
(Kp,uu).
Distributional phenomena are largely driven by high
capacity, low affinity nonspecific binding interactions
between drugs and tissue macromolecules, and macromolecular structures (e.g., phospholipid membranes).
Such interactions are mostly a function of physicochemical properties of the drug, especially lipophilicity
and ionization state. Greater lipophilicity and greater
cationic character are properties that tend to increase
nonspecific binding to tissue macromolecular structures. Since a majority of biotransformation reactions
result in decreases in lipophilicity, metabolites tend
not to partition into tissues as well as their parent
drugs. An example of an exception to this is ketoconazole, which undergoes deacetylation thereby converting a neutral amide to a basic amine, and in this case it
is the cationic metabolite that is better at binding to
membranes (Whitehouse et al., 1994).
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Fig. 19. Metabolism of irinotecan to SN-38.
Membrane penetrability is also frequently a function
of PSA, increases in which tend to debilitate penetrability. Since almost all biotransformation reaction
types result in increases in PSA, metabolites will tend
to be less membrane permeable than their respective
parent drugs. If enough hydrophilicity is introduced,
the metabolite could be completely membrane impermeable. If a metabolite is still membrane permeable,
but of lower permeability than the parent drug, then
the result is that it will take longer for the metabolite
to achieve a steady-state exposure in the tissues than
the parent drug. Increased hydrophilicity also renders
metabolites better substrates for several of the drug
efflux transport proteins, which also serves to decrease
the free tissue concentrations of metabolites relative to
parent drugs.
The effects of decreased tissue partitioning and
penetration can be offset by differences in plasma
protein binding between parent drugs and their
metabolites. Metabolites are frequently less proteinbound in plasma than their respective parent drugs,
presumably due to their decreased hydrophobicity
(Table 1). Thus, the potential decreases in membrane
permeability and Kp,uu of metabolites relative to their
parent drugs can be offset by increases in free fraction.
A potentially challenging situation for understanding
the relative importance of metabolites to pharmacological effect occurs when the metabolite is generated
within the target tissue. For example, the hydroxyl
metabolites of atorvastatin have potency at the
pharmacological target. They are generated by CYP3A
within the liver, which is the target tissue for
inhibition of cholesterol biosynthesis. Due to this, the
free concentrations within hepatocytes may not be
reflected by systemic free plasma concentrations, and
thus a true knowledge of the contribution of the
metabolites to clinical effect would be unattainable.
Estimates would need to be made from in vitro data or
data from laboratory animals.
III. Active Metabolites in Drug Research
For the purposes of this discussion, the process by
which new drugs are created can be generally broken
down into two major portions: 1) the preclinical phase,
in which medicinal chemists and pharmacologists
collaborate, along with other related discipline scientists, to identify a new molecular target and design
ligands that can bind and affect the activity of that
target (termed “drug discovery”), and 2) the clinical
Fig. 20. Metabolism of loratadine to desloratadine.
Pharmacologically Active Metabolites
Fig. 21. Metabolism of losartan to EXP-3174.
phase, wherein the selected molecule is studied first in
healthy volunteers (phase 1) and then in patients for
efficacy and safety (“drug development”). The impact of
pharmacologically active metabolites in these two
phases is different. However, it is critical to account
for active metabolites in drug research as early as possible,
as the insights that can be gleaned regarding target
structure-activity relationship (SAR), pharmacokineticpharmacodynamic (PK/PD) relationships, and toxicity
are invaluable (Fura, 2006)
A. Research Tools in Metabolite Identification
A lengthy discussion of how drug metabolites are
identified is beyond the scope of this review and the
interested reader is referred to other descriptions of
this topic for greater depth (Prakash et al., 2007; Zhang
and Comezoglu, 2007). However, to understand the
issue of pharmacologically active metabolites, a very
brief description of how metabolites are identified is
necessary.
In the early efforts (preclinical), the identification of
metabolites of new compounds is done using incubates
from in vitro liver-derived systems such as hepatocytes
and microsomes, as well as various biologic fluids (e.g.,
plasma, urine, bile) obtained from laboratory animal
species following administration of the compound of
interest. Identification of metabolites in the drug
discovery phase is done for a variety of reasons,
including 1) the identification of specific sites of
modification on a rapidly metabolized molecule so that
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drug design efforts can slow down metabolism, 2)
identification of the main routes of metabolism and
enzymes involved so that the overall clearance mechanism in humans can be understood and predicted, 3)
identification of the possibility of generation of chemically reactive metabolite types associated with toxicity, and, relevant to this article, 4) the identification of
pharmacologically active metabolites. In the early
stages of drug research, considerable effort is expended
through the use of animal models of disease to
understand the biologic effects of manipulating the
underlying pharmacological target of interest. Developing the relationship between the drug concentration and
the effect (i.e., the pharmacokinetic/pharmacodynamic
relationship) will offer insight into the duration and
degree to which the target receptor needs to be occupied
for effect. The presence of an active metabolite will
alter that relationship, and if an active metabolite is
present but unaccounted for, the pharmacokinetic/
pharmacodynamic relationship will be misinterpreted.
In the clinical stage, the same questions regarding
establishing the PK/PD relationship exist, albeit in
human patients rather than animal disease models,
and unlike the earlier work the efforts are focused on
one or maybe two compounds for any given target. In
early phase 1 studies, plasma and urine samples from
human study subjects can be used to seek metabolites.
Later in clinical development, it is typical practice to
carry out a study wherein humans are administered
a radiolabeled drug and excreta and plasma are
thoroughly interrogated for a complete and comprehensive picture of the metabolism of the new drug
(Penner et al., 2012).
The main tool used by the drug metabolism
scientist in metabolite identification is high-pressure
liquid chromatography-mass spectrometry (HPLC-MS).
HPLC-MS provides information on the molecular
weight of the metabolite (so it can be compared with
the molecular weight of the parent compound) to
discern the type of metabolic modification, and the
fragmentation of the metabolite in the MS offers
insight into the structure of the metabolite. While MS
data can sometimes yield enough information to assign
the chemical structure of a metabolite, it is frequently
the case that the information is not enough to propose
a precise structure. In those cases, additional data
using complementary methods is required, such as
NMR spectroscopy, chemical derivatization, and/or
synthesis of an authentic standard of the metabolite
for comparison.
B. Identification of Active Metabolites in
Preclinical Research
Fig. 22. Metabolism of oxcarbazepine to 10-hydroxycarbamazepine.
As stated above, knowledge of pharmacologically
active metabolites in animal models of disease is
important. Furthermore, the prediction that humans
may also be exposed to pharmacologically active
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Fig. 23. Metabolism of oxybutynin to desethyloxybutynin.
metabolites is important in predicting the efficacious
dose prior to human studies. In the preclinical stage,
efforts are ongoing to develop knowledge of the SAR for
receptor binding potency, and the identification of an
active metabolite can offer the medicinal chemist
greater insight as to the types of substituents that
can be tolerated in the ligand-receptor interaction.
Finally, an active metabolite could be a better compound to develop than the drug itself (e.g., terfenadine
versus fexofenadine).
A sequential approach to identifying metabolites in
biologic matrices—proposing chemical structures of
these metabolites, followed by organic synthesis and
testing of the metabolite at the target receptor—
represents one strategy for identifying active metabolites in preclinical drug research. Some SAR insight
may already be known for a chemical series such that
proposed metabolite structures could be ruled out from
having pharmacological activity in the absence of data.
For instance, N-demethylation of a tertiary amine drug
to a secondary amine metabolite is frequently anticipated to have minimal impact on target receptor
activity, thus it is prudent to establish this potential
activity experimentally. However, N-deamination of an
amine drug followed by oxidation to the carboxylic acid
metabolite is much less likely to yield an active
metabolite, due to the marked difference in structure
and charge.
An approach we have employed in our laboratories to
address the possibility of pharmacologically active
metabolites involves a close collaboration between the
drug metabolism scientist and pharmacologist (see Fig.
3 and Fura et al., 2004). The drug metabolism scientist
carries out an analysis of a biologic sample (in vivo or
in vitro) for metabolite identification on HPLC-MS as
Fig. 24. Metabolism of procainamide to N-acetylprocainamide.
usual. However, the HPLC eluent is split prior to
introduction into the MS. A small portion is introduced
to the MS to yield spectral data. The remaining portion
is diverted to a fraction collector. The fractions are
delivered to the pharmacologist for testing activity at
the target receptor. The HPLC-MS chromatogram is
compared with the receptor activity profile of the
fractions to create an “activitygram.” If receptor
activity is detected in fractions corresponding to where
a metabolite eluted, then the metabolite is potentially
an active metabolite. Since the amount of the metabolite in the fraction is unknown, an absolute potency
value cannot be determined. This merely offers an
early signal regarding whether an active metabolite is
even possible, and if so, then synthesis of an authentic
standard of that metabolite for testing can be undertaken. If the metabolite poses a synthetic challenge,
it can still be addressed by generating a biologic
sample containing the metabolite, purifying it by
HPLC, determining the concentration of the metabolite
using proton NMR (Walker et al., 2011; Vishwanathan
et al., 2009), and using that solution as a concentrated
stock solution from which to dilute the metabolite into
the activity assay.
C. Identification of Active Metabolites in
Clinical Research
As humans are the target species, knowledge of
active metabolites in human is essential to proper dose
selection and understanding determinants of interpatient variability in drug response. By the time of
administration to humans in phase 1 studies, the
presence (or not) of active metabolites in laboratory
Fig. 25. Metabolism of propoxyphene to norpropoxyphene.
Pharmacologically Active Metabolites
593
Fig. 28. Metabolism of roflumilast to roflumilast-N-oxide.
Fig. 26. Metabolism of remacemide to desglycylremacemide.
animals will have already been determined. In addition, human in vitro systems will have been used to
predict the metabolites that will be present in vivo.
Thus, a good understanding of the potential for active
metabolites in humans should already have been
obtained. Nevertheless, it is still possible for an active
metabolite to be revealed for the first time in a clinical
study.
From animal models, a prediction of the human
concentration-effect relationship can be made (either
for a biomarker or an effect on disease). If that
relationship does not hold upon first examination in
human (either the magnitude or duration of effect, or
both), then the possibility of pharmacologically active
metabolites must be entertained. The same type of
approach described above can be performed using
human plasma samples. An example of this is
illustrated in Fig. 4 (Hagen et al., 2008). In this
Fig. 27. Metabolism of risperidone to paliperidone.
instance, the parent drug was targeting a subtype of
the serotonin receptor family. During the phase 1 dose
escalation study, suprapharmacological effects were
observed at parent drug concentrations that were far
lower than anticipated. Plasma samples that had
already been used for quantitative analysis of the
Fig. 29. Metabolism of sibutramine to its N-demethylated metabolites.
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Fig. 30. Metabolism of tamoxifen to its hydroxylated and N-demethylated metabolites.
parent drug were subsequently used in the “activitygram” approach, and the presence of two peaks of high
receptor binding activity was observed, with one
activity peak corresponding to the retention time of
the parent drug and an earlier eluting peak corresponding to where a hydroxylated metabolite eluted.
This showed the likelihood of a circulating active
metabolite that could explain the clinical observations.
Subsequent structure elucidation of the metabolite was
done, and synthesis of an authentic standard showed
that the metabolite had intrinsic binding potency
equivalent to the parent drug.
In addition to those instances wherein pharmacodynamic observations trigger an aggressive search for
pharmacologically active metabolites, the determination
of the metabolites present in human plasma and excreta is done for other purposes, such as gaining an
understanding of the important clearance pathways and
determining whether human metabolites are also
observed in laboratory animal species that are used to
explore the toxicology of the drug. As those metabolites
are structurally identified, a judgment can be made
regarding whether they would potentially be active,
based on the known structure-activity relationship for
the target receptor. Even if not contributing substantially to the efficacy of the drug, discovering a new active
metabolite in clinical samples can potentially offer
a new lead for a second-generation drug.
D. Data Needed for Active Metabolites
Once a metabolite is identified that has intrinsic
binding potency to the target receptor, the following
information needs to be gathered to properly characterize it:
Pharmacologically Active Metabolites
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Fig. 31. Metabolism of terfenadine to fexofenadine.
1. In addition to target binding potency, functional
activity measurement is needed (antagonist,
agonist, partial agonist, inhibitor, activator, etc.).
2. Plasma protein binding in humans and laboratory animal species.
3. Penetration into the target tissue(s) and prediction of the free tissue–to–free plasma concentration ratio (i.e., Kp,uu).
4. Pharmacokinetics in humans and laboratory
animal species, and the underlying clearance
pathways for the active metabolite in humans.
Essentially, this set of information reflects the same
set that is needed to understand the pharmacology of
the parent drug. For example, if an active metabolite is
responsible for the majority of the pharmacological
effect, knowledge of what can cause a change in the
exposure to the metabolite (to either increase or
decrease it) must be known. Drug interactions that
alter the exposure to an active metabolite must be
known. For example, tamoxifen is converted to the
more potent metabolite endoxifen by CYP2D6, so the
concomitant use of potent CYP2D6 inhibitors in
patients receiving tamoxifen should be avoided (Mannheimer and Eliasson, 2010).
IV. Drugs with Active Metabolites
In this section, a description of numerous examples
of drugs with active metabolites is included. The
molecular targets, indications, and metabolic reaction
types span a broad array, indicating that there is no
single complement of circumstances that favor or
disfavor the possibility of an active metabolite
phenomenon. As described in section III.D, the
Fig. 32. Metabolism of thiocolchicoside to its glucuronide.
information needed to truly assess the contribution of
an active metabolite to clinical effect is challenging and
requires knowledge of intrinsic receptor potency, free
fraction, target tissue penetrability, and plasma pharmacokinetics. It is rare that all of these properties are
known for metabolites (especially plasma protein
binding and target tissue penetrability), so in many
cases inferences must be made and assumptions
accepted when making such projections. Additionally,
it is not uncommon that the reported intrinsic potency
values measured for active metabolites may have been
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made using nonhuman-derived reagents and tissues,
so it must be assumed that relative potency values
between parent and metabolite(s) are similar across
species. From the assessments of these examples, it is
clear that in many cases there is considerable data
gathering that needs to be done to fully understand the
action of active metabolites. Many of the estimates of
contributions of metabolites to efficacy relative to the
parent drug described below were made using the
properties listed above that were obtained from the
scientific literature.
The examples have been divided into four categories:
1) active metabolites wherein the metabolite contributes the majority of the activity, even though the
parent drug has intrinsic target potency; 2) active
metabolites that contribute to target activity at a level
comparable to the parent drug; 3) active metabolites
that possess target affinity but would be estimated to
contribute little to in vivo effect, relative to the parent;
and 4) metabolites that have activity at alternate
pharmacological targets closely related to the intended
target that the parent drug interacts with, and
putatively contribute to clinical effect via this alternate
target. Section IV.E describes a few examples wherein
the delineation of metabolite and parent contributions
is highly ambiguous.
