Operativa del Sistema .1 Descripción General

Rory P. Remmel and Jin Zhou

Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, U.S.A.

Upendra A. Argikar

Novartis Pharmaceuticals, Cambridge, Massachusetts, U.S.A.

INTRODUCTION

The uridine diphosphate (UDP)-glycosyltransferases (EC2.4.21.17) are a group of enzymes that catalyze the transfer of sugars (glucuronic acid, glucose, and xylose) to a variety of acceptor molecules (aglycones). The sugars may be attached at aromatic and aliphatic alcohols, carboxylic acids, thiols, primary, secondary, tertiary, and aromatic amino groups, and acidic carbon atoms. In vivo, the most common reaction occurs by transfer of glucuronic acid moiety from UDP glucuronic acid (UDPGA) to an acceptor molecule. This process is termed either glucuronidation or glucuronosylation. When the enzymes catalyze this reaction, they are also referred to as UDP-glucuronosyltransferases (UGTs). The structure and function of the enzymes have been the subject of several reviews (1 4). This chapter reviews the role of these enzymes in drug-drug interactions that occur in humans.

Glucuronidation is an important step in the elimination of many important endogenous substances from the body, including bilirubin, bile acids, steroid hormones, thyroid hormones, retinoic acids, and biogenic amines such as serotonin. Many of these compounds are also substrates for sulfonyltransferases (SULTs) (2). The interplay between glucuronidation and sulfonylation (sulfation) of steroid and thyroid hormones and the corresponding hydrolytic enzymes, b-glucuronidase and sulfatase, may play an important role in development and regulation. The UGTs are expressed in many tissues, including liver, kidney, intestine, colon, adrenals, spleen, lung, skin, testes, ovaries, olfactory glands, and brain. Interactions between drugs at the enzymatic level are most likely to occur during the absorption phase in the intestine and liver or systemically in the liver, kidney, or intestine.

Given the broad array of substrates and the variety of molecular diversity, it is not surprising that there are multiple UGTs. The UGTs have been divided into two families (UGT1 and UGT2) on the basis of their sequence homology. All members of a family have at least 50% sequence identity to one another (3). The UGT1A family is encoded by

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a gene complex located on chromosome 2. The large UGT1A gene complex contains 13 variable region exons that are spliced onto four constant region exons that encode for amino acids on the C-terminus of the enzyme. Consequently, all enzymes in the UGT1 family have an identical C-terminus (encoding for the UDPGA binding site), but the N-terminus is highly variable, with a sequence homology of only 24 49% (3). The UGT1A enzymes are generally named in order of their proximity to the four constant region exons, i.e., UGT1A1 through UGT1A13. The arrangement (Fig. 1) appears to be conserved across all mammalian species studied to date (5). In humans, all of the gene products are functions except for pseudogenes UGT1A2, UGT1A11, UGT1A12, and UGT1A13. Pseudogenes encoding for inactive proteins vary from species to species. For example, UGT1A6 is a pseudogene in cats (6), whereas UGT1A3 and UGT1A4 are pseudogenes in rats and mice. The UGT1A gene complex is located on human chromosome 2 at 2q.37. Nomenclature for these enzymes in other species can be found on the UGT Web site at http://som.flinders.edu.au/FUSA/ClinPharm/UGT/.

The UGT2A subfamily represents olfactory UGTs and will not be discussed further in this review. Human UGT2A was originally cloned by Burchell and coworkers (7). The UGT2B subfamily is encoded in a series of complete UGT genes located at 4q12 on chromosome 4. Like the UGT1A enzymes, the C-terminus is highly conserved among all members of the UGT2B genes, with greater variation in the N-terminal half of the protein.

Several human UGT2B enzymes have been cloned, expressed, and characterized for a variety of substrates. The nomenclature for the UGT2B genes has been assigned on the basis of the order of their discovery and submission to the nomenclature committee similar to that for CYP2 and CYP3 family enzymes. The human UGT2B enzymes are UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B43.

Inhibitory interactions involving glucuronidation have been described in a number of clinical and in vitro studies and have been recently reviewed (8). Apparent decreases in the amount of glucuronide excreted in urine or bile or apparent increases in the AUC (decreased clearance) have been demonstrated in clinical studies. These apparent effects on glucuronidation could occur via several different mechanisms as follows:

1. Direct inhibition of the enzyme by competition with substrate or with UDPGA 2. Induction of the individual UGT enzymes resulting in increased clearance 3. Depletion of the UDPGA cofactor

4. Inhibition of the transport of UDPGA into the endoplasmic reticulum (ER) 5. Inhibition of the renal excretion of the glucuronide, with subsequent

reconver-sion to the parent aglycone byb-glucuronidases (futile cycling)

6. Alteration of ER transport, sinusoidal membrane transport, or bile canalicular membrane transport of the glucuronides

7. Inhibition of the intestinal microflora, resulting in interruption of enterohepatic recycling and increased fecal excretion of the glucuronide metabolite.

