Phase II Drug Metabolism

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Sponsored by:

Thanks to Hypha Discovery for their support of this independently written article.

Dr. Julia Shanu-Wilson of Hypha Discovery says, “The biotransformation “maps” resulting from the study of drug metabolism are intriguing and sometimes surprising. The reactions described herein, and in the previous Phase I drug metabolism poster are a valuable reminder of the complexity that is possible.”

Hypha Discovery is a specialist CRO supporting pharma companies worldwide through the production, purification, and identification of metabolites.

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This article explains what Phase II drug metabolism is, contains a poster summarizing common enzyme-mediated bioconjugation reactions, and provides interesting examples of Phase II transformations and their impact on drug pharmacology.

"As any biotransformation scientist will know, metabolism is not a binary function, and drugs can be cleared through multiple mechanisms," says Dr. Julia Shanu-Wilson of Hypha Discovery. "For instance, epacadostat presents an interesting case where, in addition to gut microbiome-facilitated enterohepatic circulation of the parent, major circulating metabolites derive from extensive Phase II metabolism, reductive metabolism by gut microbiota, and secondary systemic Phase I metabolism of the absorbed gut metabolite."

What is Phase II Metabolism

The metabolism of drugs and other xenobiotics often involves biotransformations of molecules to less lipophilic, more water-soluble, and more quickly eliminated products. This typically occurs in two phases:

• Phase I drug metabolism: polar groups are added or exposed (e.g., hydroxylation)

• Phase II drug metabolism: conjugation of molecules to polar ionic groups (e.g., glucuronidation)

The poster below shows examples of Phase II metabolism of drugs and other xenobiotics. It includes examples of the most common Phase II biotransformations, such as:

• Glucuronidation – mediated by uridine 5’-diphospho (UDP)-glucosyltransferases (UGTs)

• Sulfonation – mediated by sulfotransferases (SULTs)

• Glutathione conjugation – mediated by glutathione S-transferases (GSTs)

• Methylation – mediated by methyltransferases (MTs)

• Acetylation – mediated by N-acetyltransferases (NATs)

• Amino acid conjugation – mediated by amino acid N-acyltransferases (AATs)

Phase II Drug Metabolism Poster

Glucuronidation

Ezetimibe

Ezetimibe lowers cholesterol by selectively inhibiting cholesterol absorption by the small intestine. This drug undergoes UGT1A1, UGT1A3, and UGT2B15 mediated glucuronidation and extensive extrahepatic recirculation (EHC). EHC lowers the systemic exposure of ezetimibe and contributes to its safety and its efficacy as a novel cholesterol-lowering drug.

Kosoglou T, Statkevich P, Johnson-Levonas AO, Paolini JF, Bergman AJ, Alton KB. Ezetimibe: a review of its metabolism, pharmacokinetics, and drug interactions. Clin. Pharmacokinet. 2005, 44, 467–494.

Ghosal A, Hapangama N, Yuan Y, Achanfuo-Yeboah J, Iannucci R, Chowdhury S, Alton K, Patrick JE, Zbaida S. Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia). Drug Metab. Dispos. 2004, 32, 314–320.

MK-8666

Developed for the treatment of type 2 diabetes, MK-8666 was discontinued in Ph. I clinical trials due to liver safety concerns. It was determined that glucuronidation of the carboxylic acid moiety of MK-8666 forms a reactive acyl glucuronide which can covalently modify proteins. Speaking to the importance of carboxylic acid groups for phase II metabolism, a CoA thioester also forms at this moiety which can also lead to protein adduction. Protein adduction is thought to be one causative mechanism for MK-8666-mediated liver toxicity.

Shang J, Tschirret-Guth R, Cancilla M, Samuel K, Chen Q, Chobanian HR, Thomas A, Tong W, Josien H, Buevich AV, Mitra K. Bioactivation of GPR40 Agonist MK-8666: Formation of Protein Adducts in Vitro from Reactive Acyl Glucuronide and Acyl CoA Thioester. Chem. Res. Toxicol. 2020, 33, 191–201.

“Glucuronide Synthesis.” Hypha Discovery, 5 Nov. 2021

Gemfibrozil

The major metabolite of gemfibrozil is an acyl-glucuronide (gemfibrozil 1-O-β-glucuronide). In vitro, gemfibrozil inhibits CYP2C9 more potently than CYP2C8. In vivo, this trend is reversed. Interestingly, this incongruity was resolved when it was determined that gemfibrozil 1-O-β-glucuronide is a potent and specific CYP2C8 metabolism-dependent inhibitor while the parent compound is a competitive inhibitor for CYP2C9.