A. Drugs with Active Metabolites That Dominate
the Activity
In some instances, while the parent drug possesses
affinity for the intended target protein, a metabolite
can be present that actually dominates the in vivo
effect. Such cases are not truly prodrugs, since the
parent drug has target activity, but their behavior can
resemble those of prodrugs.
1. Albendazole. Albendazole offers an interesting
example of site-dependent effects of parent drug versus
its S-oxide metabolite (Fig. 5). Albendazole is an
anthelminthic agent used in the treatment of various
parasitic diseases and its mechanism of action is
through the binding of tubulin in nematodes. The
parent is about 6-fold more intrinsically potent at
tubulin binding than the S-oxide metabolite (Lubega
and Prichard, 1991). For systemic parasitic infection it
is the metabolite that is believed to exhibit the effect
because metabolite plasma concentrations achieved
are in the range of 3 mM while the parent drug is
below the limit of quantitation of plasma assays (Jung
et al., 1992; Medina et al., 1999; Mirfazaelian et al.,
2003). It can be used in the treatment of neurocysticercosis because of the brain penetrability of the
S-oxide metabolite. However, for parasitic infections
of the gastrointestinal tract, it is likely the parent
drug has the effect since the S-oxide is generated
postabsorption.
2. Allopurinol. Allopurinol is oxidized to oxypurinol
by aldehyde oxidase (Fig. 6). Oxypurinol is approximately
Fig. 33. Metabolism of thioridazine to its S-oxidized metabolites.
10-times less potent an inhibitor of the target enzyme,
xanthine oxidase (Tamta et al., 2006); however, the
circulating concentrations of oxypurinol in humans
following allopurinol administration are far greater and
sustained for a longer time. Maximal concentrations of
oxypurinol are about 4- to 5-fold greater, and the half-life
of parent and metabolite are 1 and 20 hours, respectively
(Turnheim et al., 1999; Day et al., 2007). Thus, the
contribution of oxypurinol to xanthine oxidase inhibition
activity dominates at later timepoints.
Pharmacologically Active Metabolites
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Fig. 34. Metabolism of tibolone to hydroxylated and isomer metabolites.
3. Artesunate. Artesunate is an ester-containing
derivative containing a hydroperoxide that is believed
to form toxic adducts with heme as hemoglobin is being
digested by the Plasmodium species that causes
malaria (Creek et al., 2008). It is more water-soluble
than other artemisinin derivatives and thus offers
multiple dosing approaches. Upon oral administration
it is entirely cleaved to dihydroartimisinin, an active
metabolite (Fig. 7), but after intravenous administration both parent drug and the active metabolite are
present in circulation (Nealon et al., 2002; Morris et al.,
2011). The intrinsic potency of the metabolite is
approximately 4-fold greater than the parent (Creek
et al., 2008), and after correction for differences in
plasma free fraction and plasma concentrations, it can
be estimated that the metabolite contributes about 4fold greater activity than the parent.
4. Astemizole. Astemizole is a histamine-1 receptor
antagonist that was withdrawn from clinical use due
to occurrence of cardiac arrhythmia caused by blockade of the human ether-a-go-go-related gene potassium channel. It has been described to be converted
to O-desmethylastemizole (Fig. 8) which has a much
longer t1/2 than astemizole (Heykants, 1984). Detailed
information on the human H1 receptor potency of this
metabolite is not available in the literature, but it
has been described as being equipotent (Richards
et al., 1984). It can thus be anticipated that the
metabolite would contribute the majority of the
pharmacological activity, especially upon accumulation at steady state. Other metabolites, such as 6-
hydroxydesmethylastemizole, have been shown to
have activity in guinea pig-derived models of antihistamine, but this may be due to alterations in
histamine release (Kamei et al., 1991).
Fig. 35. Metabolism of tolterodine to desfesoterodine.
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Fig. 36. Metabolism of tramadol to O-desmethyltramadol.
5. Buspirone. Buspirone is an anxiolytic agent
purported to act via binding to the 5-HT1A (serotonin)
receptor. It has long been known to undergo Ndealkylation to 1-(2-pyrimidyl)piperazine (1-PP), which
has weak activity at the 5-HT1A receptor, and that this
metabolite could also play a role in the effect of
buspirone (Fig. 9). However, in a clinical pharmacokinetic study, it was shown that 6-hydroxybuspirone is
present at much greater concentrations than buspirone
and may contribute to activity (Dockens et al., 2006;
Wong et al., 2007). If it is assumed that the free
fraction of this metabolite is equivalent to buspirone
itself, then it can be estimated that the metabolite
contributes seven times the activity of the parent.
Reports in the literature on the process of making 6hydroxybuspirone on a large scale suggest that there is
interest in this metabolite as a potential drug itself.
Investigation of 1-PP as an active metabolite has
revealed that it also could contribute considerably to
the action of buspirone by virtue of higher exposure. In
addition, 1-PP has activity at the a2 receptor (Caccia
et al., 1986), the role of which is undefined.
6. Chlordiazepoxide. Chlordiazepoxide generates
several active metabolites via sequential demethylation, deamination, reduction, and hydroxylation reactions (Fig. 10). Which metabolite contributes the
greatest activity cannot be discerned, but it is clear
that metabolites, especially desmethyldiazepam, contribute to activity. The affinities of the entities with the
N–O bond (chlordiazepoxide, norchlordiazepoxide, and
demoxapam) for the benzodiazepine receptor are 10- to
20-fold lower than the desmethyldiazepam and oxazepam metabolites (Richelson et al., 1991). Concentrations of oxazapam after chlordiazepoxide were not
available, but unbound concentrations of desmethyldiazepam were comparable to its binding affinity
Fig. 37. Metabolism of trimethadione to dimethadione.
(Dixon et al., 1976; Boxenbaum et al., 1977; Greenblatt
et al., 1978; Ito et al., 1997).
7. Clomiphene. Similar to tamoxifen (section IV.
A26.), clomiphene is metabolized by hydroxylation at
the para position of one of the phenyl rings by CYP2D6
to yield a metabolite, 4-hydroxyclomiphene (Fig. 11),
that has two orders of magnitude greater potency at
binding to the estrogen receptor (Ruenitz et al., 1983;
Mürdter et al., 2012). Thus, CYP2D6 extensivemetabolizer (EM) and poor-metabolizer (PM) subjects
exhibit different responses, with the EM subjects
expected to enjoy greater efficacy due to 7-fold greater
exposure (Mürdter et al., 2012). Clomiphene also
undergoes N-deethylation, but N-desethylclomiphene
has similarly low activity to the parent drug. The 4hydroxy metabolite of N-desethylclomiphene also has
activity and would be estimated to contribute 50-fold
more than the parent drug. Furthermore, the picture is
complicated by the fact that the drug is administered
as two geometric isomers, with the E-isomers possessing greater activity.
8. Codeine. Codeine is converted to the active Odemethylated metabolite morphine by CYP2D6 (Fig.
12). After oral administration, the exposure to codeine
is almost 40-fold greater than that of morphine
(Quiding et al., 1986). However, the potency of
morphine at the m-opioid receptor is 600-fold greater
(Volpe et al., 2011), thus the pharmacological effects of
codeine administration are almost exclusively attributable to the active metabolite morphine (and its
subsequent pharmacologically active glucuronide
metabolites; see Section IV.B.15.). In CYP2D6 poor
metabolizers, morphine concentrations are very low
(;50-fold lower than in extensive metabolizers; Kirchheiner et al., 2007), and there is not enough generated
to elicit an effect. Codeine still has other clearance
pathways besides CYP2D6, and thus exposures are not
different between EM and PM subjects. This supports
the notion that in EM subjects, codeine itself does not
contribute to pharmacological effect.
9. Dihydroergotamine. Dihydroergotamine is a vasodilator effective in the treatment and prophylaxis of
migraine, with the serotonin 1A receptor considered as
the target. The oral bioavailability of dihydroergotamine itself is very low, yet the drug is effective at low
parent drug concentrations. Investigation led to the
identification of the metabolite 89-hydroxydihydroergotamine as the putative active agent (Fig. 13). Its
intrinsic potency is about thrice that of the parent
(Hanoun et al., 2003), but more importantly, the
plasma concentrations of the metabolite are 6-times
greater (Wyss et al., 1991; Chen et al., 2002). It can be
estimated that the metabolite possesses 20-fold greater
in vivo activity than the parent, assuming that free
fractions and brain penetrability properties are similar. Considering that the addition of one hydroxyl
group to a molecule with a molecular weight of nearly
Pharmacologically Active Metabolites
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Fig. 38. Metabolism of venlafaxine to O-desvenlafaxine.
600 would alter the physicochemical properties to only
a 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 as
an antidiarrheal agent that acts by agonizing the
opiate receptor. The carboxylic acid metabolite is
somewhat more potent than the parent drug (as
assessed using a rat brain homogenate binding assay),
and systemic concentrations of the metabolite are
greater than for the parent (Karim et al., 1972). The
target of action is the gut, so it cannot be necessarily
stated that the metabolite bears the greater role in
activity since the effect could be due to local exposures
to parent drug upon oral administration (Wüster and
Herz, 1978). In a comparison, dosing the metabolite
directly to humans showed that five-times less was
needed to achieve a constipatory effect the same as the
parent ester (Rubens et al., 1972).
11. Dolasetron. Dolasetron is a 5-HT3 receptor
antagonist used in the treatment of nausea and
vomiting caused by emetogenic anticancer agents
(Anzemet). It undergoes reduction to hydrodolasetron
and this metabolite dominates the contribution to
activity (Fig. 15). When used orally, dolasetron is not
measurable, thus it has been concluded that the
activity is dominated by the alcohol metabolite (Keung
et al., 1997). The metabolite molecule has 20- to 60times greater affinity at the 5-HT3 receptor than the
parent (Gregory and Ettinger, 1998). Even when
dolasetron is intravenously administered, hydrodolasetron is generated rapidly and exceeds the parent in
concentration by over 10-fold. Thus, even though
dolasetron possesses intrinsic affinity for the 5-HT3
receptor, it offers no contribution to efficacy in vivo; the
activity resides with the reduced metabolite.
12. Ebastine. Ebastine is a second-generation antihistamine that contains a t-butyl substituent that
undergoes extensive conversion to a carboxylic acid
metabolite, carebastine (Fig. 16). After administration
of ebastine, carebastine concentrations are 30-fold
greater (Sastre, 2008; Shon et al., 2010). Furthermore,
carebastine is intrinsically more potent at the H1
receptor (Anthes et al., 2002). The exact values for
plasma protein binding have not been reported as both
parent and metabolite are highly bound and merely
reported as .95%. On the basis of these measurements, it can be estimated that the metabolite
contributes virtually all of the antihistamine activity
following oral administration of ebastine.
13. Flutamide. Flutamide is an antiandrogen that
is converted to the active metabolite 2-hydroxyflutamide (Fig. 17). The intrinsic potency and plasma
protein binding values of the parent and metabolite
are not very different (Feau et al., 2009), but what
drives estimation of a high contribution of the
metabolite to efficacy are the 20- to 40-fold greater
Fig. 39. Metabolism of acebutolol to diacetolol.
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TABLE 2
Examples of drugs for which active metabolites are now used as drugs themselves
Original Drug
Amitriptyline
Metabolite That Is Used as a Drug
Nortriptyline
Mechanism/Indication
Antidepressant
Differential effect at neurotransmitter transport
proteins
Bromhexine
Ambroxol
Diazepam
Diazepam
Etretinate
Hydroxyzine
Imipramine
Temazepam
Oxazepam
Acitretin
Cetirizine
Desipramine
Mucokinetic agent
and topical anesthetic
Anxiolysis
Anxiolysis
Antipsoriatic
Antihistamine
Antidepressant
Loratadine
Loxapine
Desloratadine
Amoxapine
Antihistamine
Antidepressant
Primidone
Procainamide
Phenobarbital
Acecainide
Antiseizure
Antiarrythmic
Risperidone
Paliperidone
Antipsychotic
Spironolactone
Terfenadine
Thioridazine
Tolterodine
Canrenone
Fexofenadine
Mesoridazine
Fesoterodine
Diuretic
Antihistamine
Antipsychotic
Urinary incontinence
Venlafaxine
Desvenlafaxine
Antidepressant
plasma concentrations of the metabolite (Belanger
et al., 1988).
14. Hydroxyzine. Hydroxyzine is a first-generation
antihistamine. Its use in the treatment of allergy has
fallen out of favor because it causes sedation, unlike
more recently available antihistamines. However,
because of this side effect, it has found use as a sedative
and potentially as an anxiolytic. Hydroxyzine contains
a 1° alcohol that is metabolized by oxidation to the
carboxylic acid metabolite cetirizine (Fig. 18). Cetirizine also has intrinsic H1 potency that is about 3-fold
weaker than the parent drug (Chen, 2008), yet this
potency was enough to permit introduction of this
Rationale for the Usefulness of the Metabolite as a Drug
Improved pharmacokinetics
Lower brain penetration, less sedation
Differential effect at neurotransmitter transport
proteins
Lower brain penetration, less sedation
Differential effect at neurotransmitter transport
proteins
Molecular mechanism resulting in different
electrophysiological effect
Decreased impact of CYP2D6 clearance, lower
intersubject variability
Safety; reduced potassiumr channel activity
Decreased impact of CYP2D6 clearance, lower
intersubject variability
Decreased impact of CYP2D6 clearance, lower
intersubject variability
metabolite as a drug itself. Cetirizine is zwitterionic
and therefore not necessarily as good at penetrating
the brain as the parent hydroxyzine, and this serves as
an advantage by offering antihistimic activity peripherally while causing much less sedation. With hydroxyzine dosing, the contribution of cetirizine to
antihistamine activity is likely high, due to higher
concentrations of the metabolite (Simons et al., 1989).
15. Irinotecan. Irinotecan is a topoisomerase 1
inhibitor structurally derived from natural products
from Camptotheca. It undergoes hydrolysis of the
carbamate bond to the active metabolite SN-38 (Fig.
19), which is over 1000-times more potent at inhibition
Fig. 40. Metabolism of acetohexamide to hydroxyhexamide and structures of analogs.
Pharmacologically Active Metabolites
601
Fig. 41. Metabolism of amiodarone to desethylamiodarone.
of the enzyme (Kawato et al., 1991). While SN-38 is
present in circulation, it is only there at about a 40-fold
lower concentration than the parent drug, and its free
fraction is about 10-times lower (Wiseman and Markham,
1996; Iyer and Ratain, 1998; Mathijssen et al., 2001;
Camptosar, 2012). Combining the inhibition potency
and free plasma concentrations, it would be estimated
that SN-38 contributes 10-fold more than the parent.