Figure 1 The UGT1 gene complex.

Major interactions involving individual UGT enzymes will be discussed in detail along with a brief discussion of the function of each enzyme. A table of substrates, inducers, and inhibitors for the UGT enzymes is provided in the appendix to this chapter.

UGT1A1

UGT1A1 is an important enzyme that is primarily responsible for the glucuronidation of bilirubin in the liver. Cloned, expressed UGT1A1 is a glycosyltransferase that is also capable of catalyzing the formation of bilirubin xylosides and glycosides in the presence of UDP-xylose and UDP-glucose, respectively (9). In vivo, glucuronidation predominates, but bilirubin xylosides and glucosides have been identified in human bile. Polymorphisms in the UGT1A1 gene have been extensively studied because of a rare inborn error of bilirubin metabolism resulting in Crigler-Najjar syndrome. Type I Crigler-Najjar patients typically require liver transplantation, whereas Type II patients can be treated with UGT1A1 inducers such as phenobarbital. Gilbert’s syndrome is an asymptomatic unconjugated hyperbilirubinemia that is most often caused by a genetic polymorphism in the promoter region of the UGT1A1 gene in Caucasians and Africans. Decreased expression of UGT1A1 in Gilbert’s patients is a result of the presence of a (TA)7TAA allele (UGT1A1*28) in place of the more prevalent (TA)6TAA allele (10,11). Persons who are homozygous for the (TA)₇TAA express approximately 70% less UGT1A1 enzyme in the liver. A second mutation at 3279 C>T in a phenobarbital response enhancer module (PBREM) also is linked with Gilbert’s syndrome and is often in linkage disequilibrium with UGT1A1*28 in Caucasians and Japanese (12 14). Larger screening studies have demonstrated that this regulatory defect occurs in approximately 2 19% of various populations (11). In Asian patients, other mutations in the UGT1A1 gene besides the (TA)₇TAA genotype contribute significantly to hyperbilirubinemia, including UGT1A1*6 (211 G>A, G71R) (15,16). Drugs that are substrates for or inhibit UGT1A1 may cause a further increase of unconjugated bilirubin concentrations, especially in patients with Gilbert’s syndrome. For example, the HIV protease inhibitors atazanavir and indinavir are known to increase bilirubin levels (17). Lankisch et al.

recently found that atazanavir treatment increased median bilirubin concentrations from 10 to 41mM (p ¼ 0.001) (18). Bilirubin levels exceeding 43 mM were observed in 37% of the 106 patients. Hyperbilirubinemia>43 mM was significantly associated with three non-1A1 mutations UGT1A3-66C, UGT1A7-57G, and UGT1A7*2 along with UGTnon-1A1*28, although these variants are not typically in linkage disequilibrium in other populations.

Six patients expressing all four mutations had bilirubin levels>87 mM, a level that may require discontinuation or dosage adjustment. UGT1A3 is a weak catalyst of bilirubin glucuronidation, whereas UGT1A7 would not be expected to contribute given its extrahepatic tissue distribution.

Older studies in persons with mild hyperbilirubinemia (meeting the criteria for Gilbert’s syndrome, but not genetically determined) demonstrated a decreased clearance rate for drugs that are glucuronidated. Clearance of acetaminophen (APAP; also catalyzed by other UGT enzymes, especially UGT1A5) was decreased by 30% in six subjects with Gilbert’s syndrome (19). In contrast, a small study by Ullrich et al. demonstrated no difference in the APAP-glucuronide/acetaminophen ratio in urine of 11 persons with Gilbert’s syndrome (20). A more recent study in genotyped patients also found no difference in the glucuronide/acetaminophen urinary ratio (21). Racemic (S/R) lorazepam clearance (catalyzed by UGT2B7 and UGT2B15) was 30 40% lower in persons with Gilbert’s syndrome (22). A modest decrease (32%) in lamotrigine oral clearance was

observed in persons with Gilbert’s syndrome (23). However, lamotrigine is glucuroni-dated by cloned, expressed UGT1A3 and UGT1A4, but not by UGT1A1 (24,25). In general, these studies were conducted in a small number of Gilbert’s syndrome subjects.