Ogilvie BW, Zhang D, Li W, Rodrigues AD, Gipson AE, Holsapple J, Toren P, Parkinson A. Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions. Drug. Metab. Dispos. 2006, 34, 191–197.

Molidustat

Molidustat stimulates red blood cell proliferation by inhibiting HIF prolyl-hydroxylase. The major metabolite of molidustat is its N-glucuronide. UGT1A1 and UGT1A9 have been identified as the isoforms predominantly responsible for molidustat glucuronidation. Molidustat is almost entirely eliminated in urine as its N-glucuronide.

Lentini S, van der Mey D, Kern A, Thuss U, Kaiser A, Matsuno K, Gerisch M. Absorption, distribution, metabolism and excretion of molidustat in healthy participants. Basic Clin. Pharmacol. 2020, 127, 221–233.

Olanzapine

Olanzapine is a second-generation antipsychotic used to treat schizophrenia, bipolar disorder, and treatment-resistant depression. There is significant interindividual variability in olanzapine clearance, and studies suggest that clinical outcomes and plasma concentrations are related. The major route of metabolism for olanzapine is N-glucuronidation. Two N-glucuronide metabolites have been identified, a tertiary amine N-glucuronide and a unique, human-specific, N-glucuronide at the benzodiazepine moiety. Polymorphisms in UGT1A4 and UGT2B10 significantly alter glucuronidation in vitro and could contribute to inter-individual differences in olanzapine metabolism.

Erickson-Ridout KK, Zhu J, Lazarus P. Olanzapine metabolism and the significance of UGT1A448V and UGT2B1067Y variants. Pharmacogenet. Genom. 2011, 21, 539–551.

Nicotine

Nicotine, widely known as the active compound in cigarette smoke, is a good example of a compound that undergoes pyridine N-glucuronidation in vivo. UGT2B10 is the enzyme responsible for the majority of nicotine glucuronidation. Polymorphisms of UGT2B10 are associated with reductions in the levels of glucuronidated nicotine in the urine of smokers. Alterations in the clearance of nicotine or shifts in exposure to nicotine and its metabolites and metabolite-conjugates could explain differences in addiction and carcinogen exposure across the population.

Chen G, Giambrone NE Jr, Dluzen DF, Muscat JE, Berg A, Gallagher CJ, Lazarus P. Glucuronidation genotypes and nicotine metabolic phenotypes: importance of functional UGT2B10 and UGT2B17 polymorphisms. Cancer Res. 2010, 70 (19), 7543–7552.

Malotilate

This compound is an early example of thiol S-glucuronidation, a major biliary metabolite of the intermediate dithiol formed by ring opening. This compound has been used in liver disease studies and has been shown to have a modest beneficial effect on the liver in cases of cirrhosis.

Nakaoka M, Suzuki W, Hakusui H. Identification of a dithiol intermediate metabolite of malotilate in rats. Xenobiotica 2009, 20, 91–98.

Bührer M, Le Cotonnec JY, Wermeille M, Bircher J. Treatment of liver disease with malotilate. A pharmacokinetic and pharmacodynamic phase II study in cirrhosis. Eur. J. Clin. Pharmacol. 1986, 30, 407–416.

Sulfinpyrazone

Sulfinpyrazone (SFZ) is a uricosuric agent indicated for the treatment of gout. SFZ is a potent and highly selective inhibitor of CYP2C9 and inhibits most UGT1A subfamily enzymes but is only glucuronidated by UGT1A9. The glucuronide metabolite of SFZ is an uncommon C-glucuronide that forms at the alpha carbon of SFZ’s diketone moiety.

Kerdpin O, Elliot DJ, Mackenzie PI, Miners JO. Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9. Drug Metab. Dispos. 2006, 34, 1950–1953.

Sabia H, Sunkara G, Ligueros-Saylan M, Wang Y, Smith H, McLeod J, Prasad P. Effect of a selective CYP2C9 inhibitor on the pharmacokinetics of nateglinide in healthy subjects. Eur. J. Clin. Pharmacol. 2004, 60, 407–412.