However, it is likely that irinotecan is cleaved inside the tumor cell and bioactivated there, and SN-38
would then make a much larger contribution (Kawato
et al., 1991).
16. Loratadine. Loratadine was one of the first
nonsedating antihistamines and as such was a popular
alternative to the older generation antihistamines that
readily penetrated the blood-brain barrier and caused
sedation. It is extensively metabolized to desloratadine
via hydrolysis of the carbamate moiety (Fig. 20). The
intrinsic potency of desloratadine far exceeds that of
the parent (Anthes et al., 2002), the free fraction is
higher by 5-fold, and the circulating concentrations are
also higher (Ramanathan et al., 2007). When these
three factors are combined, it suggests that loratadine
Fig. 42. Metabolism of aripiprazole to dehydroaripiprazole.
itself is responsible for none of the in vivo antihistaminic activity. Desloratadine is now used as a drug
itself. The picture is further complicated by the fact
that 3-hydroxydesloratadine is also listed as an active
metabolite; however, the intrinsic potency value is not
available in the scientific literature. 3-Hydroxydesloratadine has a comparable free fraction and circulates
at about half the concentration of desloratadine.
17. Losartan. Losartan is an antihypertensive
agent that acts by binding to the angiotensin II
receptor. It is a primary alcohol that is oxidized in
two steps to a carboxylic acid (EXP3174) by CYP3A and
CYP2C9 (Fig. 21). An integrated examination of the
relative potency (Le et al., 2003), free fraction in
plasma (Cozaar, 2011), and plasma exposures (Lo
et al., 1995) yields the projection that the carboxylic
acid metabolite would contribute approximately 14times the activity than the parent drug. This is
primarily driven by the approximately 25-fold greater
potency of the metabolite. The relevance of the
metabolite to efficacy is reinforced by clinical observations. As CYP2C9 has a major role in the generation of
EXP3174, subjects that are genotyped as containing
CYP2C9*3 have shown lower metabolite concentrations (Yasar et al., 2002). Such subjects have also been
shown to have a lower therapeutic response to losartan
(Lajer et al., 2007; Joy et al., 2009).
18. Oxcarbazepine. Oxcarbazepine is reduced to 10hydroxycarbamazepine (Fig. 22) and it is the metabolite that has been claimed to carry the majority, if not
all, of the antiepileptic activity. Exposure to the active
metabolite is considerably greater than the parent
drug and it is somewhat less protein-bound (Dickinson
Fig. 43. Metabolism of carbamazepine to the epoxide metabolite.
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Fig. 44. Metabolism of clarithromycin to 14-hydroxyclarithromycin.
et al., 1989; Patsalos et al., 1990). Although the
mechanism of action may not be definitively proven,
both compounds block sodium channels in vitro with
equivalent potency (Schmutz et al., 1994). Considering the relative potency, plasma exposure, and
free fraction values, and assuming equivalent brain
penetrability, it can be estimated that the metabolite carries 17-times the in vivo activity of the
parent drug.
19. Oxybutynin. Oxybutynin is an antimuscarinic
agent used in the treatment of urinary incontinence. It
undergoes an N-deethylation reaction to yield a major
circulating metabolite present at 8-fold greater concentrations (Fig. 23) (Reiz et al., 2007). Interestingly,
this metabolite has shown a higher protein binding
(Mizushima et al., 2007), which is counter to most cases
wherein secondary amine metabolites have higher free
fractions than their corresponding tertiary amine
parent drugs. Both parent and metabolite are equally
active in vitro (Waldeck et al., 1997). Thus, it can be
estimated from the combination of these data that
desethyloxybutynin has about 4-fold the in vivo
activity as the parent drug. It should be noted that
these estimates are based on data for the racemate,
and somewhat different estimates could be possible by
considering the stereoisomers independently.
20. Procainamide. Procainamide is a class 1 antiarrythmic agent that blocks the sodium channel. It
undergoes acetylation to the active metabolite Nacetylprocainamide (Fig. 24), which also has been
shown to bind to this channel (Sheldon et al., 1994b).
Combining the circulating concentrations, plasma protein binding (which is low for both), and the sodium
channel affinity leads to the conclusion that the
acetylated metabolite contributes twice as much to
the receptor activity as the parent drug. Despite this
estimation, there are other data to suggest that Nacetylprocainamide does not make this contribution
and actually works as a class III antiarrythmic via
potassium channel block (Harron and Brogden, 1990).
Direct intravenous administration showed that Nacetylprocainamide concentrations needed to be
greater than what would be expected from findings
when the metabolite was measured after administration of procainamide and that the electrophysiological
profile 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-acetyltransferase 2 enzyme, thus the ratio of parent to
metabolite in vivo differs between extensive and poor
metabolizers (Reidenberg et al., 1975).
Fig. 45. Metabolism of clobazam to N-desmethylclobazam.
Pharmacologically Active Metabolites
603
Fig. 46. Metabolism of disopyramide to its desisopropyl metabolite.
21. Propoxyphene. Propoxyphene is a m-opioid
agonist that was recently removed from clinical use
in the United States due to cardiac toxicity. It undergoes N-demethylation to norpropoxyphene (Fig. 25),
which has nearly equivalent intrinsic potency and
plasma binding (Giacomini et al., 1978; Neil and
Terenius, 1981). However, it is present in nearly 10fold excess to parent after steady-state dosing, with
nonstationary clearance for both parent and metabolite
resulting in high accumulation (Inturrisi et al., 1982).
This would suggest that norpropxyphene carries the
bulk of the target activity. However, in rats it was
shown that the norpropoxyphene metabolite does not
partition into brain as readily as the parent drug (Way
and Schou, 1979) and that propoxyphene itself is a
P-glycoprotein substrate in mouse (Doran et al., 2005).
Thus, the relative central effects of the two agents are
unclear. The cardiotoxicity may be largely due to the
active metabolite (Way and Schou, 1979; Ulens et al.,
1999).
22. Remacemide. Remacemide is an antiepileptic
agent that undergoes loss of a glycine to an active
metabolite (Fig. 26). While a complete examination of
all input parameters necessary for assessing the
relative contributions to efficacy of parent drug and
active metabolite are not available, it has been shown
that the metabolite is over 100-fold more potent at the
target ion channel and achieves greater free exposure
in the brain than the parent drug (Palmer et al., 1992;
Schachter and Tarsy, 2000). Assuming equivalent
plasma protein binding and target tissue penetration
leads to the estimate that the desglycine metabolite
actually possesses nearly all of the pharmacological
effects, essentially making the parent a prodrug.
23. Risperidone. Risperidone is an effective antipsychotic agent active at several human neurotransmitter receptors, particularly the serotonin-2A and
Fig. 47. Metabolism of fluoxetine to norfluoxetine.
Fig. 48. Metabolism of itraconazole to hydroxyitraconazole.
dopamine-2 types (Schotte et al., 1996). It is extensively metabolized to the active metabolite 9-hydroxyrisperidone (paliperidone) by CYP2D6 (Fig. 27)
(Huang et al., 1993; Fang et al., 1999). The metabolite
possesses intrinsic potency similar to the parent.
Deconvolution of the relative contributions of each to
efficacy is challenging because of potential differences
in brain penetrability caused by P-glycoprotein, for
which both parent and metabolite are substrates
(Wang et al., 2004; Ejsing et al., 2005). In mouse,
risperidone and 9-hydroxyrisperidone brain penetration appear to be hampered 10- and 20-fold, respectively, as shown by comparison of brain/plasma ratios
in multidrug resistance knockout mice. Since CYP2D6
is polymorphically expressed, risperidone demonstrates different exposure levels in EM and PM
subjects; however, this difference does not appear to
manifest itself in different levels of efficacy and this is
attributed to the fact that 9-hydroxyrisperidone is also
an active antipsychotic agent. In EM subjects, it can be
projected that the metabolite carries the large share of
Fig. 49. Metabolism of ketamine to norketamine.
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study done at steady-state roflumilast dosing, along
with values of 0.8 and 2.0 nM for PDE4 inhibition in
vitro (Sanz et al., 2007), with free fractions yields
values for the two terms of:
AUCp •fu;p
IC50;p •t
35:2 mg hr=l•0:01 1000 nmol mmol
•
•
¼ 0:046
¼
0:8 nM•24h
mmol
400mg
roflumilast contribution :
AUCm •fu;m
IC50;m •t
417 mg hr=l•0:03 1000 nmol mmol
•
•
¼ 0:62
¼
2:0 nM•24h
mmol
416mg
roflumilast N-oxide contribution :
Fig. 50. Metabolism of levosimendan to OR-1896.
receptor occupancy (maybe as much as 10-fold), even
with a potentially greater P-glycoprotein-catalyzed
efflux. The efficacy of 9-hydroxyrisperidone has been
leveraged in that this compound is now used as a drug
itself.
24. Roflumilast. The phosphodiesterase (PDE) 4
inhibitor roflumilast is used for chronic obstructive
pulmonary disease (COPD) and it possesses an active
N-oxide metabolite (Fig. 28). A thorough analysis of the
pharmacokinetics of these two and their variability, as
they relate to contribution to activity, has been done
(Lahu et al., 2010). In this analysis the following
equation was used:
tPDE4i ¼
AUCp •fu;p AUCm •fu;m
þ
IC50;p •t
IC50;m •t
in which tPDE4i refers to a unitless value for inhibition of the target enzyme, which is related to the
sum of a parent term and a metabolite term, each of
which contain values for the total exposure, free
fraction, potency, and dosing interval. The contribution
of the N-oxide metabolite was modeled to be greater
than the parent, and interestingly, based on comparisons of healthy volunteers and COPD patients, this
contribution was modeled to be even greater in the
patients. Using exposure values from Böhmer et al.,
(2009) during the control phase of a drug interaction
thus projecting that the N-oxide metabolite contributes 93% of the activity, provided it can penetrate the
target tissue equivalently to the parent.
25. Sibutramine. Sibutramine offers an interesting
example of two active metabolites and an instance
wherein the parent drug probably contributes little
effect. It was used as a weight-loss agent, although
safety problems led to its removal from clinical
practice. Its mechanism of action is purported to be
via action on serotonin, norepinenephrine, and possibly
dopamine transporters as well, although it does not
work in models of depression in which other drugs
possessing these activities work. The most potent
binding is to the norepinephrine transporter, but the
desmethyl and didesmethyl metabolites (Fig. 29)
possess 100- and 60-times greater intrinsic potency
(Cheetham et al., 1996). Furthermore, the metabolites
are present in circulation at concentrations in the same
range as the parent drug (Kim et al., 2009), and the
parent drug is more bound in plasma (fu = 0.03 versus
0.06 for both metabolites) (Meridia, 2010). Thus, the
metabolites are really driving the in vivo activity.
Understanding the relationship among the active
entities can be further confounded by the fact that
sibutramine is administered as a racemate and that
the metabolism can show quantitative differences
between the enantiomers.
26. Tamoxifen. Tamoxifen offers a particularly interesting example of active metabolites in that it had
been used clinically for many years before it was
uncovered that a secondary metabolite, endoxifen, may
contribute the majority of the antiestrogenic activity.
Fig. 51. Metabolism of metoprolol to a-hydroxymetoprolol.
Pharmacologically Active Metabolites
Fig. 52. Metabolism of metronidazole to hydroxymethylmetronidazole.
Endoxifen arises via sequential hydroxylation and
N-demethylation reactions that are catalyzed by
CYP2D6 and CYP3A, respectively (Fig. 30). CYP2D6
generates 4-hydroxytamoxifen, which is also pharmacologically active. In CYP2D6 poor metabolizers or
patients taking CYP2D6 inhibitors, like paroxetine,
the levels of endoxifen will be lower and may reduce
efficacy in these patients (Goetz et al., 2005; Jin et al.,
2005), although this may not be that clear cut (Nowell
et al., 2005; Regan et al., 2012). An examination of the
intrinsic potency of the metabolites and circulating
concentrations would suggest that endoxifen is important in efficacy. While a side-by-side comparison of
tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen,
Fig. 53. Metabolism of morphine to morphine-6-glucuronide.
605
and endoxifen has not been reported, assembling the
data from several reports on these compounds suggests
that 4-hydroxytamoxifen and endoxifen are two orders
of magnitude more potent than their respective nonhydroxylated counterparts (Furr and Jordan, 1984;
Johnson et al., 1989, 2004). Endoxifen is present in
circulation at approximately 1/10th that of tamoxifen,
whereas 4-hydroxytamoxifen is present at about 1/
100th that of tamoxifen. Summing these up, and
assuming similar free fractions (Bourassa et al., 2011),
since affinities for albumin are nearly equivalent, it can
be projected that endoxifen contributes approximately
10 times the receptor activity as tamoxifen, whereas the
contributions of 4-hydroxytamoxifen and tamoxifen are
about equal. N-Desmethyltamoxifen, which quantitatively is the major metabolite present in circulation at
concentrations in the same range as tamoxifen and in
20- to 100-fold greater levels than endoxifen and 4hydroxytamoxifen, would have a contribution similar to
tamoxifen itself. One final interesting point regarding
tamoxifen active metabolites is that a sophisticated
analytical method was developed whereby an estrogen
receptor binding assay was placed in-line with an HPLC
for the facile detection of active estrogen receptor
compounds, and it can be used to detect active
metabolites (Oosterkamp et al., 1996; Kool et al., 2006).
27. Terfenadine. Terfenadine was the first of the
nonsedating antihistamines; however, it was withdrawn from clinical use due to serious cardiac toxicity,
particularly when coadministered with CYP3A inhibitors such as azole antifungals. Terfenadine undergoes
a very high first-pass extraction that results in much
higher exposures to its major carboxylic acid metabolite, fexofenadine (Fig. 31), while terfenadine itself is
barely measurable (Lalonde et al., 1996). Fexofenadine
has about 4-fold lower intrinsic potency at the H1
receptor (Anthes et al., 2002); however, due to its very
high relative exposure, it contributes extensively to in
vivo efficacy. Essentially, terfenadine was a prodrug.
When terfenadine was removed from clinical use, it
was replaced with fexofenadine. The exposure values
for fexofenadine that are efficacious after direct
administration are similar to those achieved following
administration of efficacious doses of terfenadine,
lending support to the notion that terfenadine
exhibited its effect primarily via the active metabolite.
28. Thiocolchicoside. Thiocolchicoside (Fig. 32) is
an unusual derivative of a natural product that has
muscle-relaxant activity believed to be mediated by
binding to GABA receptors. It possesses a glucose
substituent that undergoes deglycosylation to the
phenol intermediate, which is subsequently glucuronidated. After an oral dose that shows efficacy, the
glucuronide is present but the glucose-containing
parent drug is undetectable (Trellu et al., 2004).
