Phase II Biotransformation

First examples of drug conjugates possessing sulfate, glucuronic acid, methyl and acetylated moieties were discovered in the mid-1800s, realizing that they are less toxic compared to parent compound and metabolites of phase I. Phase II biotransformation is catalyzed by the enzymes which are collectively known as "transferases” as they catalyze the transfer of a moiety from a donor molecule to the drug recipient (uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), sulfotransferases, N- acetyltransferases (arylamine /V-acety transferase; NATs), glutathione (GSH) S-transferases (GSTs) and various methyltransferases) (Omiecinski et al., 2011). Phase II enzymes can directly interact with xenobiotics but more commonly interact with the newly formed functional group of metabolites produced by phase I reactions (Croom, 2012). Since many metabolites of phase I are too lipophilic to be retained in the kidney tubules, a subsequent conjugation reaction with an endogenous substrate, such as glucuronic acid, acetic acid, sulfuric acid or an amino acid, results in formation of more polar and hydrophilic compounds that are sufficiently polar to be excreted by the kidneys. These reactions represent the real drug detoxification pathways since the most common outcome of phase II reactions is the production of therapeutically inactive conjugates (Omiecinski et ah, 2011). An important exception is morphine-6-glucuronide, a widely used opioid analgesic and the most cited example of a pharmacologically active glucuronide which is twice as potent as parent morphine in many models of analgesia (Christrup, 1997). Although phase II reactions most commonly follow phase I, not all drugs undergo reactions in that order. For example, the major pathways of isoniazid metabolism include acetylation, in the first place, forming acetyil-isoniazid by means of NAT, followed by hydrolysis to produce isonicotinic acid by means of amidase (Wang et al., 2016).

Hydrolysis of procainamide to p-aminobenzoic acid by amidase

Figure 6.11 Hydrolysis of procainamide to p-aminobenzoic acid by amidase.

Different types of conjugation reactions along with the enzymes involved and examples of drugs undergoing the relevant reaction are listed in Table 6.4 (Taxak and Bharatam, 2014).

Type of reaction

Enzyme

Endogenous conjugating agent

Examples of drugs metabolized by the same pathway

Glucuronidation

UDP-

glucuronosyltransferase

UDP-Glucoronic acid

Chloramphenicol, morphine, paracetamol, salicylic acid, propofol fenoprofen, diazepam

Sulfation

Sulfotransferases

sulfate

Paracetamol, salbutamol

Glycine/

glutamine

conjugation

JV-Acyl transferases

Glycine

Glutamine

Cholic acid, salicylic acid, nicotinic acid, phenylacetic acid

Glutathione

conjugation

Glutathione S transferases

Glutathione

Adriamycin, busulfan, chlorambucil, cis-platin, cyclophosphamide, ethacrynic acid

Acetylation

N- Acetyl transferases

Acetyl-CoA

Histamine, procainamide, sulfonamides, isoniazid, hydralazine

Methylation

Methyl

transferase

S-Adenosyl-L- methionine

Morphine, norephedrine, nicotine, histamine, isoprenaline, propylthiouracil

Uridine diphosphate-glucuronosyltransferases

Glucuronidation is the most common and the most important conjugation reaction. The UGTs catalyze the transfer of glucuronic acid from donor molecule, uridine-a-glucuronide to a variety of substrates, both endogenous compounds and xenobiotics (Foti and Dalvie, 2016). UGTs are located predominantly in the endoplasmic reticulum of liver, but may be also found in other organs such as kidney, intestine, lungs, prostate, mammary glands, skin, brain, spleen, and nasal mucosa (Penner et al., 2012). Well-characterized endogenous substrates of UGTs include bilirubin, estradiol and serotonin (Foti and Dalvie,

2016). Glucuronidation is an important metabolic pathway for many anesthetic drugs; for example it is the major route of propofol metabolism (Le Guellec et ah, 1995). Although the majority of glucuronic conjugates are inactive metabolites, the important exception is morphine-6-glucuronide, as previously mentioned, and the reaction of activation is shown in Fig. 6.12. Regarding the glucuronidation of morphine, two metabolites are formed, morphine-3-glucuronide, which is the major metabolite (45-55%) but inactive and morphine-6-glucuronide (20-30%), which is the more potent analgesic than its parent compound (Christrup, 1997).