Ethchlorvynol

Ethchlorvynol is a central nervous system depressant that is now rarely prescribed in favor of safer sedative-hypnotics. The major metabolite of ethchlorvynol is a rare example of C-glucuronidation at an acetylenic moiety.

Abolin CR, Tozer TN, Craig JC, Gruenke LD. C-Glucuronidation of the acetylenic moiety of ethchlorvynol in the rabbit. Science 1980, 209, 703–704.

Horwitz JP, Brukwinski W, Treisman J, Andrzejewski D, Hills EB, Chung HL, Wang CY. Ethchlorvynol: potential of metabolites for adverse effects in man. Drug Metabolism. Dispos. Biological Fate Chem. 1980, 8, 77–83.

Sulfonation

Apixaban

Apixaban is an orally bioavailable, direct inhibitor of activated factor X (FXa), a key serine protease in the coagulation cascade. It is used for the treatment and prevention of several thromboembolic disorders. While unchanged apixaban is the primary excretion product, roughly 25% of the dose is excreted in the urine and feces as metabolites. The sulfation of O-desmethyl apixaban by the sulfotransferase SULT1A1 forms O-desmethyl apixaban sulfate, the most abundant circulating metabolite in humans.

Raghavan N, Frost CE, Yu Z, He K, Zhang H, Humphreys WG, Pinto D, Chen S, Bonacorsi S, Wong PC, Zhang D. Apixaban Metabolism and Pharmacokinetics after Oral Administration to Humans. Drug Metab. Dispos. 2009, 37, 74–81.

Byon W, Garonzik S, Boyd RA, Frost CE. Apixaban: A Clinical Pharmacokinetic and Pharmacodynamic Review. Clin. Pharmacokinet. 2019, 58, 1265–1279.

Wong PC, Pinto DJP, Zhang D. Preclinical Discovery of Apixaban, a Direct and Orally Bioavailable Factor Xa Inhibitor. J. Thromb. Thrombolys. 2011, 31, 478–492.

Minoxidil

Minoxidil is a hair growth promoter and antihypertensive. Better known by the trade name Rogaine, this compound is given as an N-oxide, which is sulfonated to form its active N-oxide sulfate metabolite. The bioactivation of minoxidil by sulfonation is a unique biotransformation that is facilitated by hepatic thermolabile phenol sulfotransferase (SULT1A1). An interesting example of a drug that is activated by sulfonation, minoxidil activity is correlated with SULT1A1 activity, and thus its effectiveness may be impacted by copy number and polymorphisms in the gene.

Anderson RJ, Kudlacek PE, Clemens DL. Sulfation of minoxidil by multiple human cytosolic sulfotransferases. Chem.-Biol. Interact. 1998, 109, 53–67.

Buhl AE, Waldon DJ, Baker CA, Johnson GA. Minoxidil sulfate is the active metabolite that stimulates hair follicles. J. Invest. Dermatol. 1990, 95, 553–557.

Goren A, Castano JA, McCoy J, Bermudez F. Lotti T. Novel enzymatic assay predicts minoxidil response in the treatment of androgenetic alopecia. Dermatol. Ther. 2014, 27, 171–173.

Hebbring SJ, Adjei AA, Baer JL, Jenkins GD, Zhang J, Cunningham JM, Schaid DJ, Weinshilboum RM, Thibodeau SN. Human SULT1A1 gene: copy number differences and functional implications. Hum. Mol. Genet. 2006, 16, 463–470.

Glutathione Conjugation

Aflatoxin B1-exo-8,9-epoxide

Aflatoxin (AFB1) is a highly potent carcinogen that is activated by CYPP3A4 to form Aflatoxin B1-Exo-8,9-epoxide (AFB1-Epoxide). The epoxide is highly reactive with DNA and protein nucleophiles. GST-mediated glutathionylation of AFB1-Epoxide is thought to be an important route of detoxification. Interspecies variability in AFB1 carcinogenicity is reflected in differences in GST expression. Compared to rat GSTs, human GSTs are poor catalysts for glutathione conjugation of AFB1-Epoxide. Based on in vitro experiments, GSTM1-1 is likely responsible for this conjugation reaction in humans.

Johnson WW, Ueng YF, Widersten M, Mannervik B, Hayes JD, Sherratt PJ, Ketterer B, Guengerich FP. Conjugation of highly reactive aflatoxin B1 Exo-8,9-epoxide catalyzed by rat and human glutathione transferases: estimation of kinetic parameters. Biochemistry 1997, 36, 3056–3060.