A distinct heterogeneity may be present in persons exhibiting mild hyperbilirubinemia that could include patients with Crigler-Najjar Type II syndrome who have mutations in the UGT1A1-coding region, persons who are homozygous for UGT1A1*28, or in patients with a higher than normal breakdown of heme.

The role of UGT1A1*28 polymorphism and irinotecan toxicity has been extensively investigated in Japan by Ando et al. (26) and in the United States by Innocenti et al. (27).

Irinotecan is a prodrug that is rapidly converted by esterases to active phenolic compound, SN-38. SN-38 glucuronidation is catalyzed primarily by UGT1A1 in studies with cloned, expressed enzymes. Iyer et al. compared the liver microsomal glucuronidation rate of SN-38 and bilirubin in 44 patients genotyped for the (TA)₇TAA allele (UGT1A1*28) and found a high correlation (r ¼ 0.9) (28). Patients with the UGT1A1*28 allele who take irinotecan have a significantly higher risk for neutropenia, and the FDA has recently recommended that patients should be genotyped prior to use of irinotecan.

Evidence for drug-drug or herb-drug interactions involving UGT1A1 and irinotecan are limited (29). Case reports have suggested that inducers (e.g., phenytoin, carbamazepine, or rifampin) acting via the constitutive androstane receptor (CAR) or pregnenolone-16a-nitrile-X-receptor (pregnane-X-receptor; PXR) reduce exposure to SN-38; however, this could be due to enhanced CYP3A4-mediated metabolism of irinotecan to 7-ethyl-10-[4-N-[(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC) (30) or by glucuronidation (31,32). Similar findings by Mathijssen et al. have implicated induction of SN-38 metabolism by St. John’s wort (contains hyperforin, a potent PXR ligand) (33); however, evidence of increased glucuronidation in humans is lacking even though UGT1A1 is inducible by both PXR and CAR activation. Milk thistle (sylibinin) had no effect on SN-38 or SN-38 glucuronide levels (34). Sylibinin is metabolized by UGT1A1, but bioavailability is low and circulating levels are probably not high enough to affect glucuronidation. Gefitinib enhances irinotecan (SN-38) bioavailability in mice apparently via inhibition of the ABCG2 transporter (BCRP) (35).

In a small study of etoposide and irinotecan, Ohtsu reported that all three patients receiving the combination had grade 3 or 4 toxicities (one neutropenia, one hepatotoxicity, and one hyperbilirubinemia) (36). Etoposide was recently shown to be a UGT1A1 substrate (37,38), so this combination should be avoided. In a single patient case report, an interaction between lopinavir/ritonavir and irinotecan was reported resulting in increased SN-38 AUC, most likely because of inhibition of CYP3A4 to APC (29,39). No reports of interactions between atazanavir or indinavir (known inhibitors of UGT1A1) and irinotecan have surfaced.

UGT1A3 AND UGT1A4

UGT1A3 and UGT1A4 appear to be important enzymes involved in the catalysis of many tertiary amine or aromatic heterocycles to form quaternary ammonium glucuronides (24,25). UGT1A3, UGT1A4, and UGT1A5 share a high nucleic acid sequence homology of 93 94% in the first variable-region exon and probably have arisen by gene duplication.

The first exon of this group of enzymes appears to have diverged considerably from UGT1A1 (58% homology to 1A4), UGT1A5, and UGT1A7-10. UGT1A4 is expressed in human liver, intestine, and colon, although the level of expression of UGT1A4 mRNA is lower than that of UGT1A1 mRNA. UGT1A3 is expressed in liver, biliary epithelium,

colon, and gastric tissue. UGT1A4 has low activity for bilirubin compared with UGT1A1 and has sometimes been designated as a minor bilirubin form. Although the N-glucuronidation of UGT1A3 and UGT1A4 for a variety of tertiary amines such as imipramine, cyproheptadine, amitriptyline, tripelennamine, and diphenhydramine over-laps (K_m¼ 0.2 2 mM), some differences have been observed. UGT1A3 catalyzes the glucuronidation of buprenorphine, norbuprenorphine (low K_m values), morphine (3-position only), and naltrexone. Only UGT1A3 is capable of forming carboxyl-linked glucuronides of bile acids and nonsteroidal anti-inflammatory drugs (NSAIDs) (25).