The activation of morphine to morphine-6-glucuronide by UGT

Figure 6.12 The activation of morphine to morphine-6-glucuronide by UGT.

Neonates are deficient in this conjugating system making them vulnerable to drugs such as chloramphenicol, which is inactivated by glucuronidation, resulting in "gray-baby” syndrome characterized by cyanosis, abdominal distention, vomiting, hypothermia, and cardiovascular collapse (Mulhall et al., 1983). Insufficient conjugation of bilirubin in neonates leads to increased serum concentration of lipid-soluble unconjugated bilirubin that may easily cross poorly established blood-brain barrier and cause kernicterus and neurotoxicity (Amin and Lamola, 2011).

Glutathione S-transferases

GSTs are involved in the metabolism of some endogenous substances such as leukotrienes, prostaglandins and testosterone, but also in the metabolism of many drugs and xenobiotics play an important role in cellular protection against oxidative stress. Three major families of glutathione transferases are widely distributed: cytosolic as the largest family, mitochondrial and microsomal. These enzymes catalyze nucleophilic attack of reduced glutathione (GSH) on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom leading to formation of thioether conjugates. Therefore, conjugation with GSH is an important protective mechanism against reactive electrophilic xenobiotics, such as environmental pollutants, chemical carcinogens, antitumor agents (adriamycin, busulfan, chlorambucil, c/s-platin, cyclophosphamide, ethacrynic acid) preventing their covalent binding to proteins. GSH plays a crucial role in the protection of some toxic drug metabolites, such as paracetamol oxidized metabolite /V-acetyl-p-benzoquinone imine. However, if metabolites are generated in an amount that exceeds the amount of available GSH, metabolites may exhibit toxic effects (Fig. 6.13). A large number of epoxides, such as the antibiotic fosfomycin and those derived from environmental carcinogens, are detoxified by GST. It has been shown that overexpression of GST in mammalian tumor cells has been implicated with resistance to various antitumor agents and chemical carcinogens (Chasseaud, 1979; Hayes et al., 2005).

Mechanism of detoxification of paracetamol metabolite, JV-acetyl-p-benzo-quinone imine by conjugation with glutathione

Figure 6.13 Mechanism of detoxification of paracetamol metabolite, JV-acetyl-p-benzo-quinone imine by conjugation with glutathione.

Methyltransferases

Methylation is a relatively minor conjugative pathway in drug metabolism and is engaged more with the metabolism of some endogenous compounds such as melatonin, histamine, serotonin, and dopamine. This reaction is catalyzed by methyltransferases, which transfer a methyl group from the methyl donor S-adenosyl- L-methionine (SAM) to their substrates. Opposite to other phase II reactions, methylation reduces the polarity of substrates. The most common outcome of methylation is deactivation of a compound. Depending of substrates, there are different types of methyltransferases: S-methyltransferases, O-methyltransferases, and N-methyltransferases.

Regarding the drug metabolism, S-methyltransferase catalyzes S-methylation of aromatic heterocyclic sulfhydryl compounds including anticancer and immunosuppressive thiopurines such as 6-mercaptopurine, 6-thioguanine and azathioprine (Fig. 6.14).

S-Methylation of 6-mercaptopurine to 6-methylmercaptopurine by S-methyltransferases

Figure 6.14 S-Methylation of 6-mercaptopurine to 6-methylmercaptopurine by S-methyltransferases.

Catechol O-methyltransferases are responsible for transfer of a methyl group from S-adenosylmethionine to catecholamines such as norepinephrine, epinephrine and dopamine, being one of the major degradative pathways of the catecholamine transmitters. Some drugs having a catechol structure, used in the treatment of hypertension, asthma and Parkinson's disease, can be also O-methylated by the COMT, thus preventing the formation of toxic quinones (Vereczkey et al., 1998; Jancova et ah, 2010). In Fig. 6.15, O-methylation of antiparkinsonian drug, L-dopa is shown.

IV-methyltransferase catalyses the /V-methylation of nicotinamide, pyridines, and other structurally related compounds.

This enzyme is involved in the major route of nicotinamide catabolism, using the universal methyl donor SAM to produce S-adenosyl-L-homocysteine (SAH) and Ah-methylnicotinamide (Fig. 6.16) (Rini etal., 1990).