Stewart RK, Serabjit-Singh CJ, Massey TE. Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B1 in rabbit lung and liver. Toxicol. Appl. Pharm. 1996, 140, 499–507.

Guengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H, Langouët S. Activation and detoxication of aflatoxin B1. Mutat. Res. Fundam. Mol. Mech. Mutagen. 1998, 402, 121–128.

Kew MC. Aflatoxins as a cause of hepatocellular carcinoma. J. Gastrointest. Liver Dis. 2013, 22, 305–310.

2-(2,3-dimethyl-4-(2-methylenebutanoyl)phenoxy)acetic acid

2-(2,3-dimethyl-4-(2-methylenebutanoyl)phenoxy)acetic acid was initially developed in the 1960s as part of a new class of diuretics but never advanced to clinical use. It causes the rapid excretion of nearly equivalent amounts of sodium and chloride and a slight increase in potassium excretion. Metabolism has been shown to proceed via glutathionylation.

Schultz E M, Cragoe EJ, Bicking JB, Bolhofer WA, Sprague JM. α, β-Unsaturated ketone derivatives of aryloxyacetic acids, a new class of diuretics. J. Medicinal. Pharm. Chem. 1962, 5, 660–662.

Baer JE, Michaelson JK, McKinstry DN, Beyer KH. A new class of diuretic-saluretic agents, the α, β-unsaturated ketone derivatives of aryloxyacetic acids. P. Soc. Exp. Biol. Med. 1964, 115, 87–90.

Chasseaud LF. Distribution of enzymes that catalyse reactions of glutathione with α, β-unsaturated compounds. Biochem. J. 1973, 131, 765–769.

Bromisoval

Bromisoval has been used as a probe drug to assess GSH conjugation activity in vivo. Bromisoval metabolites are excreted in the urine as mercapturic acid conjugates. Taken as a racemic mixture, bromisoval glutathione conjugation appears to be selective for (R)-bromisoval.

Niederwieser A, Steinmann B, Matasovic A. New bromisoval (bromural) metabolites in human urine: α-(cystein-S-yl)isovalerylurea, α-(N-acetylcystein-S-yl)isovalerylurea, and α-(cysteamin-S-yl)isovaleric acid. J. Chromatogr. A 1978, 147, 163–176.

Mulders TM, Venizelos V, Schoemaker R, Cohen AF, Breimer DD, Mulder GJ. Characterization of glutathione conjugation in humans: stereoselectivity in plasma elimination pharmacokinetics and urinary excretion of (R)- and (S)-2-bromoisovalerylurea in healthy volunteers. Clin. Pharmacol. Ther. 1993, 53, 49–58.

Cisplatin

Cisplatin glutathione conjugation is an example of this conjugation pathway, leading to both detoxification and bioactivation. Glutathione conjugated cisplatin can be metabolized to a cysteinyl-glycine-conjugate, to a cysteine conjugate, and finally to a reactive thiol that is highly nephrotoxic. Alternatively, it is thought that glutathione conjugation, mediated by GSTA1, is a detoxification pathway responsible for cisplatin resistance in solid tumors, although this may be reversible.

Yasuyuki S, Yoshihiko S, Yoshio T, Sadao H. Protection against cisplatin-induced nephrotoxicity in the rat by inducers and an inhibitor of glutathione S-transferase. Biochem. Pharmacol. 1994, 48, 453–459.

Yasuyuki S, Yoshihiko S, Yoshio T. Role of glutathione S-transferase isoenzymes in cisplatin-induced nephrotoxicity in the rat. Toxicol. Lett. 1994, 70, 211–222.

Zou M, Hu X, Xu B, Tong T, Jing Y, Xi L, Zhou W, Lu J, Wang X, Yang X, Liao F. Glutathione S‑transferase isozyme alpha 1 is predominantly involved in the cisplatin resistance of common types of solid cancer. Oncol. Rep. 2019, 41, 989–998.

Niu B, Zhou Y, Liao K, Wen T, Lao S, Quan G, Pan X, Wu C. “Pincer Movement”: Reversing cisplatin resistance based on simultaneous glutathione depletion and glutathione S-transferases inhibition by redox-responsive degradable organosilica hybrid nanoparticles. Acta Pharm. Sinica. B 2022, 12, 2074–2088.