Administration of the metabolite directly to rats
yielded the same effect on polysynaptic reflex as the
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Fig. 54. Metabolism of pioglitazone to its hydroxy and ketone metabolites.
same dose of parent drug. Data comparing the in vitro
activity of parent and metabolite was not found, so the
importance of the metabolite to effect in humans must
be inferred.
29. Thioridazine. Thioridazine is an old antipsychotic agent that is still used, but not as much as the
more modern antipsychotic agents. It undergoes Soxidation reactions on the thiomethyl side chain to
form mesoridazine (sulfoxide) and sulforidazine (sulfone) (Fig. 33). Mesoridazine has also been used as
a drug itself. Mesoridazine and thioridazine possess
affinity for the dopamine D2, D3, and D4 receptors
(Richtand et al., 2007), with mesoridazine showing
somewhat greater affinity. After administration of
thioridazine, total exposure to thioridazine is about
6-fold greater than the mesoridazine; however, it has
been shown that the parent drug is vastly more
protein-bound than the metabolite. Values for fu have
been reported to be 0.0016 and 0.09 for parent and
mesoridazine, respectively, in one report (Freedberg
et al., 1979) and 0.0015 and 0.017 in a second report
Fig. 55. Metabolism of praziquantel to trans-4-hydroxypraziquantel.
(Nyberg et al., 1978); the reasons for the difference
reported for mesoridazine are not apparent. However,
in either case, it can be estimated that mesoridazine
contributes virtually all of the receptor occupancy
relative to parent. Additionally, the second S-oxidation
to mesoridazine yields the other active metabolite,
sulforidazine. This metabolite has similar potency and
free fraction as mesoridazine, but it circulates at about
a third the concentration. Nevertheless, it is estimated
that this metabolite also plays a more important role in
efficacy than thioridazine.
30. Tibolone. Tibolone is a steroidal agent that has
activity at the a-estrogen receptor and the androgen
receptor and is used in osteoperoisis and breast cancer
prevention. It undergoes reduction of the 3-keto group
to two stereoisomeric alcohols and isomerization of the
double bond to the 4-position (Vos et al., 2002; Fig. 34).
The alcohols are more potent than parent at the
estrogen receptor, while the alkene isomer is more
potent at the androgen receptor (Escande et al., 2009).
The 3a-hydroxy isomer is present at more than 9-fold
greater concentrations than tibolone (Timmer et al.,
2002) and thus can be estimated to have a 20-fold
greater contribution than tibolone itself, assuming
other distributional aspects are similar between parent
and metabolite. The 3b isomer is also present at
greater concentrations, but not as much as the other
isomer. The alkene isomer metabolite is estimated to
contribute about twice as much androgen receptor
binding as the parent.
31. Tolterodine. Tolterodine, a muscarinic antagonist used to treat urinary incontinence and overactive
bladder, offers an interesting example wherein
a marked difference in plasma protein binding probably contributes to the metabolite contributing a greater
Pharmacologically Active Metabolites
607
Fig. 56. Metabolism of quazepam to 2-oxoquazepam.
share to the efficacy. Tolterodine is metabolized to 5hydroxymethyltolterodine (desfesoterodine; Fig. 35) by
CYP2D6, as demonstrated by marked differences in
exposure to both the parent and the metabolite
between CYP2D6 EM and PM subjects. CYP2D6 EM
subjects have high metabolite and low parent; viceversa for CYP2D6 PM subjects (Brynne et al., 1998).
Both parent and metabolite bind to the target with
similar potency (Yono et al., 1999) and have similar
exposures in EM subjects; however, the metabolite is
10-fold less protein-bound (Pahlman and Gozzi, 1999;
Olsson et al., 2001). Thus, it can be extrapolated that in
EM subjects, the metabolite contributes the lion’s
share of the activity, while in PM subjects the parent
drug would be the major contributor. Furthermore, it
was shown in animal studies that 5-hydroxymethyltolterodine has a lower capability to penetrate the
brain (as assessed using Kp,uu values; Callegari et al.,
2011), thus it could be proposed that the metabolite
would have lower propensity to cause side effects
caused by antimuscarinic activity in the brain, such as
memory impairment. The lack of an impact of CYP2D6
on the metabolism of 5-hydroxymethyltolterodine
served as a rationale for the development of fesoterodine, an ester prodrug of the metabolite (Malhotra
et al., 2009).
32. Tramadol. Tramodol is an opiate used in pain
treatment. It is dosed as a racemate and undergoes Odemethylation to an active metabolite (Fig. 36). The (+)
enantiomer of the active metabolite possesses m-opioid
receptor binding that is over 100-fold greater than the
parent [as a racemate or the (+)enantiomer] (Lai et al.,
Fig. 57. Metabolism of quinidine to 3-hydroxyquinidine.
1996). This biotransformation reaction is catalyzed by
CYP2D6, thus poor metabolizers do not form appreciable amounts of the metabolite and as a consequence
do not experience the same antinociceptive effect
(Paulsen et al., 1996). This also suggests that the
metabolite may be entirely responsible for action,
essentially making tramadol itself a prodrug.
33. Trimethadione. Trimethadione is an old oxazolidinedione antiepileptic agent that undergoes Ndemethylation to dimethadione (Fig. 37). It offers an
interesting example in that N-demethylation results in
the generation of an acidic 2° imide which substantially alters the physicochemical properties of the
molecule. At the neuromuscular junction the effect of
trimethadione and dimethadione differ (Alderdice and
McMillan, 1982). By use of frog tissue, the parent drug
was shown to alter the postjunctional sensitivity,
whereas the metabolite acts by altering acetylcholine
release. The concentrations at which these effects are
demonstrated are relevant to those measured in vivo
for the metabolite; however, the potency of the parent
is not as great and the antiepileptic effects may be due
mostly to the metabolite.
34. Venlafaxine. Venlafaxine is an inhibitor of the
serotonin reuptake transporter that has been used as
an antidepressant. It has an active metabolite, Odesmethylvenlafaxine (Fig. 38) that is also now used as
a drug itself (Deecher et al., 2006). Venlafaxine is
metabolized to the metabolite by CYP2D6, thus
exposure values differ between CYP2D6 EM and PM
subjects with the former having higher metabolite
Fig. 58. Metabolism of saxagliptin to 5-hydroxysaxagliptin.
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Fig. 59. Metabolism of spironolactone to canrenone.
concentrations and the latter having higher parent
drug concentrations (Fukuda et al., 1999). Thus, the
contribution of parent versus metabolite to in vivo
efficacy will differ between the two populations.
Considering the free plasma levels and in vitro
affinities (Owens et al., 1997), in EM subjects the
metabolite can be calculated to contribute almost 4times the activity relative to parent, whereas for PM
subjects the parent can be estimated to contribute
about twice the activity relative to the metabolite. Both
parent and metabolite have been shown to have
equivalent brain partitioning and impact of Pglycoprotein (in mouse; Karlsson et al., 2010), thus it
is not anticipated that different metabolite-versusparent ratios would alter this relationship. The affinity
of both for the norepinephrine transporter is considerably less and a low occupancy of this protein would be
predicted from pharmacokinetic data.
B. Drugs with Active Metabolites That Contribute
Comparably to the Parent
1. Acebutolol. Acebutolol is an antagonist of the
b-adrenergic receptor used in the treatment of
Fig. 60. Metabolism of triamterene to 4-hydroxtriamterene sulfate.
Fig. 61. Metabolism of verapamil to norverapamil.
hypertension and cardiac arrythmias. It is metabolized
by hydrolysis of a butyl amide group followed by
acetylation to diacetolol (Fig. 39), a pharmacologically
active metabolite with a longer half-life than the
parent (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 direct
administration to humans (Ohashi et al., 1981). While
data on the in vitro potency at the human receptor are
not reported, considerable exploration of the relative
effects of acebutolol and diacetolol has been done in
animals and using animal tissues. In a guinea pig
cardiac preparation, the metabolite was shown to have
about 3-fold lower potency (Basil and Jordan, 1982).
At early timepoints following oral administration,
acebutolol and diacetolol plasma concentrations are
Fig. 62. Metabolism of zolmitriptan to N-desmethylzolmitriptan.
Pharmacologically Active Metabolites
609
Fig. 63. Metabolism of alprazolam to 19-hydroxyalprazolam.
about equal, but at later timepoints diacetolol is
present at much greater concentrations. Thus, it is
likely that the parent contributes more activity at early
timepoints (up to Tmax ;4 hours) and the metabolite
contributes at the later timepoints.
2. Acetohexamide. Acetohexamide is reduced to
hydroxyhexamide (Fig. 40), which is also active at
stimulating insulin secretion and lowering blood
glucose. The glucose-lowering effect is better correlated
to the pharmacokinetics of the metabolite, which has
a longer average half-life (4.6 versus 1.3 hours) and
later Tmax (4 hours versus 1) (Smith et al., 1965). The
importance of knowing that an active metabolite can be
responsible for effect is illustrated by the observation
that coadministration of phenylbutazone results in an
enhancement of the effect of acetohexamide, despite no
change in acetohexamide exposure (Field et al., 1967).
Hydroxyhexamide is excreted in urine, and phenylbutazone is known to affect renal clearance of anionic
drugs; thus it can be proposed that the phenylbutazoneacetohexamide interaction is due to an increase in
hydroxyhexamide exposure caused by inhibition of the
renal clearance of the metabolite. The activity of
hydroxyhexamide can be rationalized by comparison
with other members of the sulfonylurea class of drugs.
Tolbutamide, chlorpropamide, and tolazemide possess
Fig. 64. Metabolism of buprenorphine to N-dealkyl and glucuronide metabolites.
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Fig. 65. Metabolism of carisoprodol to meprobamate.
similar structures, with a site of structural variation
being on the 4-position of the central phenyl ring, which
is the site of reduction of acetohexamide to hydroxyhexamide (Gopalakrishnan et al., 2000) (Fig. 40).
3. Amiodarone. Amiodarone is an unusual antiarrhythmic agent that putatively has multiple mechanisms of action, thus delineating the relative roles of
the parent drug and its active desethyl metabolite (Fig.
41) is challenging. Multiple cation channels are
affected by both parent and metabolite as well as
inhibition of the conversion of thyroxine to triiodothyronine (Kodama et al., 1997). Plasma concentrations of
amiodarone and desethylamiodarone associated with
efficacy are about 2 and 1.5 mM, respectively, and due
to high lipophilicity the plasma protein binding is high
(Stäubli et al., 1985; Ujhelyi et al., 1996). In vivo
studies suggest that desethylamiodarone has greater
effects in animal models at equivalent total concentrations and that these effects may be due to a higher
free fraction (Nattel and Talajic, 1988).
4. Aripiprazole. Aripiprazole is a dopamine-receptor
partial agonist used in the treatment of various
psychiatric disorders. It undergoes dehydrogenation of
a dihydroquinolone ring to generate quinolone (Fig. 42).
The metabolite has intrinsic affinity and functional
activity at the dopamine receptors equivalent to the
parent drug (Tadori et al., 2011) and steady-state
concentrations in patients have been shown to be
comparable, with somewhat greater concentrations of
the metabolite (Molden et al., 2006). Protein binding is
very high for both (Abilify, 2012), making a precise
estimate challenging, but if assumed to be equal to the
parent (which is reasonable considering the very
moderate difference in physicochemical properties between the two), it could be estimated that the dehydro
metabolite contributes slightly more than the parent.
Both are substrates of P-glycoprotein and this may alter
estimates of the relative contributions of parent and
active metabolite to efficacy in vivo (Kirschbaum et al.,
2010).
5. Carbamazepine. Carbamazepine is used in the
treatment of epilepsy, as well as some other central
nervous system (CNS) disorders. It is oxidized by
cytochrome P450 enzymes to the active 10,11-epoxide
metabolite (Fig. 43), which subsequently undergoes
hydrolysis to the inactive diol. The mechanism of
action is believed to arise via blockage of voltagedependent sodium channels in the brain, and the in
vitro activity of carbamazepine and the epoxide are
similar (McLean and Macdonald, 1986). Circulating
concentrations of the parent exceed those of the
metabolite by about 5-fold, while the metabolite has
a free fraction about twice that of the parent.
Cerebrospinal fluid (CSF) concentrations have been
measured in humans for both, and consistent with the
total plasma concentrations and free fractions, the
metabolite is present at about one-third of the parent
(Johannessen et al., 1976), suggesting equilibration of
free plasma and CSF levels. Thus, it can be estimated
that the epoxide metabolite will contribute about
a third of the activity in vivo. Interestingly, in a study
in neuralgia patients, blinded replacement of effective
carbamazepine therapy with carbamazpine-10,11epoxide dosing resulted in no alteration in reported
effect on pain (Bertilsson and Tomson, 1984).
6. Clarithromycin. Clarithromycin is metabolized
by CYP3A to 14-hydroxyclarithromycin, which also has
antibacterial activity (Fig. 44). The plasma concentration of the metabolite is about half of that of the parent
Fig. 66. Metabolism of chloroquine to desethylchloroquine.
Pharmacologically Active Metabolites
Fig. 67. Metabolism of citalopram to demethyl metabolites.
drug (Rodvold, 1999), but its in vitro minimally
inhibitable concentration for Haemophilus influenzae
is also about half that of parent (Hardy et al., 1990).
Thus, it can be estimated that the parent and
metabolite contribute equally, depending on the potential for differential tissue penetration.
7. Clobazam. Clobazam is a benzodiazepine drug
that is used as an anxiolytic and anticonvulsant. It
undergoes N-demethylation (Fig. 45) to a metabolite
with one-fifth of the potency (Onfi Product Label). The
metabolite has a longer t1/2 and accumulates to greater
exposures than the parent (Ochs et al., 1984; Bun
et al., 1986). Taking into consideration a 2-fold
611
difference in free fraction, it can be predicted that the
metabolite and parent drug would have approximately
equal contributions to efficacy.
8. Disopyramide. Disopyramide is a class I antiarrythmic agent that blocks the cardiac sodium
channel. It possesses two isopropyl groups on an amine
and undergoes N-dealkylation of one of these to the
active metabolite N-desisopropyldisopyramide (Fig.
46). The metabolite possesses comparable potency for
the channel, but the free plasma concentrations of the
metabolite are about a third of those of the parent (Hill
et al., 1988; Sheldon et al., 1994b). [The parent drug
has been shown to be subject to saturable plasma
protein binding (Hinderling et al., 1974), so the
contribution of the metabolite may increase as the
parent drug concentration decreases.] An estimate can
be made from these data that the metabolite contributes about a third of the activity of the parent drug
(Hinderling and Garrett, 1976).
9. Fluoxetine. Fluoxetine has a major active metabolite norfluoxetine (Fig. 47). The half-life of the
metabolite is about a week, as compared with the
parent drug, which has a half-life of about 2 days
(Aronoff et al., 1984). CYP2D6 has been shown to have
a role in the conversion of fluoxetine to norfluoxetine,
and poor metabolizers have less of the metabolite in
circulation (Llerena et al., 2004; Scordo et al., 2005).