O-Methylation of L-dopa to 3-0-methyl dopa by 0- methyltransferases

Figure 6.15 O-Methylation of L-dopa to 3-0-methyl dopa by 0- methyltransferases.

iV-Methylation of nicotinamide to ^-methylnicotinamide by JV-methyltransferase

Figure 6.16 iV-Methylation of nicotinamide to ^-methylnicotinamide by JV-methyltransferase.

N-Acetyltransferases

Acetylation is an important route of metabolism for certain drugs and a range of food-derived and environmental carcinogens with an aromatic amine (R-NH2) or a hydrazine structure (R-NH- NH2), which are converted to aromatic amides (R-NH-COCH3) and hydrazides (R-NH-NH-COCH3), respectively. Aryl N- acetyltransferases (NAT) are cytosolic conjugating enzymes which transfer an acetyl group from donor acetyl coenzyme A (AcCoA) to arylamine and arylhydrazines. These enzymes commonly catalyse drug deactivation. Similar to products of methylation, /V-acetylated metabolites are less water-soluble compared to parent compounds. There are two known human isoenzymes known as NAT2, which is responsible for isoniazid (Fig. 6.17), hydralazine and procainamide metabolism and NAT1, which is more specific for p-aminosalicylate, p-aminobenzoic acid and the folate metabolite, p-aminobenzoylglutamate (Sim et al., 2014; Grant et al., 1997). Both NAT1 and NAT2 activities have been described also in the intestine (Hickman et al., 1998). In humans, NAT1 has a much wider tissue distribution than NAT2 (Sim et al., 2014). These enzymes were the first discovered drug metabolizing enzymes with genetic polymorphisms, thus establishing the basics of pharmacogenetics. Pharmacogenetic variations have important clinical implications related to therapeutic efficacy and occurrence of adverse drug reactions. Pharmacogenomics is known to play the key role in the metabolism of isoniazid, a first-line drug for the treatment of tuberculosis. There are great interindividual variations in the rate of acetylation due to differences in the concentrations of NAT2 enzyme in the liver and gut mucosa. Therefore, patients may be characterized phenotypically as being either rapid or slow acetylators. The slow acetylators more commonly experience toxicity from drugs due to higher blood levels of the drug, whilst fast acetylators may not respond adequately to isoniazid in the treatment of tuberculosis (Ramachandran and Swaminathan, 2012; Roy et al., 2008).

ЛГ-Acetylation of isoniazid to N-acetyl isoniazid by N- acetyltransferases

Figure 6.17 ЛГ-Acetylation of isoniazid to N-acetyl isoniazid by N- acetyltransferases.

Sulfotransferases

Sulfortranferasese are cytosolic conjugating metabolizing enzymes which use З'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor of sulfate groups and transfer them to an acceptor substrate compounds. In that way PAPS is transformed to adenosine phosphosulfate (APS). In the majority of cases, the final outcome of the sulfation reaction is a molecule with increased hydrophilic properties and inactivation of biologically active compounds (Gamage et al., 2006). Sulfation is a major conjugation pathway for some endogenous compounds (steroid/ thyroid hormones, bile acids and catecholamines), procarcinogens (heterocyclic aromatic amines), some dietary compounds (flavonoids), and certain drugs which have an alcoholic, phenolic, hydroxylamino or amino function in their structure. For example, sufotranferases catalyze the metabolism of phenolic drugs such as paracetamol, isoproterenol, salbutamol and dobutamine, and some steroid drugs such as budesonide and tibolone, and some opioid drugs such as morphine, hydromorphone, oxymorphone, butorphanol, levorphanol, nalorphine, and naltrexone (Kurogi et al., 2014). In addition to glucuronidation that is responsible for metabolism of approximately 55% of paracetamol as the major metabolic pathway, approximately 30% of circulating paracetamol may be also conjugated by sulfotransferase, as shown in Fig. 6.18. As already mentioned, the minor part of this drug (5-10%) can be oxidized by a CYP450 (particularly CYP2E1) to the toxic free radical N-acetyl-p-benzoquinone imine (Mazaleuskaya et ah, 2015).

Sulfatation of paracetamol to paracetamol sulfonate by sulfoltransfe rases

Figure 6.18 Sulfatation of paracetamol to paracetamol sulfonate by sulfoltransfe rases.

 
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