Acetaminophen

The toxic bioactivation of acetaminophen might be one of the most well-taught biotransformations in the field of pharmaceutical sciences. Acetaminophen goes through two rounds of P450-mediated oxidation to become the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is highly electrophilic and will readily form conjugates with essential biomolecules. GST-catalyzed glutathione conjugation is a major route of NAPQI detoxification, but in cases of overdose, the GST/GSH detoxification pathway can become saturated, leading to NAPQI mediated liver toxicity.

Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR, Brodie BB. Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Ther. 1973, 187 (1), 185–194.

McGarry DJ, Chakravarty P, Wolf CR, Henderson CJ. Altered protein S-glutathionylation identifies a potential mechanism of resistance to acetaminophen-induced hepatotoxicity. J. Pharmacol. Exp. Ther. 2015, 355, 137–144.

Methylation

Ethinylestradiol

Compounds that come to mind as COMT substrates are small endogenous catechol-amines like dopamine. The methylation of 2-OH-ethinylestradiol (2-OH-EE) by COMT highlights the fact that larger catechols like catecholestrogens are also substrates of COMT. Furthermore, COMT-mediated methylation of 2-OH-EE reduced the carcinogenicity of these metabolites by preventing catechol-specific redox cycling and free radical generation.

Zhu BT, Roy D, Liehr JG. The carcinogenic activity of ethinyl estrogens is determined by both their hormonal characteristics and their conversion to catechol metabolites. Endocrinology 1993, 132, 577–583.

N-Acetylserotonin

N-Acetylserotonin (NAS), or normelatonin, is a precursor to melatonin and an endogenous substrate for the O-methyltransferase acetylserotonin methyltransferase (ASMT). Melatonin, an endogenous neurotransmitter, is commonly taken as an herbal supplement to aid in falling asleep. Abnormal melatonin synthesis, attributed to polymorphisms in the gene that codes for ASMT, is considered a risk factor for autism spectrum disorder and is associated with several other neuropsychiatric disorders.

Oxenkrug G, Ratner R. N-Acetylserotonin and aging-associated cognitive impairment and depression. Aging Dis. 2012, 3, 330–338.

Melke J, Goubran Botros H, Chaste P, Betancur C, Nygren G, Anckarsäter H, Rastam M, Ståhlberg O, Gillberg IC, Delorme R, Chabane N, Mouren-Simeoni MC, Fauchereau F, Durand CM, Chevalier F, Drouot X, Collet C, Launay JM, Leboyer M, Gillberg C, Bourgeron T. Abnormal melatonin synthesis in autism spectrum disorders. Mol. Psychiatry 2008, 13, 90-8.

Moskaleva PV, Shnayder NA, Nasyrova RF. Association of polymorphisms of DDC genes (AADC), AANAT and ASMT encoding melatonin synthesis enzymes, with a risk of developing psychoneurological disorders. Zh. Nevro.l Psikhiatr. Im. S. S. Korsakova. 2021; 121, 151-157.

Norepinephrine

Norepinephrine is the precursor to the fight-or-flight hormone adrenaline. PNMT is abundant in the cytosol of endocrine cells of the adrenal medulla and methylates norepinephrine’s primary amine to form epinephrine.

Baetge EE, Behringer RR, Messing A, Brinster RL, Palmiter RD. Transgenic mice express the human phenylethanolamine N-methyltransferase gene in adrenal medulla and retina. Proc. Natl. Acad. Sci. 1988, 85, 3648–3652.

Ziegler MG, Bao X, Kennedy BP, Joyner A, Enns R. Location, development, control, and function of extraadrenal phenylethanolamine N-methyltransferase. Ann. NY Acad. Sci. 2002, 971, 76–82.

Nicotine

N-methylation of nicotine is a minor pathway catalyzed by NNMT. However, it has been hypothesized that N-methylation contributes to the toxic effects of exposure to nicotine. Nicotine methylation is also considered an important event in some cancer progression. It is thought that N-methylation of xenobiotics might channel SAM away from critical physiological processes like DNA methylation. This brings up the interesting point that many drug-metabolizing methyltransferases have clear endogenous metabolites. Because of the potential impact it could have on endogenous metabolite homeostasis, it is important to consider the physiological effects of introducing a xenobiotic that is a good substrate for drug-metabolizing methyltransferases.

Crooks PA, Godin CS. N-methylation of nicotine enantiomers by human liver cytosol. J. Pharm. Pharmacol. 1988, 40, 153–154.