Also, after repeated dosing, CYP2D6 in EM subjects
becomes inhibited so that CYP2C9, another enzyme
involved in fluoxetine metabolism, plays an increasing
role in fluoxetine clearance as shown by comparison of
pharmacokinetics in subjects containing a defective
CYP2C9 allele (Scordo et al., 2005). Affinity for the
human serotonin uptake transporter is almost the
same between the parent and metabolite, and concentrations of the two are also similar (Owens et al., 1997;
Scordo et al., 2005). Initially, fluoxetine was approved
as a once-per-day drug—recognition that the norfluoxetine metabolite possesses the same activity while
having a longer half-life led to the introduction of
a once-per-week formulation. Brain permeation for
both parent drug and metabolite was high and the
residence time was long with a half-life the same as in
plasma (as assessed with 19F NMR for combined
parent and metabolite; Bolo et al., 2000).
10. Itraconazole. The antifungal agent itraconazole
undergoes metabolism to hydroxyitraconazole (Fig.
48). In vitro the metabolite possesses similar antifungal activity (Mikami et al., 1994; Odds and Bossche,
2000). It also circulates in plasma at levels similar to
parent (Barone et al., 1998), so it can be estimated that
the metabolite and parent drug will have a similar
contribution 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 also
possesses N-methyl-D-alanine–receptor binding activity
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Fig. 68. Metabolism of cyclosporine to active hydroxylated metabolites.
(Fig. 49) (Ebert et al., 1997). After oral administration,
norketamine exposure values are greater than ketamine values and it could be estimated that the
metabolite may contribute almost equally to effect
(Hijazi and Boulieu, 2002). However after intravenous
administration, exposures are about equal, and the
contribution to activity would be mostly due to the
parent compound (Clements et al., 1982). Ketamine is
thus illustrative of the potential impact the route of
administration may have on metabolite exposures and
hence contribution of a metabolite to pharmacological
effect. For drugs that undergo high first pass metabolism (hepatic and/or intestinal), there can be a large
amount of active metabolite generated following oral
administration. Metabolite/parent ratios can differ, and
this is illustrated by the example of ketamine.
12. Levosimendan. Levosimendan is an intravenously administered inotropic agent for heart failure
patients, believed to cause its effect via sensitization of
troponin-C to Ca2+ binding, although the mechanism is
not definitively proven. It possesses a propanedinitrilesubstituted hydrazinylidene substituent that is removed via reduction of the N–N bond by gut microflora
to yield an intermediate aniline metabolite that subsequently undergoes N-acetylation to the active metabolite OR-1896 (Fig. 50). While the half-life of
levosimendan is short (;1 hour), the active metabolite
has a half-life of a few days, and the beneficial effect is
observed for considerably longer than what the parent
half-life would indicate, suggesting an important role
for OR-1896 in the extended duration of activity
(Antoniades et al., 2007). In contrast to this, no
apparent difference in efficacy has been observed
between poor and extensive acetylators despite a difference in exposure to OR-1986 between the two
groups (Antila et al., 2004; Kivikko et al., 2011).
Delineating the relative contributions of OR-1896 to
levosimendan is not straightforward; the pharmacokinetics are such that during the intravenous infusion,
the parent drug concentrations are far greater than the
Pharmacologically Active Metabolites
613
Fig. 69. Metabolism of dabigatran to its acyl glucuronide.
metabolite and cardiovascular effect is observed
(Kivikko et al., 2002). Upon cessation of the infusion,
levosimendan concentrations drop rapidly. OR-1896
concentrations slowly increase to levels about 10% of
those that were observed for the parent drug during
the infusion phase, presumably due to biliary secretion
of parent, reduction in the gut by microflora, absorption of the reduced intermediate, and acetylation to
yield OR-1896. OR-1896 concentrations are measureable for days, while levosimendan levels are undetectable. Thus, it may be the case that levosimendan
parent is responsible for the hemodynamic effects
during infusion and the metabolite takes over
postinfusion.
13. Metoprolol. Metoprolol is a b-blocker that is
subject to a pharmacokinetic difference in CYP2D6 EM
and PM subjects. It undergoes extensive metabolism to
mostly inactive metabolites with the exception of the
hydroxylation to a-hydroxymetoprolol, an active metabolite by CYP2D6 (Kaila and Iisalo, 1993) (Fig. 51).
Delineation of the temporal relationship between the
pharmacokinetics of metoprolol (short t/1/2, 3–4 hours),
a-hydroxymetoprolol (t/1/2, 7–10 hours), and the effect
on tremor measured over time in humans suggests
that 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 observed
that the pharmacodynamic responses in EM versus PM
Fig. 70. Metabolism of dasatinib to dealkyldasatinib.
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Fig. 71. Metabolism of diazepam and its metabolites.
subjects do not differ to the extent that the pharmacokinetic differences in parent drug exposure between
these two groups would suggest; i.e., the PK/PD
relationship for metoprolol differs between EM and
PM subjects in that EM subjects have lower metoprolol
concentrations but the efficacy is not diminished
(Jonkers et al., 1991). This also supports the presence
and importance of an active metabolite.
14. Metronidazole. The nitroimidazole drug metronidazole is an antibacterial that is metabolized by
hydroxylation of the methyl substituent and this
metabolite also has bacteriocidal activity (Fig. 52).
The relative activities depend on the species and
strain. For Gardnerella vaginalis, the metabolite has
been reported to be 8-fold more efficacious (as determined by comparison of minimum inhibitory
concentration; Ralph and Amatnieks, 1980), whereas
for Bacteroides fragilis the parent is more potent than
metabolite (Ralph and Amatnieks, 1980; Pendland
et al., 1994). The parent is present at greater
circulating concentrations and neither entity has
appreciable plasma protein binding (Easmon et al.,
1982). For G. vaginalis, an estimate that the metabolite and parent contribute nearly equally can be
made, whereas for B. fragilis it is the parent drug that
should dominate the efficacy. Fractional inhibitory
concentration values were calculated using in vitro
activity and in vivo exposure data and were about 0.7
for the effect of the metabolite for B. fragilis (Pendland et al., 1994).
15. Morphine. Morphine represents a fascinating
example of the potential for an active metabolite to
contribute to the pharmacological activity of the parent
drug because it has been well studied, and the reader
interested in a greater level of detail is directed to
a thorough review by Kilpatrick and Smith (2005).
Morphine is metabolized by glucuronidation to the 3and 6-glucuronides (Fig. 53). The 3-glucuronide is the
major circulating metabolite in humans but does not
possess activity at the m-opioid receptor. Morphine-6glucuronide possesses nearly equivalent in vitro receptor activity (binding and agonism) as the parent
drug. Morphine-6-glucuronide is present in human
circulation at about the same concentrations as
morphine after intravenous administration of the
latter, and it has been estimated that about 10% of
morphine is cleared by the 6-glucuronidation route
(Lotsch et al., 1996). The analgesic effects of morphine-
Pharmacologically Active Metabolites
615
Fig. 72. Metabolism of diltiazem to desmethyl and desacetyl metabolites.
6-glucuronide have been studied directly in humans
following intravenous administration of the metabolite
and such data offer the best approach to delineate
whether a metabolite can contribute to activity in vivo.
(This represents a rather unique situation as it is rare
that a metabolite is administered directly to humans,
unless the metabolite is being tested as a drug itself.)
Morphine-6-glucuronide showed good efficacy at standard endpoints of opioid activity, albeit the central
effects occurred later (6–8 hours), relative to morphine
itself (2–3 hours) (Lotsch et al., 2001; Murthy et al.,
2002; Skarke et al., 2003). This could be due to either
a slow uptake into human brain or a delayed egress of
the metabolite from the brain. Based on physicochemical attributes, high penetration of glucuronide
metabolites into the brain would be unexpected;
however in the case of morphine-6-glucuronide it has
been suggested that a folded conformation may be
adopted that reduces hydration and increases lipophilicity (Gaillard et al., 1994). In later work, the
partitioning of unbound drug into brain (Kp,uu), a metric
used to assess the potential for free-plasma and freebrain (hence efficacious) concentrations, showed a value
for morphine-6-glucuronide that was far lower than
that for morphine (in rat) (Friden et al., 2009). Animal
studies suggest that drug transport may be involved in
morphine-6-glucuronide brain disposition, but data in
humans are lacking or inconclusive. Furthermore,
interpretation can be confounded by the possibility
that morphine is glucuronidated within the brain, as it
Fig. 73. Metabolism of donepezil to 6-O-desmethyldonepezil.
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Fig. 74. Metabolism of granisetron to 7-hydroxygranisetron.
has been shown in vitro that human brain tissue is
capable of this (Yamada et al., 2003).
16. Pioglitazone. Pioglitazone is a peroxisome
proliferator-activated receptor–g agonist useful in the
treatment of diabetes. It is metabolized on the two
benzylic positions adjacent to the pyridine to hydroxyl
and ketone metabolites (Fig. 54) (Krieter et al., 1994;
Actos, 2011). The potencies of these two metabolites
have been measured in a cellular lipogenesis assay and
mouse glucose lowering assay (Sohda et al., 1995;
Tanis et al., 1996; Young et al., 1998) and shown to be
equivalent to the activity of pioglitazone itself. Protein
binding is comparable and the metabolites are present
in circulation at concentrations of 10–50% of parent
(Kalliokoski et al., 2008). As such, it can be estimated
that these two metabolites combined can make
a considerable contribution to the activity of pioglitazone, 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 vitro
potency of the metabolite at killing the parasite is 60fold less than parent (Ronketti et al., 2007); however, it
is present at over 20-fold greater exposure (Westhoff
and Blaschke, 1992; Cioli et al., 1995). If free fractions
are assumed to be nearly equal, it can be estimated
that the metabolite may contribute about a third of the
effect of the parent drug.
18. Quazepam. Quazepam is a benzodiazepine anxiolytic with an unusual thioamide structure. Metabolism results in the replacement of the =S to =O, the 2oxoquazepam 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-thirds
of the parent (Chung et al., 1984). As such, it can be
estimated that 2-oxoquazepam contributes substantially to the total activity.
19. Quinidine. Quinidine has been used for decades in the management of cardiac arrhythmias.
Despite this length of use, its molecular mechanism
is largely unknown, although it is known to interact
with several ion channels (Na+ and K+). It gives rise to
the active 3-hydroxyquinidine metabolite (Fig. 57).
Following oral quinidine administration, concentrations of the metabolite are not as great as quinidine;
however, it has lower plasma protein binding and
thus the free concentrations of quinidine and 3hydroxyquinidine are about the same (Wooding-Scott
et al., 1988). 3-Hydroxyquinidine has been administered to humans directly, with measurement of
cardiac pharmacodynamic endpoints (Vozeh et al.,
1985), which is an excellent way to gather data that
can be used in estimating the contribution of the
metabolite to the parent drug action. When administered directly, plasma concentrations of 1.5 mM were
shown to yield similar effects to those observed when
quinidine was administered, and 1.5 mM concentrations of metabolite are similar to those observed after
quinidine administration. Thus, it can be concluded
that 3-hydroxyquinidine contributes equivalently to
quinidine in effect.
20. Saxagliptan. Saxagliptan is an antihyperglycemic dipeptidyl peptidase-4 inhibitor for the treatment
of diabetes. It possesses a hydroxyl adamantyl substituent, and metabolic introduction of a second hydroxyl group yields 5-hydroxysaxagliptan (Fig. 58),
which retains about half of the intrinsic potency (Fura
et al., 2009). In consideration of the SAR for the parent
drug (Augeri et al., 2005), it is not surprising that the
addition of a second hydroxyl to this substituent does
not abolish activity. The metabolite circulates at
concentrations about twice that of the parent and
neither compound is bound to plasma proteins
(Onglyza, 2011), thus the 2-fold difference in potency
and 2-fold difference in exposure essentially cancel
each other out and lead to the conclusion that parent
Fig. 75. Metabolism of halofantrine to desbutylhalofantrine.
Pharmacologically Active Metabolites
Fig. 76. Metabolism of imatinib to N-desmethylimatinib.
and metabolite could contribute to in vivo efficacy
about equally, provided that free target tissue concentrations are similar to free plasma concentrations.
21. Spironolactone. Spironolactone is an aldosterone inhibitor used as a diuretic in hypertension. It
possesses a thioacetate group that is converted to an
alkene bond in the metabolite canrenone (Fig. 59)
(Abshagen et al., 1976). Canrenone itself is used as
a drug, as well as an analog in which the lactone ring is
open and dosed as the potassium salt (canrenoate). It is
not entirely clear regarding all the entities’ contributions to activity and whether activity is better related
to plasma or urinary concentrations; however, canrenone has been estimated to contribute to 73% of the
activity (Ramsay et al., 1977). While spironolactone
and potassium canrenoate yield similar levels of
canrenone, spironolactone is more potent, suggesting
that other spironolactone metabolites may contribute
to activity (Kojima et al., 1985; Peile, 1985). Spironolactone had been shown to generate metabolites that
lost the acetyl group but retained the sulfur (Abshagen
et al., 1976). A comparison of the concentrations of
spironolactone and canrenone (Dong et al., 2006) shows
that the metabolite is present at about 3-fold excess at
maximum plasma concentration but vastly greater
concentrations at later timepoints. When correcting for
relative free fractions (Chien et al., 1976) and intrinsic
potencies (Funder et al., 1976), it would suggest that
617
canrenone contributes a quarter of the activity of
parent. At later timepoints, this contribution would
increase.
22. Triamterene. Triamterene is used in the treatment of hypertension as a potassium-sparing antihypertensive acting at sodium channels. It is frequently
co-dosed with antihypertensives that cause loss of
potassium. A major circulating metabolite in humans
arises by sequential hydroxylation and sulfation
reactions, 4-hydroxytriamterene sulfate (Fig. 60), with
concentrations in excess of 10-times those of the parent
drug (Sörgel et al., 1985). The metabolic reactions are
catalyzed by CYP1A2 and platelet phenol sulfotransferase. Accounting for differences in free fraction and
estimates of intrinsic potency (Busch et al., 1996), it
can be estimated that the metabolite contributes
approximately half of the activity of the parent drug.
The metabolite has been shown to be active in animal
models (Voelger, 1991).
23. Verapamil. Verapamil undergoes extensive metabolism, including a major N-demethylation pathway
that yields the active circulating metabolite norverapamil (Fig. 61). The metabolite has been shown to be active in vivo and an estimate of its intrinsic potency at the
Ca2+ channel places it approximately 5-fold less active
(Ferry et al., 1985; Johnson et al., 1991). It circulates at
a greater concentration than parent and is slightly less
protein-bound in plasma (Yong et al., 1980; Powell et al.,
1988), thus it can be estimated that it contributes about
a third of the activity of the parent drug.
