Phase I or functionalization reactions do not always produce hydrophilic or pharmacologically inactive metabolites. Various phase II or conjugation reactions, however, can convert these metabolites to more polar and water-soluble products. Many conjugative enzymes accomplish this objective by attaching small, polar, and ionizable endogenous molecules, such as glucuronic acid, sulfate, glycine, and glutamine, to the phase I metabolite or parent xenobiotic. The resulting conjugated products are relatively water soluble and readily excretable. In addition, they generally are biologically inactive and nontoxic. Other phase II reactions, such as methylation and acetyla-tion, do not generally increase water solubility but mainly serve to terminate or attenuate pharmacological activity. The role of GSH is to combine with chemically reactive compounds to prevent damage to important biomacromol-ecules, such as DNA, RNA, and proteins. Thus, phase II reactions can be regarded as truly detoxifying pathways in drug metabolism, with a few exceptions.
A distinguishing feature of most phase II reactions is that the conjugating group (glucuronic acid, sulfate, methyl, and acetyl) is activated initially in the form of a coenzyme before transfer or attachment of the group to the accepting substrate by the appropriate transferase enzyme. In other cases, such as glycine and glutamine conjugation, the substrate is activated initially. Many endogenous compounds, such as bilirubin, steroids, catecholamines, and histamine, also undergo conjugation reactions and use the same coenzymes, although they appear to be mediated by more specific transferase enzymes. The phase II conjugative pathways
Figure 3.11 • Formation of UDPGA and ß-glucuronide conjugates.
Figure 3.11 • Formation of UDPGA and ß-glucuronide conjugates.
discussed include those previously listed in this chapter. Although other conjugative pathways exist (e.g., conjugation with glycosides, phosphate, and other amino acids and conversion of cyanide to thiocyanate), they are of minor importance in drug metabolism and are not covered in this chapter.
Glucuronidation is the most common conjugative pathway in drug metabolism for several reasons: (a) a readily available supply of d-glucuronic acid (derived from d-glucose), (b) numerous functional groups that can combine enzymat-ically with glucuronic acid, and (c) the glucuronyl moiety (with its ionized carboxylate [pKa 3.2] and polar hydroxyl groups), which, when attached to xenobiotic substrates, greatly increases the water solubility of the conjugated product.117,359-361 Formation of f-glucuronides involves two steps: synthesis of an activated coenzyme, uridine-5'-diphospho-a-d-glucuronic acid (UDPGA), and subsequent transfer of the glucuronyl group from UDPGA to an appropriate substrate.117,360,361 The transfer step is catalyzed by microsomal enzymes called UDP-glucuronyltransferases. They are found primarily in the liver but also occur in many other tissues, including kidney, intestine, skin, lung, and brain.360,361 The sequence of events involved in glucuronidation is summarized in Figure 3.11.117,360,361 The synthesis of the coenzyme UDPGA uses a-d-glucose-1-phosphate as its initial precursor. Note that all glu-curonide conjugates have the S-configuration or S-linkage at C-1 (hence, the term ff-glucuronides). In contrast, the coenzyme UDPGA has an a-linkage. In the enzymatic transfer step, it appears that nucleophilic displacement of the a-linked UDP moiety from UDPGA by the substrate RXH proceeds with complete inversion of configuration at C-1 to give the f-glucuronide. Glucuronidation of one functional group usually suffices to effect excretion of the conjugated metabolite; diglucuronide conjugates do not usually occur.
The diversity of functional groups undergoing glu-curonidation is illustrated in Table 3.3 and Figure 3.12.
Metabolic products are classified as oxygen-, nitrogen-, sulfur-, or carbon-glucuronide, according to the heteroatom attached to the C-1 atom of the glucuronyl group. Two important functionalities, the hydroxy and carboxy, form O-glucuronides. Phenolic and alcoholic hydroxyls are the most common functional groups undergoing glucuronidation in drug metabolism. As we have seen, phenolic and alcoholic hydroxyl groups are present in many parent compounds and arise through various phase I metabolic pathways. Morphine,362,363 acetaminophen,364 and p-hydroxyphenytoin (the major metabolite of phenytoin)49,50 are a few examples of phenolic compounds that undergo considerable glucuronidation. Alcoholic
TABLE 3.3 Types of Compounds Forming Oxygen, Nitrogen, Sulfur, and Carbon Glucuronidesa
Phenols: morphine, acetaminophen, p-hydroxyphenytoin Alcohols: tricholoroethanol, chloramphenicol, propranolol Enols: 4-hydroxycoumarin W-Hydroxyamines: W-hydroxydapsone W-Hydroxyamides: W-hydroxy-2-acetylaminofluorene
Aryl acids: benzoic acid, salicylic acid
Arylalkyl acids: naproxen, fenoprofen
Tertiary amines: cyproheptadine, tripelennamine
Sulfhydryl groups: methimazole, propylthiouracil, diethylthiocarbamic acid
3,5-Pyrazolidinedione: phenylbutazone, sulfinpyrazone aFor structures and site of ß-glucuronide attachment, see Figure 4.12.
hydroxyls, such as those present in trichloroethanol (major metabolite of chloral hydrate),288 chloramphenicol,365 and propranolol,366,367 are also commonly glucuronidated. Less frequent is glucuronidation of other hydroxyl groups, such as enols,368 N-hydroxylamines,226 and N-hydroxyl-amides.241 For examples, refer to the list of glucuronides in Table 3.3.