Dutta AD, Kumar A, Lokhande K, Mitruka M, Pal JK, Sarode SC, Sharma NK. Detection of oncometabolites 1-methylnicotinamide, nicotine imine, and N-Methylnicotinium in nails of oral cancer patients and prediction of them as modulators of DNMT1. Medrxiv 2020, 2020.09.20.20198101.

BI 187004

BI 187004 is an 11-β-hydroxysteroid dehydrogenase 1 inhibitor intended to treat type 2 diabetes. It is a curious example of potential TMT-mediated methylation of an imidazole moiety. TMT is uniquely microsomal, while most other drug-metabolizing methyltransferases are cytosolic. The two arguments for TMT’s role in BI 187004 methylation are that the methylation occurs in human liver microsomes (HLMs) and it is potently inhibited by the TMT inhibitor 2,3-dichloro-α-methylbenzylamine (DCMB) hydrochloride.

Maw HH, Zeng X, Campbell S, Taub ME, Teitelbaum AM. N-Methylation of BI 187004 by Thiol S-Methyltransferase. Drug Metab. Dispos. 2018, 46, 770-778.

Clopidogrel

Clopidogrel, vicagrel, and prasugrel are a class of novel thienopyridine prodrugs that are bioactivated by P450-mediated oxidation to reveal a reactive thiol that covalently forms a disulfide bond with the protein target P2Y12. Thiol S-methylation by TMT is a major metabolic route of clearance and deactivation of the active metabolites of thienopyridines.

Smith RL, Gillespie TA, Rash TJ, Kurihara A, Farid NA. Disposition and metabolic fate of prasugrel in mice, rats, and dogs. Xenobiotica 2008, 37, 884–901.

Nishihara M. Inhibitory Effect of Vonoprazan on the Metabolism of [14C]Prasugrel in Human Liver Microsomes. Eur. J. Drug Metab. Ph. 2019, 44, 713–717.

Farid NA, Smith RL, Gillespie TA, Rash TJ, Blair PE, Kurihara A, Goldberg MJ. The disposition of prasugrel, a novel thienopyridine, in humans. Drug Metab. Dispos. 2007, 35, 1096–1104.

Liu C, Lu Y, Sun H, Yang J, Liu Y, Lai X, Gong Y, Liu X, Li Y, Zhang Y, Chen X, Zhong D. Development and validation of a sensitive and rapid UHPLC-MS/MS method for the simultaneous quantification of the common active and inactive metabolites of vicagrel and clopidogrel in human plasma. J. Pharmaceut. Biomed. 2018, 149, 394–402.

Dansette PM, Libraire J, Bertho G, Mansuy D. Metabolic oxidative cleavage of thioesters: evidence for the formation of sulfenic acid intermediates in the bioactivation of the antithrombotic prodrugs ticlopidine and clopidogrel. Chem. Res. Toxicol. 2009, 22, 369–373.

6-Mercaptopurine (6MP)

6MP and other mercaptopurine drugs are antimetabolites for purines and are used to treat cancers and autoimmune diseases. TPMT S-methylation inactivates 6MP. TPMT is highly polymorphic, and poor metabolizers are at risk of elevated levels of thiopurine nucleotides which leads to life-threatening myelosuppression.

Nguyen CM, Mendes MA, Ma JD. Thiopurine methyltransferase (TPMT) genotyping to predict myelosuppression risk. PLoS Curr. 2011, 3, RRN1236.

Arsenic Trioxide

Arsenic trioxide is used as a second-line treatment for leukemia. Arsenic trioxide is predominantly eliminated as dimethylarsinic acid. The dimethylarsinic acid is much less toxic than the parent compound arsenic trioxide; therefore, As3MT has an important role in the detoxification of this unique chemotherapeutic agent.

Ren X, Aleshin M, Jo WJ, Dills R, Kalman DA, Vulpe CD, Smith MT, Zhang L. Involvement of N-6 Adenine-Specific DNA Methyltransferase 1 (N6AMT1) in Arsenic Biomethylation and Its Role in Arsenic-Induced Toxicity. Environ. Health Persp. 2011, 119, 771–777.

Zhang H, Ge Y, He P, Chen X, Carina A, Qiu Y, Aga DS, Ren X. Interactive Effects of N6AMT1 and As3MT in Arsenic Biomethylation. Toxicol. Sci. 2015, 146 (2), 354–362.