24. Zolmitriptan. Zolmitriptan is a serotonin receptor agonist used in the treatment of migraine. It
undergoes N-demethylation to yield a secondary amine
metabolite that is claimed to be twice as intrinsically
potent (Fig. 62) (Jandu et al., 2001). Following oral
administration, the metabolite circulates at about half
the level of the parent drug (Dixon et al., 1997; Peck
et al., 1998), thus it can be projected that the
contributions of zolmitriptan and the N-desmethyl
metabolite are about equal.
C. Drugs with Metabolites That Possess Target
Potency But Contribute Little to In Vivo Effect
In some cases, metabolites are identified in laboratory animals or in vitro systems and are then shown
to have some affinity to the receptor targeted by the
Fig. 77. Metabolism of lidocaine to its deethylated metabolites.
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Fig. 78. Metabolism of lumefantrine to desbutyllumefantrine.
parent drug. However, when examined in humans in
vivo, it is shown that the metabolite is estimated to
actually contribute little effect. This can be due to low
in vivo concentrations, higher plasma protein binding,
or a lower ability to enter the target tissue.
1. Alprazolam. Alprazolam is a highly used anxiolytic agent that is cleared by oxidative metabolism and
renal excretion. Metabolism is on the methyl group on
the triazole (19-hydroxyalprazolam) and on the diazepine (4-hydroxyalprazolam), with the 19-hydroxyalprazolam possessing intrinsic binding potency that is
about 3-fold lower than the parent drug (Richelson
et al., 1991; Fig. 63). The metabolite is present at far
lower concentrations in plasma (Smith and Kroboth,
1987; Garzone and Kroboth, 1989) and it is thus
unlikely to contribute significantly to the pharmacological effects of alprazolam.
2. Buprenorphine. Buprenorphine is an opioid
agent that acts as a partial agonist at the m receptor
and also binds the k subtype as well. Data regarding
the contribution to activity of the metabolites norbuprenorphine and buprenorphine 3-glucuronide (Fig.
64) have only recently emerged (Brown et al., 2011),
Fig. 79. Metabolism of macitentan to ACT-132577.
and thus the full picture of data needed to assess the
relative contributions of parent and metabolites to in
vivo effect in humans has not emerged. The drug is
typically administered intravenously, sublingually, or
transdermally, and plasma exposure to the metabolites
is less than that for the parent drug (Polettini and
Huestis, 2001). Protein binding values have not been
reported for the metabolites. In postmortem brain
samples, it has been shown that buprenorphine is
present in the brain, but the desmethyl and glucuronide metabolites were undetectable (Elkader and
Sproule, 2005). Furthermore, it has been shown that
norbuprenorphine is a substrate of P-glycoprotein,
which may diminish brain exposure (Tournier et al.,
2010). Thus, while buprenorphine metabolites may
show intrinsic receptor activity, in vivo data suggest
that only the parent drug contributes to the effect.
3. Carisoprodol. Carisoprodol is a sedative and
muscle relaxant that yields an active metabolite
meprobamate, which itself was a drug used as an
anxiolytic in the 1960s (Fig. 65). The mechanism of
action is not entirely clear, although some activity may
be mediated through the GABAA receptor (Gonzalez
et al., 2009). The conversion of carisprodol to meprobmate is partially catalyzed by CYP2C19 (Dalen et al.,
1996), an enzyme subject to genetic polymorphism, and
thus concentrations of parent drug and metabolite
differ between extensive and poor metabolizers (Olsen
et al., 1994). If GABAA is the receptor responsible for
the activity, it can be estimated that meprobamate
contributes about 10% of the activity; less in CYP2C19
poor metabolizers.
4. Chloroquine. The antimalarial agent chloroquine
is converted to an active N-desethyl metabolite (Fig.
66). The metabolite is present in human circulation at
about a third of the concentration of the parent drug
following a single dose. The mean residence time for
the metabolite is longer than for the parent drug, so
after repeated administration the metabolite concentrations will increase more than the parent. Incorporating the differences in free fraction and intrinsic
potency, it can be estimated that the desethyl
Pharmacologically Active Metabolites
Fig. 80. Metabolism of mexiletine to hydroxylated metabolites.
metabolite contributes about 20% to activity (OforiAdjei 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 in
both its racemic forms and as the S-isomer alone
(escitalopram). It is metabolized via sequential Ndemethylation reactions to yield N-desmethylcitalopram and didesmethylcitalopram (Fig. 67). The metabolites have lower affinity for the reuptake transporter
than the parent (6- to 8-fold) (Owens et al., 1997;
Deupree et al., 2007) and circulate at lower steadystate concentrations than the parent (Sidhu et al.,
1997). Thus, the metabolites contribute very little to
the antidepressant effect of citalopram.
6. Cyclosporine. The immunosuppressant cyclosporine undergoes complex metabolism, and considerable attention has been paid to whether it is important
to measure metabolites during therapeutic monitoring
of this drug (Santori et al., 1997; Ozbay et al., 2007).
Among the metabolites identified, two hydroxylated
metabolites possess measureable in vitro activity
Fig. 81. Metabolism of mianserin to N-desmethylmianserin.
619
(Copeland et al., 1990; Fig. 68). Combining blood
concentrations (Christians et al., 1991) and in vitro
potencies, and assuming similar tissue penetrability of
the metabolites and cyclosporine [which, considering
the very minor alteration introduced by metabolism
into such a large molecule, along with little difference
between plasma binding (Kodobayashi et al., 1995), is
likely a reasonable assumption], it can be estimated
that the two metabolites probably only contribute
5–15% to efficacy.
7. Dabigatran. Dabigatran is a thrombin inhibitor
intended to prevent deep vein thrombosis. It is orally
administered as a carbamate ester of an amidine that
yields dabigatran, which is the major entity in
circulation. Dabigatran undergoes glucuronidation of
its carboxylic acid group and the resulting 1-Oacylglucuronide (Fig. 69) as well as its rearrangement
products were all shown to cause prolongation of
thromboplastin time. It was hypothesized that the
COOH group is remote from the amidine group that is
essential for target binding, thus alteration of the
COOH, even with a change as large as esterification of
glucuronic acid, would not affect activity (Ebner et al.,
2010). However, in humans the circulating concentrations of glucuronides are less than a tenth of that for
dabigatran itself (Blech et al., 2008; Stangier et al.,
2008), thus while this represents an interesting
example of a glucuronide conjugate that retains in
vitro potency, its relevance to in vivo effect is minimal.
8. Dasatinib. Dasatinib is a second-generation
kinase inhibitor affecting the Bcr-Abl Src family of
enzymes and is used in the treatment of leukemia. It
undergoes extensive metabolism (including an N-dealkylation of an ethanol side chain) to yield a metabolite
with equivalent intrinsic potency (Christopher et al.,
2008) (Fig. 70). The free fraction of the metabolite is
almost twice as high (Sprycel, 2011); however, the
circulating concentrations of the metabolite are far
lower than the parent (Furlong et al., 2012), thus the
metabolite likely contributes less than 10% to the
activity.
9. Diazepam, Temazepam, and Oxazepam. Diazepam is metabolized to several metabolites via oxidation
reactions, and three of these metabolites have
been used as drugs themselves. Diazepam undergoes N-demethylation and this metabolite is the major
circulating metabolite, relative to temazepam (a
hydroxyl metabolite) and oxazepam (the corresponding
hydroxyl metabolite of desmethyldiazepam) (Ghabrial
et al., 1986; Rouini et al., 2008) (Fig. 71). All three
metabolites have lower affinity for the benzodiazepine
receptor (4- to 7-fold; Richelson et al., 1991) and
circulate at concentrations much lower than the parent
drug (8- to 50-fold lower). With only small differences
in free fraction, it is unlikely that any of these
metabolites contribute substantially to the effects of
diazepam; even when summed the contributions of the
620
Obach
Fig. 82. Metabolism of midazolam to 19-hydroxymidazolam and its glucuronide.
active metabolites are less than 10% of that of parent
drug. The half-lives for temazepam and oxazepam are
shorter than diazepam, indicating formation-rate limitation, while the half-life for N-desmethyldiazepam is
similar to that of diazepam.
10. Diltiazem. Diltiazem represents an interesting
example of active metabolites. The N-desmethyl and
desacetyl metabolites have been a focus as contributing
to activity (Fig. 72). These circulate at concentrations
that are lower than the parent (Montamat and
Abernethy, 1987) and are comparably protein-bound
(Boyd et al., 1989). The in vitro potency values of the
metabolites relative to the parent drug are nominally
lower (Li et al., 1992), thus overall the contribution of
metabolites to the efficacy of diltiazem is probably
quite low. It is interesting to note that in a single-dose
study a clockwise hysteresis was observed for diltiazem
concentrations (i.e., decreased effect at the same
concentrations at a later time), and the authors
proposed the possibility of metabolites that antagonize
the P-R interval–prolongation effect of diltiazem,
among other possibilities (Boyd et al., 1989). Such an
effect of the desmethyl and desacetyl metabolites
would not be consistent with their diminished activity
at the Ca2+ channel but would need to be due to an
alternate target.
Fig. 83. Metabolism of mirtazepine to N-desmethylmirtazepine.
11. Donepezil. Donepezil, an acetylcholinesterase
inhibitor used in the treatment of dementia, possesses
an active metabolite that arises via O-demethylation of
the 6-methoxy substituent on the indanone ring (Fig.
73). The metabolite is reported as possessing equivalent intrinsic potency (Sugimoto et al., 1995); however,
when plasma concentrations have been measured, they
are far below those measured for the parent compound
(at least 150-fold), thus it is unlikely that 6-Odesmethyldonepezil actually contributes to in vivo
efficacy (Okereke et al., 2004; Patel et al., 2008).
12. Granisetron. Granisetron is a serotonin-3 receptor antagonist used as an antiemetic in cancer
patients undergoing treatment with emetogenic chemotherapeutics (Fig. 74). It undergoes hydroxylation
on the indazole ring at the 7-position (Clarke et al.,
1994) to yield a metabolite with slightly greater target
potency than the parent drug (Vernekar et al., 2010).
After both oral and intravenous administration, the
metabolite is present at about 10% of that of the parent
(Clarke et al., 1994; Boppana, 1995). Assuming similar
free fractions, it can be estimated that the metabolite
would contribute about 20% of the activity of the
parent.
13. Halofantrine. Halofantrine is an antimalarial
agent that is not used very much due to QT-interval
prolongation. However, it has a major circulating
metabolite (Milton et al., 1989; Veenendaal et al.,
1991) that possesses some in vitro activity as well,
desbutylhalofantrine, in which one of the two butyl
substituents are removed (Fig. 75) (Bhattacharjee and
Karle, 1998). While protein binding of desbutylhalofantrine has not been reported, if potency and total
concentrations are compared, the metabolite could
have about a quarter of the activity.
14. Imatinib. Imitinib is a protein–tyrosine kinase
inhibitor selective for the v-ABL type as well as
potency for the platelet-derived growth factor kinase.
Pharmacologically Active Metabolites
Fig. 84. Metabolism of primidone to phenobarbital.
It undergoes N-demethylation by CYP3A4 (Fig. 76),
and the metabolite circulates at approximately 10–15%
of levels of the parent drug. Other attributes (free
fraction, intrinsic potency) are the same between the
parent and metabolite, thus it can be projected that the
parent will dominate the contribution to activity by
virtue of its greater exposure (Kretz et al., 2004; Le
Coutre 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 reactions to yield putatively active metabolites monoethylglycine xylidide (MEGX) and glycine xylidide (Fig. 77).
The metabolites are not as active at sodium channel
block as the parent drug using rat in vitro data
(Sheldon et al., 1991); however, after comparison to
621
free circulating concentrations it can be estimated that
MEGX contributes about a fifth of the activity while
glycine xylidide contributes very little. MEGX is very
poorly bound to plasma proteins so free concentrations
are almost equal to parent and the potency is about
one-fourth that of parent. Given earlier animal data, it
had been concluded that MEGX might contribute more
to the activity (Drayer et al., 1983).
16. Lumefantrine. The example of lumefantrine is
very similar to that of halofantrine. Similar to
halofantrine, it undergoes N-dealkyation to desbutyllumefantrine (Fig. 78). The metabolite has 6-fold
greater in vitro potency at killing Plasmodium falciparum but is present in circulation of patients undergoing successful treatment of malaria at about 1/
20th the concentration of the parent compound (Wong
et al., 2011). Plasma binding has not been reported, but
if assumed to be equal, it can be predicted that the
metabolite carries about 30% of the activity of the
parent.
17. Macitentan. Macitentan is an endothelin antagonist being developed to treat pulmonary arterial
hypertension. It has an N-propylsulfonylurea that
undergoes N-depropylation to yield an active metabolite that possesses about an eighth of the intrinsic
activity of the parent (Fig. 79) (Iglarz et al., 2008). In
humans, the metabolite is present at about 50%
Fig. 85. Metabolism of propafenone to hydroxylated and N-dealkylated metabolites.
622
Obach
Fig. 86. Metabolism of propranolol to 4-hydroxypropranolol.
greater plasma concentrations (Sidharta et al., 2011).
The free fraction is unreported, so making an estimate
of the contribution of the metabolite relative to parent
suggests 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 is
converted to several hydroxyl metabolites (Paczkowski
et al., 1992), two of which have been shown to have in
vitro activity (p-hydroxymexiletine and hydroxymethylmexiletine) (Fig. 80) (Catalano et al., 2004; DeBellis
et al., 2006). While the free fraction for the parent is
known, there is no report of the free fraction for the
metabolites, so the comparison on contributions to
activity in vivo must be made based on total concentrations. Neither metabolite is projected to contribute
more than 10% of activity, and even if they are assumed to be entirely free, their contributions would not
exceed 20% of the parent drug.
19. Mianserin. Mianserin is a tetracyclic antidepressant that is metabolized to its N-demethyl analog
(Fig. 81). It binds many receptors, the a2-adrenergic
being one of the most important. Upon demethylation,
the intrinsic affinity for a2 decreases by about 3-fold
(Nickolson et al., 1982). Circulating concentrations of
the metabolite are about 60% of those of parent (Reis
et al., 2009). The free fraction of the parent is available
(Kristensen et al., 1985), but for the metabolite it is not
reported, so estimation of relative contributions to
activity can only be made assuming that protein
binding and central penetration are equivalent. Under
these assumptions, and also assuming that the a2adrenergic receptor is the most relevant for effect, it is
estimated that the metabolite contributes about 25% of
the efficacy of the parent.