The carboxy group is also subject to conjugation with glucuronic acid. For example, arylaliphatic acids, such as the anti-inflammatory agents naproxen369 and fenopro-fen,370,371 are excreted primarily as their O-glucuronide derivatives in humans. Carboxylic acid metabolites such as those arising from chlorpheniramine290 and propranolol289 (see "Reduction of Aldehyde and Ketone Carbonyls,") form O-glucuronide conjugates. Aryl acids (e.g., benzoic acid,372 salicylic acid373,374) also undergo conjugation with gluc-uronic acid, but conjugation with glycine appears to be a more important pathway for these compounds.
Occasionally, N-glucuronides are formed with aromatic amines, aliphatic amines, amides, and sulfonamides. Representative examples are found in the list of gluc-uronides in Table 3.3. Glucuronidation of aromatic and aliphatic amines is generally a minor pathway in comparison with N-acetylation or oxidative processes (e.g., oxidative deamination). Tertiary amines, such as the anti-histaminic agents cyproheptadine (Periactin)375 and tripelennamine,376 form interesting quaternary ammonium glucuronide metabolites.
Because the thiol group (SH) does not commonly occur in xenobiotics, S-glucuronide products have been reported for only a few drugs. For instance, the thiol groups present in methimazole (Tapazole),377 propylthiouracil,378,379 and N,N-diethyldithiocarbamic acid (major reduced metabolite of disulfiram, Antabuse)380 undergo conjugation with gluc-uronic acid.
The formation of glucuronides attached directly to a carbon atom is relatively novel in drug metabolism. Studies in humans have shown that conjugation of phenylbutazone (Butazolidin)381,382 and sulfinpyrazone (Anturane)383 yield the corresponding C-glucuronide metabolites:
Besides xenobiotics, several endogenous substrates, notably bilirubin384 and steroids,385 are eliminated as gluc-
uronide conjugates, which are excreted primarily in the urine. As the relative molecular mass of the conjugate exceeds 300 Da, however, biliary excretion may become an important route of elimination.386 Glucuronides that are excreted in the bile are susceptible to hydrolysis by ^-glucuronidase enzymes present in the intestine. The hydrolyzed product may be reabsorbed in the intestine, thus leading to enterohepatic recycling.22 S-Glucuronidases are also present in many other tissues, including the liver, the endocrine system, and the reproductive organs. Although the function of these hydrolytic enzymes in drug metabolism is unclear, it appears that, in terms of hormonal and ccendocrine regulation, j-glucuronidases may liberate active hormones (e.g., steroids) from their inactive gluc-uronide conjugates.22
In neonates and children, glucuronidating processes are often not developed fully. In such subjects, drugs and endogenous compounds (e.g., bilirubin) that are metabolized normally by glucuronidation may accumulate and cause serious toxicity. For example, neonatal hyperbiliru-binemia may be attributable to the inability of newborns to conjugate bilirubin with glucuronic acid.387 Similarly, the inability of infants to glucuronidate chloramphenicol has been suggested to be responsible for the gray baby syndrome, which results from accumulation of toxic levels of the free antibiotic.388
Conjugation of xenobiotics with sulfate occurs primarily with phenols and, occasionally, with alcohols, aromatic amines, and N-hydroxy compounds.389-391 In contrast to glucuronic acid, the amount of available sulfate is rather limited. The body uses a significant portion of the sulfate pool to conjugate numerous endogenous compounds such as steroids, heparin, chondroitin, catecholamines, and thyroxine. The sulfate conjugation process involves activation of inorganic sulfate to the coenzyme 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Subsequent transfer of the sulfate group from PAPS to the accepting substrate is catalyzed by various soluble sulfotransferases present in the liver and other tissues (e.g., kidney, intestine).392 The sequence of events involved in sulfoconjugation is depicted in Figure 3.13. Sulfate conjugation generally leads to water-soluble and inactive metabolites. It appears, however, that the O-sulfate conjugates of some N-hydroxy compounds give rise to chemically reactive intermediates that are toxic.241
Phenols compose the main group of substrates undergoing sulfate conjugation. Thus, drugs containing phenolic moieties are often susceptible to sulfate formation. For example, the antihypertensive agent a-methyldopa (Aldomet) is metabolized extensively to its 3-O-sulfate ester in humans.393,394 The jS-adrenergic bronchodilators salbutamol (albuterol)395 and terbutaline (Brethine, Bricanyl)396 also undergo sulfate conjugation as their o o
II ATv JPi II I \ 0 Adenine
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