Wood TC, Salavagionne OE, Mukherjee B, Wang L, Klumpp AF, Thomae BA, Eckloff BW, Schaid DJ, Wieben ED, Weinshilboum RM. Human arsenic methyltransferase (AS3MT) pharmacogenetics: gene resequencing and functional genomics studies. J. Biol. Chem. 2006, 281 (11), 7364–7373.

Acetylation

Procainamide

Procainamide is an anti-arrhythmic agent that induces a voltage-dependent open channel blockage of batrachotoxin (BTX)-activated sodium channels in cardiac muscle. It has a narrow therapeutic window, with many of its metabolites showing toxicity as well. However, its most common metabolite, N-acetylprocainamide, is less toxic. Interestingly, the rate of acetylation is genetically determined, with both slow and rapid acetylators known.

Reidenberg MM, Drayer DE, Levy M, Warner H. Polymorphic acetylation of procainamide in man. Clin. Pharmacol. Ther. 1975, 17, 722–730.

Gibson TP, Matusik J, Matusik E, Nelson HA, Wilkinson J, Briggs WA. Acetylation of procainamide in man and its relationship to isonicotinic acid hydrazide acetylation phenotype. Clin. Pharmacol. Ther. 1975, 17, 395–399.

Roden DM, Reele SB, Higgins SB, Wilkinson GR, Smith RF, Oates JA, Woosley RL. Antiarrhythmic efficacy, pharmacokinetics, and safety of N-acetylprocainamide in human subjects: comparison with procainamide. Am. J. Cardiol. 1980, 46, 463–468.

Isoniazid

Isoniazid is an oral antibiotic most commonly used in the treatment of tuberculosis, along with rifampicin, pyrazinamide, and either ethambutol or streptomycin. It is classified as an Essential Medicine by the World Health Organization. Metabolism occurs primarily via acetylation, with acetylation rates controlled genetically. This genetic difference causes dosing regimens to vary within the population and can cause higher rates of treatment failure in rapid acetylators if dosing is not adjusted properly. Conversely, in the slow acetylator group, elevated serum concentration can lead to adverse neurologic side effects due to an accumulation of the unmetabolized drug.

Alsultan A, Peloquin CA. Therapeutic Drug Monitoring in the Treatment of Tuberculosis: An Update. Drugs 2014, 74, 839–854.

Klein DJ, Boukouvala S, McDonagh EM, Shuldiner SR, Laurieri N, Thorn CF, Altman RB, Klein TE. PharmGKB Summary: isoniazid pathway, pharmacokinetics. Pharmacogenet. Genom. 2016, 26, 436–444.

Weber WW, Hein DW. Clinical Pharmacokinetics of Isoniazid. Clin. Pharmacokinet. 1979, 4, 401–422.

Amino Acid Conjugation

Salicylic acid

Salicylic acid is both a precursor and metabolite of acetylsalicylic acid (aspirin). Aside from its use in the production of aspirin, salicylic acid has several other medical uses, primarily as an ingredient in many skin care products where it is commonly used as an exfoliant. Salicylic acid undergoes a number of oxidative or conjugative transformations however, the major route of metabolism is via conjugation with glycine.

Hutt AJ, Caldwell J.; Smith RL. The metabolism of aspirin in man: a population study. Xenobiotica 2008, 16, 239–249.

Navarro SL.; Saracino MR.; Makar KW.; Thomas SS.; Li L.; Zheng Y.; Levy L.; Schwarz Y.; Bigler J.; Potter JD.; Lampe JW. Determinants of aspirin metabolism in healthy men and women: effects of dietary inducers of UDP-glucuronosyltransferases. Lifestyle Genom. 2011, 4, 110–118.

Bojić M.; Sedgeman CA.; Nagy LD.; Guengerich FP. Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450. Eur. J. Pharm Sci. 2015, 73, 49–56.

Mescaline

Mescaline, one of the main active constituents found in the peyote cactus, has been used by the indigenous peoples of the Americas for thousands of years in their religious, ceremonial, and medicinal rituals; it is used recreationally as a hallucinogenic drug. Historically, mescaline has been classified as a Schedule I controlled substance, drugs with no currently accepted medical purposes; however, there has been an increasing interest in using it, and other hallucinogenic drugs, as a treatment for mental health disorders such as anxiety and depression. Mescaline initially undergoes oxidative deamination to an inactive intermediate, which is then conjugated to glutamine by glutamine-N-acetyltransferase. However, this is not the only route of metabolism as different biotransformations occur in other tissues.