20. Midazolam. Midazolam is used as an anesthetic
agent frequently by the intravenous route. It is initially
metabolized to 19-hydroxy and 4-hydroxy metabolites
(Fig. 82), with the former possessing intrinsic binding
activity to the benzodiazepine receptor similar to that
of the parent drug (Richelson et al., 1991). Furthermore, the glucuronide conjugate of 19-hydroxymidazolam may also contribute to activity, based on the
observation of prolonged activity in renal insufficiency
patients who are less able to renally clear this
metabolite (Bauer et al., 1995). Plasma protein binding
of the glucuronide metabolite is considerably lower
than midazolam; however, the ability of the glucuronide metabolite to penetrate the brain relative to the
parent is unknown, thus precluding a true assessment
of the relative contributions of parent and metabolite to
effect. The 19-hydroxymidazolam metabolite has been
shown to have activity in humans following its direct
administration, which is a highly valuable approach to
understanding the activity of a metabolite. However, it
is infrequently carried out (Mandema et al., 1992). This
activity was comparable to midazolam itself. However,
the clearance of 19-hydroxymidazolam is high and thus
Fig. 87. Metabolism of rifampin to 23-deacetylrifampin.
Pharmacologically Active Metabolites
623
Fig. 88. Metabolism of rizatriptan to monodesmethylrizatriptan.
exposure to this metabolite after administration of
midazolam is a lot lower than exposure to midazolam
itself. Thus, the contribution of 19-hydroxymidazolam
to the sedative/anesthetic properties of midazolam is
likely to be low.
21. Mirtazapine. Mirtazepine, a close analog of
mianserin described above, also undergoes N-demethylation to an active metabolite with reported 3- to 4fold lower intrinsic potency than the parent drug (Fig.
83) (Stimmel et al., 1997). Mirtazepine acts by
antagonizing the a2-adrenergic receptor, which results
in downstream effects on serotonin and norepinephrine
release (de Boer et al., 1988). It also binds to some
serotonin receptors and the H1 receptor. The metabolite is present at about half the exposure of the parent
drug (Shams et al., 2004), but the role that the
metabolite can play in all of these activities has not
been described in the literature. For the a2-adrenergic
receptor activity, if it is assumed that the free fraction
Fig. 89. Metabolism of rosuvastatin to N-desmethylrosuvastatin.
does not differ between parent and metabolite and
brain penetration is equal, it would be anticipated that
the metabolite would contribute less than 20% of the
activity. A more accurate assessment would require
further investigation.
22. Primidone. Primidone is a relatively old drug
that is used in the treatment of epilepsy. It is oxidized
to phenobarbital (Fig. 84), a drug itself, but a metabolite when primidone is the parent drug. Affinity for the
GABA receptor is approximately 2.5-fold greater for
primidone (Ticku and Olsen, 1978), and phenobarbital
concentrations following oral primidone are lower than
the parent drug (Martines et al., 1990). Plasma protein
binding is comparable between the two compounds, so
if brain penetration is equivalent it can be estimated
that phenobarbital contributes about a quarter of the
activity. A second metabolite, phenylethylmalonamide
is present in circulation but does not contribute to in
vivo activity.
23. Propafenone. Propafenone is a sodium channel
blocker used in the treatment of cardiac arrhythmias.
It is metabolized by the polymorphic enzyme CYP2D6
to 5-hydroxypropafenone (Fig. 85), which is an active
metabolite (Zoble et al., 1989). Propafenone is subject
to a supraproportional exposure-dose relationship in
CYP2D6 extensive metabolizers that is not shown in
poor metabolizers, consistent with saturation of
CYP2D6-catalyzed first-pass metabolism (Siddoway
Fig. 90. Metabolism of sertraline to N-desmethylsertraline.
624
Obach
Fig. 91. Metabolism of triazolam to 19-hydroxytriazolam.
et al., 1987). At the higher doses, EM and PM
differences are about 2-fold and no dose adjustment
is made between the two populations. 5-Hydroxypropafenone, which is also subject to nonlinear clearance
(Vozeh et al., 1990), has about half the intrinsic
potency as the parent and is present at about 4-fold
lower unbound exposure. Thus, it does not contribute
much to the efficacy of propafenone. The N-despropyl
metabolite is also weakly active in vitro (Oti-Amoako
et al., 1990) but is present at even lower concentrations
in vivo and therefore would not be expected to
contribute to in vivo effect.
24. Propranolol. Propranolol is oxidized to 4hydroxypropranolol (Fig. 86) and this metabolite is
present in human circulation following administration
of 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-adrenergic
receptor (compared using turkey as the source species
for receptor; Bilezikian et al., 1978), so it is unlikely
that the metabolite has any substantive contribution to
activity.
25. Rifampin. Rifampin (rifampicin) is an antibiotic
drug that has been used for decades to treat tuberculosis. It undergoes hydrolysis of an acetyl group at the
23-position to yield a metabolite that has similar
efficacy against Mycobacterium (Fig. 87; Tenconi
et al., 1970) and increased potency against many other
bacteria (Nakazawa et al., 1970). After oral administration, 23-desacetylrifampicin is present in circulation
at about 1/12th the concentrations (Loos et al., 1985;
Tenconi et al., 1970). Plasma protein binding for the
Fig. 92. Metabolism of valproic acid to 2-propyl-2(E)-pentenoic acid.
metabolite is not reported, but if assuming a similar
value (i.e., 81% bound; Kochansky et al., 2008), then it
can be assumed that the metabolite contributes less
than 10% to the antibacterial activity in vivo.
26. Rizatriptan. Rizatriptan is an antimigraine
drug that acts by binding serotonergic receptors (5HT1B and 5-HT1D) with high affinity (Hargreaves,
2000). The mono N-desmethyl metabolite (Fig. 88) has
intrinsic potency that is about 3-fold lower. Its
contribution to in vivo efficacy is probably very low or
negligible, since circulating concentrations are about
one-tenth those of the parent drug (Goldberg et al.,
2000). Consideration of any potential free-fraction
differences are not necessary to understanding the
effect of the metabolite since rizatriptan itself is mostly
free in plasma (Maxalt, 2011).
27. Rosuvastatin. Rosuvastatin is a widely used
lipid-lowering agent that inhibits 3-hydroxy-3methylglutaryl-coenzyme A (HMGCoA) reductase,
with an inhibitory potency greater than other clinically
used compounds of this class (McTaggart et al., 2001).
It undergoes N-demethylation of a sulfonamide nitrogen to yield N-desmethylrosuvastatin, which is
claimed to have 2- to 6-fold less potency at inhibiting
the enzyme (Crestor, 2012; Fig. 89). It also is present in
circulation at concentrations 7-fold less than the
parent drug (Schneck et al., 2004). Thus it is projected
to have very little contribution to the activity of
rosuvastatin.
28. Sertraline. Sertraline is a serotonin reuptake
inhibitor widely used in the treatment of depression. It
undergoes N-demethylation to desmethylsertraline
(Fig. 90), which has only about 1/50th the binding
affinity of the parent drug (Owens et al., 1997). It
circulates at about 1.5-fold greater exposure at steady
state (Ronfeld et al., 1997) but the free fraction is
unknown. Because of the considerably lower potency, it
is doubtful that desmethylsertraline contributes to in
vivo efficacy.
29. Triazolam. Similar to midazolam and alprazolam described above, triazolam is also metabolized on
the methyl group of the azole ring to the 19-hydroxy
Pharmacologically Active Metabolites
625
Fig. 93. Metabolism of zopiclone to desmethyl and N-oxide metabolites.
metabolite (Fig. 91), and also similar to the other two
drugs, this metabolite possesses intrinsic potency that
is approximately 3- to 5-fold lower than the parent
(Richelson et al., 1991). However, less is known about
this metabolite regarding pharmacodynamics, and the
human free fraction is not reported. Combining the
plasma exposure relative to parent (Eberts et al., 1981;
O’Connor-Semmes et al., 2001) and the relative potency
values, it can be estimated that 19-hydroxytriazolam
would only possess a minor contribution to the efficacy
of triazolam. However, if the relationship between the
free fractions of triazolam and 19-hydroxytriazolam
follow those for midazolam and 19-hydroxymidazolam,
then the contribution of 19-hydroxytriazolam to efficacy
could be greater.
30. Valproic Acid. Valproic acid is an antiepileptic
agent that has been used successfully in the control of
seizures for many years. While its molecular target
may be ambiguous, studies in systems in vitro of
nerve cell firing suggest that its dehydrogenated
metabolite trans-2-propyl-2-pentenoic acid (Fig. 92)
also possesses this activity at about half of the potency
(Löscher, 1981). However, valproic acid concentrations are extremely high (;0.1 mM) and considerably
exceed those of this metabolite (Addison et al., 2000).
Furthermore, in a unique study wherein human brain
samples were able to be taken from patients on
valproic acid therapy who were also undergoing brain
surgery, it was shown that the brain concentrations
were a lot less than those of valproic acid itself and
that the free fraction for the metabolite was lower
than that of valproic acid (Adkison et al., 1995). It can
be concluded from these data that it is unlikely that
valproic acid metabolites actually contribute much to
the efficacy in vivo, despite the in vitro potency shown
for trans-2-propyl-2-pentenoic acid. This example
illustrates the importance of gathering dispositional
data for metabolites that are shown to have in vitro
activity.
31. Zopiclone. Zopiclone is a sedative agent active
at the benzodiazepine receptor family. In one paper it
was claimed that the N-oxide metabolite possessed
activity 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 less
potent than parent (Trifiletti and Snyder, 1984), and
the desmethyl was one order of magnitude less potent
(Fig. 93). Subsequently, a closer examination of the Senantiomer of the N-desmethyl metabolite showed that
it possesses pharmacological activity by binding better
to GABAA receptors possessing the g2 subunit (Fleck,
2002). Nevertheless, after administration of zopiclone,
neither the N-oxide nor desmethyl metabolites are
detected in plasma (Fernandez et al., 1993), and
626
Fig. 94. Metabolism
hydroxynortriptyline.
Obach
of
amitriptyline
to
nortriptyline
and
10-
therefore, it is unlikely that any drug-related effect in
vivo can be attributed to metabolites.
D. Drugs with Metabolites Possessing Activity at
Related Targets
In some instances, changes in structure introduced
by metabolism can cause a metabolite to have increased affinity at a receptor other than the one
targeted by the parent drug. It is typically the case
that the receptor or enzyme that the metabolite binds
is one among those in a family that the parent binds (e.
g., alternate G-protein coupled receptors). This can
cause subtle alterations in clinical effect. Examples of
this are below.
1. Amitriptyline and Nortriptyline. Amitriptyline,
a tricyclic antidepressant, is metabolized by N-demethylation to nortriptyline, which undergoes further
metabolism to several hydroxyl metabolites including
E-10-hydroxynortriptyline (Fig. 94). The activity profile
contribution of these entities is complex. Amitriptyline
is more potent at the serotonin transporter than the
norepinephrine transporter, whereas the reverse is true
for nortriptyline (Owens et al., 1997). Consideration of
the affinity and free plasma concentrations associated
with efficacy with amitriptyline dosing suggests that for
the serotonin transporter, amitriptyline would contribute to about 50% occupancy and norytriptyline about
another 10%. For the norepinephrine transporter,
nortriptyline free concentrations are about the same
as the potency value, while for amitriptyline the free
concentrations are about a fifth of the reported affinity
suggesting a similar level of occupancy for the norepinephrine transporter with the metabolite contributing
the greater share (Breyer-Pfaff et al., 1982). This is all
further complicated by the observation that 10-hydroxynortriptyline also can contribute to effect (Nordin and
Bertilsson, 1995). After amitriptyline administration,
this metabolite has a concentration that is above its IC50
for the norepinephrine transporter (measured in whole
human plasma). Relative to amitriptyline, it can be
estimated that 10-hydroxynortriptyline contributes almost twice as much to norepinephrine transport inhibition than the parent drug, but still less than
nortriptyline.
When nortriptyline is administered as the parent
drug the picture is somewhat simpler because the
serotonin transporter is no longer of much importance
(since that activity was driven by amitriptyline). Using
the respective in vitro potency values of nortriptyline
and the 10-hydroxy metabolite (which were generated
using human plasma as the matrix) and the in vivo
concentrations after repeated administration (Dahl
et al., 1996) suggests that the metabolite contributes
to about a third of the activity. 10-Hydroxynortriptyline is generated by CYP2D6 and research on the
polymorphism in this enzyme has shown that there are
differences among CYP2D6 extensive and poor metabolizers, with lower concentrations of the metabolite in
the latter. Examination of CSF concentrations have
shown that there may be a partial barrier to brain
penetration of 10-hydroxynortriptyline, since concentrations are lower than corresponding plasma ultrafiltrate concentrations (Nordin et al., 1985; Bertilsson
et al., 1991). Finally, it has been suggested that 10hydroxynortriptyline could itself be an antidepressant
that would have a better side-effect profile because it
has less activity at muscarinic receptors.
2. Clomipramine. Clomipramine is a tricyclic antidepressant agent that is metabolized by N-demethylation and hydroxylation to produce three putatively
active metabolites: norclomipramine, 8-hydroxyclimipramine, and 8-hydroxynorclomipramine (Fig. 95).
Intrinsic potency comparisons have been made for the
serotonin and norepeinephrine reuptake transporters,
as well as the cholinergic receptors (which may be
responsible for side effects). N-Demethylation seems to
Pharmacologically Active Metabolites
627
Fig. 95. Metabolism of clomipramine by N-demethylation and hydroxylation.
favor increased norepinephrine transporter potency
while retention of the N-methyl favors serotonin
transporter potency (Thomas and Jones, 1977; Linnoila
et al., 1982; Nunez and Perel, 1995; Agnel et al., 1996;
Tatsumi et al., 1997). Plasma concentrations among
the four entities are relatively comparable (;50–300
ng/ml), but unfortunately the lack of free fraction data
on these entities precludes reliable estimations of the
relative contributions of each (Bertilsson et al., 1979).
If free fractions and CNS penetration are considered
comparable, it would suggest that norclomipramine
and 8-hydroxyclomipramine contribute 10-times the
activity of parent drug at the norepinephrine transporter. For the serotonin transporter, norclomipramine
is only projected to contribute less than 20% of the
parent, while 8-hydroxyclomipramine would be expected to contribute very substantially (about equally
to parent).
3. Clozapine. Clozapine is N-demethylated to norclozapine and while both entities possess activity at the
5-HT2A and 5-HT2C receptors, they appear to possess
differing activities at the various dopamine receptors
which will help to describe the efficacy profile of the
drug as an antipsychotic agent (Fig. 96). Norclozapine
and clozapine circulate at about the same free plasma
concentrations, albeit the total concentrations and free
fractions differ somewhat (Schaber et al., 1998).