Charalampous KD, Walker KE, Kinross-Wright J. Metabolic fate of mescaline in man. Psychopharmacologia 1966, 9, 48–63.

Dinis-Oliveira RJ, Pereira CL, Dias da Silva D. Pharmacokinetic and Pharmacodynamic Aspects of Peyote and Mescaline: Clinical and Forensic Repercussions. Curr. Mol. Pharmacol. 2019, 12, 184–194.

Agin-Liebes G, Haas TF, Lancelotta R, Uthaug MV, Ramaekers JG, Davis AK. Naturalistic use of mescaline is associated with self-reported psychiatric improvements and enduring positive life changes. ACS Pharmacol. Transl. Sci. 2021, 4, 543–552.

Obeticholic acid

Obeticholic acid (OCA) is a first-in-class farnesoid X receptor (FXR) agonist developed for the treatment of various chronic liver diseases. Inspired by chenodeoxycholic acid, which is an endogenous FXR agonist, OCA is more metabolically stable and is x100 more potent. FXR plays a crucial role in plasma cholesterol metabolism, especially in HDL-cholesterol (HDL-C) homeostasis. OCA has been shown to induce a time-dependent reduction of serum HDL-C levels, with a concomitant increase in its fecal elimination. Metabolism of OCA occurs predominantly in the liver, where it is conjugated to either glycine or taurine, and ultimately eliminated (87%) in the feces. Conjugation results in active metabolites (glyco- and tauro-obeticholic acid) which have activity similar to the parent drug. These undergo enterohepatic recirculation and conversion by intestinal microbiota back to obeticholic acid that is reabsorbed or excreted. A third metabolite, the 3-glucuronide, has minimal pharmacological activity.

Dong B, Young M, Liu X, Singh AB, Liu, J. Regulation of lipid metabolism by obeticholic acid in hyperlipidemic hamsters. J. Lipid Res. 2017, 58, 350–363.

Polyzos SA, Kountouras J, Mantzoros CS. Obeticholic acid for the treatment of nonalcoholic steatohepatitis: expectations and concerns. Metabolism 2020, 104, 154144.

Gai K, Huang Y, Liu B, Zhang Y. Synthesis of obeticholic acid, a farnesoid X receptor agonist, and its major metabolites labeled with deuterium. J. Label. Compd. Radiopharm. 2020, 61, 799–804.

Final thoughts

"Aside from the well-known routes such as glucuronidation, there is a fascinating array of Phase II biotransformations to which drugs can be subjected," says Dr. Julia Shanu-Wilson of Hypha Discovery. There is also the potential for interplay with Phase I metabolic pathways, such as the formation of GSH adducts following the bioactivation of a drug to reactive intermediates by CYP enzymes.

"At Hypha, we have seen an increasing need for the synthesis of all types of glucuronides," Julia continued. "The prevalence of N-glucuronides is possibly due to the incorporation of nitrogen-containing heterocycles, and a deviation away from clearance via CYP mechanisms during the drug design phase."

List of Enzymes and Molecules Commonly Involved in Phase II Metabolism:

UDPGA – Uridine diphosphate glucuronic acid

UGT – Glucuronosyltransferase

SULT – Sulfotransferases

GST – Glutathione S-transferases

LAP3 – Leucine aminopeptidase 3

ANPEP – Aminopeptidase

NAT – N-acetyltransferase

COMT – Catechol-O-methyltransferase

ASMT – N-acetylserotonin O-methyltransferase

TEMT – Thioether S-methyltransferase

PNMT – Phenylethanolamine N-methyltransferase

GNMT – Glycine N-methyltransferase

NNMT – Nicotinamide N-methyltransferase

HNMT – Histamine N-methyltransferase

CARNMT1 – Carnosine N-methyltransferase

TMT – Thiol methyltransferase

TPMT – Thiopurine S-methyltransferase

AS3MT – Arsenite methyltransferase

ATP – Adenosine triphosphate

ACSM – Acyl-coenzyme A synthetases

GLYAT – Glycine N-acyltransferase

GLUAT – Glutamine N-acyltransferase

BACS – Bile acyl-CoA synthetase

BAAT – Bile acid-CoA:amino acid N-acyltransferase

We hope this introduction to phase II drug metabolism is a useful drug discovery resource. Explore drughunter.com for more drug discovery articles and resources.