Clozapine binds most tightly to the D4 receptor and
is an antagonist, while it also binds with 10-fold lower
affinity to D2 and D3 receptors, where it is an inverse
agonist (Burstein et al., 2005). Norclozapine binds with
similar affinity to D2 and D3 as clozapine but is
a partial agonist at these two receptors. It binds to D4
Fig. 96. Metabolism of clozapine to norclozapine.
628
Obach
Fig. 97. Metabolism of doxepin to N-desmethyldoxepin.
as well but with ten-fold lower affinity and is an
antagonist. This offers a truly confusing picture regarding what may be happening in vivo. For the 5-HT2
activity, the contribution of the parent and metabolite
Fig. 98. Metabolism of imipramine to desipramine and 2-hydroxydesipramine.
to occupancy and activity can be considered more
straightforward and it is estimated that the norclozapine metabolite contributes about two-thirds of the
activity (Kuoppamaki et al., 1993). But for the
dopamine receptors, with the varying affinities and
functional activities of the two circulating entities, it is
much more difficult to claim what is actually occurring.
4. Doxepin. Doxepin is an unusual agent in that the
parent and N-desmethyl metabolite appear to possess
very different pharmacological activities (Mundo et al.,
1974). Doxepin is a potent antihistamine that readily
penetrates the brain and thus causes sedation and is
indicated for this purpose at low doses (Silenor, 2010).
N-Desmethyldoxepin (Fig. 97) binds to the norepinephrine transporter and thus has antidepressant properties like tricyclic antidepressants. High doses are
indicated for antidepressant activity. An estimation
of relative norepineprine transporter occupancies for
parent and metabolite suggest that the metabolite has
nearly seven-times the activity.
5. Imipramine and Desipramine. Like the tricyclic
antidepressant amitriptyline and its metabolites nortriptyline and hydroxynortriptyline, a similar situation
exists for imipramine and its metabolites desipramine
and 2-hydroxydesipramine (Fig. 98). Imipramine has
activities at both the serotonin and norepeinephrine
transporters with greater affinity for the serotonin
transporter while desipramine and 2-hydroxydesipramine have greater affinities for the norepinephrine
transporter (Owens et al., 1997). Thus, a complex
picture emerges regarding the molecular activities
occurring over the time course of drug and metabolite
exposures as well as among different individuals
(Caccia and Garattini, 1990). Following administration
of imipramine, it can be estimated that the norephinephrine transporter is almost completely occupied by
desipramine (free plasma concentration/IC50 ratio of
~20) (Borga et al., 1969; Sutfin et al., 1988; SzymuraOleksiak et al., 2001). 2-Hydroxydesipramine concentrations are approximately half of those of the parent
and is approximately 3-fold more potent at the
norepeinephrine transporter than imipramine suggesting that it also can contribute more to the occupancy of
that protein (Javaid et al., 1979; Nelson et al., 1983).
Imipramine would be predicted to drive the serotonin
transport inhibition since its potency is far greater at
that target relative to the two metabolites; however,
because of the higher free concentrations of desipramine 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 norepeinephrine transporter, with contributions from desipramine and the hydroxyl metabolite. Based on
comparative in vitro potency values and free concentration values (Cooke et al., 1984), it can be estimated
that 2-hydroxydesipramine has about 40% of the in
vivo potency. This must be interpreted with some
Pharmacologically Active Metabolites
629
Fig. 99. Metabolism of loxapine and amoxpine.
caution since it is based on potency values measured in
rat brain. Finally, since desipramine is converted to the
active 2-hydroxdesipramine metabolite by CYP2D6,
there are differences between EM and PM subjects in
exposure to desipramine and its metabolite (Shimoda
et al., 2000).
6. Loxapine and Amoxapine. The antipsychotic
agent loxapine, and its N-desmethyl metabolite amoxapine (which is also used as a drug itself) poses
a complex picture of the contribution of parent drug
and metabolites to pharmacological activity. Loxapine
undergoes N-demethylation to amoxapine as well as
hydroxylation to 7-hydroxyloxapine and 8-hydroxyloxapine. Amoxapine also undergoes these same hydroxylations to the 7- and 8-hydroxyamoxapine metabolites
(Fig. 99). The drugs and their metabolites bind to 5HT2A and D2 receptors with varying potencies, as well
as other CNS receptors to lesser extents (Ereshefsky,
1999). Following administration of loxapine, concentrations of parent drug, amoxapine, 7-hydroxyloxapine,
and 8-hydroxyloxapine are approximately 51, 11, 32,
and 180 nM, respectively (Cheung et al., 1991). Among
the four entities, 7-hydroxyloxapine has the greatest
potency and can be projected to deliver 3-times the 5HT2A receptor activity as loxapine and 11-times the D2
receptor activity. 8-Hydroxyloxapine is less potent, but
has greater concentrations and can be projected to
have 70% of the activity at 5-HT2A receptors and 1.5fold the activity at D2 receptors. Amoxapine has
similar potency as loxapine at these two receptors,
and as it circulates at about one-third of the levels, it
can be estimated to have one-third the activity.
Administration of amoxapine yields the corresponding 7- and 8-hydroxy metabolites. 7-Hydroxyamoxapine
630
Obach
Fig. 100. Metabolism of nefazodone to hydroxynefazodone, mCPP, and its triazolodione metabolites.
is more potent than amoxapine at both the 5-HT2A and
D2 receptors (Ereshefsky, 1999) and is present in
circulation at about half the concentration (Takeuchi
et al., 1993). From this, it can be estimated that 7hydroxyamoxapine contributes to 5-HT2A occupancy at
about an equal amount as parent but is the dominant
contributor to D2 occupancy. 7-Hydroxyamoxapine has
been 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 metabolite is present in greater abundance, but is 10- to 20-fold
less potent at both receptors and therefore contributes
Fig. 101. Metabolism of atorvastatin to hydroxy metabolites.
Pharmacologically Active Metabolites
631
assuming that these are similar, which is reasonable
for the hydroxynefazodone metabolite, but likely not
reasonable for mCPP. For hydroxynefazodone, it can be
estimated that it has approximately ;25% of the
activity of nefazodone itself. Protein binding data for
mCPP is available in rat (fu = 0.7), and if the value in
human is similar, it would suggest that mCPP could
have profound activity in humans [maximum plasma
concentration (Cmax) = 0.5 mM; Barbhaiya et al., 1996]
including activity at serotonin and norepinephrine
reuptake transporters (Owens et al., 1997).
Fig. 102. Metabolism of bromhexine to ambroxol.
about a third of the activity of parent. This contribution
likely increases with multiple dosing, since the t1/2 of 8hydroxyamoxapine is much longer than that of amoxapine or 7-hydroxyamoxapine (Calvo et al., 1985).
7. Nefazodone. The antidepressant nefazodone is
metabolized to three active metabolites, hydroxylation
on the ethyl side chain (hydroxynefazodone), which
undergoes further oxidative metabolism to lose the
ethyl group altogether (triazolodione metabolite), and
N-dealkylation to yield mCPP (Fig. 100). Nefazodone
and its hydroxyl metabolite have very similar receptor
binding profiles with the most potent binding occurring
at the 5-HT2A receptor (Taylor et al., 1995; Owens
et al., 1997). The triazolodione metabolite appears to
have far weaker activity. The mCPP metabolite has
a different receptor binding profile, is more potent at 5HT1A and is an agonist. While plasma protein binding
is very high for nefazodone (fu = 0.009), data are not
available for the metabolites, so estimations of contribution to activity relative to parent drug must be made
E. Drugs That Generate Active Metabolites but
Assessment of In Vivo Contribution Is Ambiguous
1. Atorvastatin. Atorvastatin is a very commonly
used lipid lowering agent for the prevention of
atherosclerotic disease. It is metabolized by CYP3A
to two metabolites claimed to be active, 2- and 4hydroxyatorvastatin (Fig. 101); however actual inhibition potencies for the HMGCoA reductase target are
not available in the scientific literature. In the product
label for atorvastatin, it is claimed that 70% of the
activity is attributable to metabolites (Lipitor, 2012).
However, examinations of the relationship between
atorvastatin efficacy and various CYP3A polymorphisms that could cause alterations in parent and
metabolite ratios have yielded mixed results (Kivistö
et al., 2004; Gao et al., 2008; Li et al., 2011). It is
important to note that since the target tissue for
inhibition of HMGCoA reductase is the liver within
which the active metabolites are generated and that it
has been shown that atorvastatin and its metabolites
are substrates for hepatic uptake transporters, it may
be difficult and even inappropriate to relate systemic
Fig. 103. Metabolism of bupropion to hydroxybupropion and stereoisomeric dihydrobupropion metabolites.
632
Obach
Fig. 104. Metabolism of etretinate to acitretin.
circulating concentrations of atorvastatin and its active
metabolites to effect.
2. Bromhexine. Bromhexine is a relatively old
agent that has been used as an expectorant, to help
make mucus less viscous and more easily expectorated.
Its active metabolite that arises from N-demethylation
and hydroxylation, ambroxol, has also been used in
that manner (Fig. 102). These agents could find use in
the treatment of serious respiratory disorders such as
cystic fibrosis or COPD (Malerba and Ragnoli, 2008).
When bromhexine is administered orally, the systemic
exposure to ambroxol is much lower (;8-fold; Liu et al.,
2010), but since the true molecular action of these
agents is not entirely known, it cannot be ascertained
whether bromhexine or ambroxol, or both, contribute
to activity. Ambroxol has also been associated with
a reduction in throat pain, which has triggered
investigation into the activity of it and bromhexine at
sodium channels (such as NaV1.8 and the tetrodotoxin
sensitive channel; Leffler et al., 2010).
3. Bupropion. Bupropion (Fig. 103) represents an
interesting challenge when attempting to discern
whether the metabolites are relevant to pharmacological effect. In the product labels for Wellbutrin and
Zyban (two products containing bupropion), it is stated
that the hydroxyl, and the two reduced metabolites are
Fig. 105. Metabolism ivabradine to N-desmethylivabradine.
active (Wellbutrin, 2011). However, even for bupropion
itself, it is not entirely clear as to the mechanism of
action as an antidepressant or smoking-cessation aid.
Bupropion does interact with dopamine and norepinephrine transporters, with somewhat greater affinity
to the former (Damaj et al., 2004). The CSF concentration of bupropion is about 40% of that in plasma,
which is slightly higher than what would be projected
when 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 active
entity and whether the dopamine transporter is the
target. The same goes for hydroxybupropion, although
the affinity is only 4-fold lower than CSF concentrations. Affinity for the reduced metabolites is not
published, but as the concentration of threohydrobupropion in CSF is nearly 1 mM and if its affinity for the
dopamine transporter is similar to that of bupropion, it
is possible that this metabolite carries the majority of
the in vivo activity. However, direct administration to
animals has suggested that bupropion itself and
perhaps the hydroxyl metabolite are actually more
active than the reduced metabolites threo- and
Fig. 106. Metabolism
metabolite.
of
mycophenolic
acid
to
its
glucuronide
633
Pharmacologically Active Metabolites
erythrohydrobupropion (Butz et al., 1982; Martin et al.,
1990). Furthermore, plasma concentrations of the
metabolites were shown to be higher in nonresponder
patients 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 metabolite acitretin that arises via hydrolysis of the ethyl
ester (Fig. 104). Etretinate had a very long half-life,
due presumably to sequestration and slow release from
deep tissue compartments. The teratogenic effect of
retinoids with this long half-life made etretinate
undesirable for theapy. Acitretin shows a formation
rate–limiting half-life when generated from etretinate,
and a much shorter half-life when given directly. The
shorter half-life is more amenable for therapy, in light
of the teratogenic effect. The molecular mechanism
(i.e., target binding protein) is unknown (Saurat, 1999),
so delineating the relative contributions of etretinate
and acitretin to activity when etretinate is administered is challenging. With etretinate dosing, acitretin
achieves concentrations of ;150 ng/ml, whereas etretinate is present at 4- to 5-fold higher concentrations
(Larsen et al., 1988). The free fraction of the ester is
also considerably greater (Urien et al., 1992; Soriatane,
2011). When acitretin is dosed directly, higher concentrations are observed (Pilkington and Brogden, 1992)
than when generated from etretinate. This suggests
that etretinate also likely had some contribution to
activity when administered directly. Finally, the
generation of etretinate by esterification in vivo has
also been observed following acitretin dosing, which
even further complicates the understanding of relative
contributions to effect (Lambert et al., 1992).
5. Ivabradine. Ivabradine is a drug used in the
treatment of angina which undergoes demethylation to
an active metabolite (Fig. 105). The delineation of the
parent drug and metabolite to effect was made using
population PK/PD modeling after oral and intravenous
administration, because the pharmacological properties of the metabolite are not published (Ragueneau
et al., 1998). The metabolite circulates at much lower
concentrations than parent. These investigators proposed an indirect PK/PD model wherein the metabolite
contributed to effect at the earlier times following
dosing and a sustained effect contributed by the parent
drug.
6. Mycophenolic Acid. The immunosuppressive
agent mycophenolic acid is used to reduce rejection of
transplanted organs. It is metabolized by glucuronidation to a phenol glucuronide and an acyl glucuronide
(Fig. 106). The latter had been tested in in vitro assays
that model immunosuppression and shown to have
potency about half that of the parent drug (Shipkova
et al., 2001); however, such a determination is
challenged by the observation that the acyl glucuronide
is 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 than
the parent drug (Shipkova et al., 2002). To consider
whether this metabolite can contribute to in vivo
activity, the free fraction needs to be assessed for
parent and metabolite. A protein binding value for the
parent is known at approximately 98% bound. However, the acyl glucuronide forms an irreversible covalent bond with albumin, which makes measurement
of the free fraction technically challenging to obtain. It
is thus not presently possible to ascribe relative
participation of the parent drug and acyl glucuronide
in the pharmacological effect.
V. Conclusions
In drug research, it is clearly the case that the
potential for pharmacologically active metabolites
must be considered. Knowledge of the properties of
active metabolites, i.e., intrinsic potency, functional
activity, pharmacokinetics, clearance mechanism and
rate, free fraction, and membrane permeability are
necessary to understand concentration-effect relationships in humans as well as interpatient variability in
response. The occurrence of active metabolites among
drugs is frequent, consistent with the notion that small
alterations in chemical structure of a drug will not
necessarily alter its biologic properties. In the research
of new drug candidates, the early identification of
active metabolites is of critical importance in developing optimal clinical study designs and correct
data interpretation. Proactively hunting for active
metaboilites in ex vivo and in vitro samples is a sound
strategy in drug research. Finally, active metabolites
may possess advantages as drugs themselves (i.e., greater
potency/efficacy, superior dispositional properties, improved safety profile), and there are many examples of
drugs used in clinical practice that were originally
observed as metabolites of other drugs.
Acknowledgments
The author greatly appreciates Dr. Doug Spracklin for critical
evaluation of this manuscript.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Obach.
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