Fulvestrant appears to be a highly selective substrate for this enzyme (40). In contrast, N-glucuronidation of trifluoperazine and tamoxifen are selectively catalyzed by UGT1A4 and the steroidal sapogenins, hecogenin, and tigogenin are low K_msubstrates (7 20mM) for 1A4, but not 1A3. UGT1A4 has good activity for progestins, especially 5 a-pregnane-3a,20a-diol and androgens such as 5a-androstane-3a,17b-diol.

Assuming that UGT1A3 and UGT1A4 are primarily responsible for the glucuronidation of tertiary amine antihistamines and antidepressants, significant drug interactions involving glucuronidation with these substrates have not been reported. This is not unexpected because<25% of the dose is excreted as a direct quaternary ammonium glucuronide in urine. The formation of quaternary ammonium glucuronides appears to be highly species specific, with the highest activity in humans and monkeys. Rats and mice are generally incapable of forming quaternary ammonium glucuronides (UGT1A3 and UGT1A4 are pseudogenes in these rodents). Lamotrigine, a novel triazine anticonvulsant, is extensively glucuronidated at the 2-position of the triazine ring in humans (>80% of the dose is excreted in human urine) (41). It is not significantly glucuronidated in rats or dogs, but 60% of the dose is excreted in guinea pig urine as the 2-N-glucuronide (42). Several significant interactions have been reported for lamotrigine in humans. Lamotrigine glucuronidation is induced in patients taking phenobarbital, phenytoin, or carbamazepine (CAR inducers), resulting in a twofold decrease in apparent half-life from 25 hours to approximately 12 hours (43). In contrast, valproic acid inhibits lamotrigine glucuronidation resulting in a two- to threefold increase in half-life (44). Valproic acid is a weak substrate for UGT1A4 and UGT1A3 (U Argikar, PhD thesis, University of Minnesota, 2006), but has higher affinity for UGT2B7. Lamotrigine had a small, but significant effect (25% increase) on the apparent oral clearance of valproic acid (44). This increase could be due to induction of the UGTs responsible for valproic acid glucuronidation, since chronic treatment with lamotrigine results in autoinduction. The interaction between APAP and lamotrigine has also been studied. Surprisingly, APAP decreased the lamotrigine AUC by approximately 20% after multiple oral doses in human volunteers. Lamotrigine clearance was 32% lower in seven patients with Gilbert’s syndrome compared with persons with normal bilirubin levels, but it does not appear to be a substrate for UGT1A1 (23).

Polymorphisms have been identified in both UGT1A3 and UGT1A4. Iwai et al.

identified four nonsynonymous single-nucleotide polymorphisms (SNPs) in the UGT1A3 sequence of a Japanese population (n¼ 100) at Q6R, W11R, R45W, and V47A (45). Five allele combinations with frequencies of 0.055 to 0.13 were identified. The intrinsic clearances of estrone glucuronidation for the cloned, expressed variants were determined and the only significant difference was in the W11R-V47A variant (UGT1A3*2) that showed an increase of 369% due to a fivefold lower K_mvalue (allele frequency¼ 0.125).

In contrast, Ehmer et al. reported that the W11R and V47A variants were much more common in German Caucasians (allele frequency ¼ 0.65 and 0.58, respectively) (46).

Chen et al. extended this work and examined activities of the variants with several other flavonoid substrates, including quercetin, luteolin, and kaempferol, and also found increased activity (47). They found that the R45W variant had 3.5 to 4.7 times higher

intrinsic clearance toward the flavonoids, whereas for estrone, activity was reduced to 70% of control (47). Regioselectivity in the glucuronidation of quercetin was also altered between variants. Two common variants in the UGT1A4 gene have also been identified, but the effect on activity appears to vary depending on the substrate. Ehmer et al. found two major variants at P24T and L48V (allele frequencies of 0.07 0.1 in Caucasians). The L48V mutant completely lost dihydrotestosterone glucuronidation activity (46), but was more efficient for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (48) and clozapine compared with wild-type UGT1A4 (49). Catalytic efficiencies for substrates such as trans-androsterone, imipramine, cyproheptadine, and tigogenin also changed (49).

Regulation of UGT1A4 and UGT1A3 has been recently investigated in a transgenic human UGT1A knock-in mouse model (50). UGT1A3 bile acid glucuronidation was highly upregulated by peroxisome proliferator activated receptor (PPAR)-a agonists (51).

UGT1A4 activity and mRNA expression was inducible by PXR and CAR agonists.

Consequently, induction interactions are likely to occur and have been demonstrated in humans as demonstrated by lamotrigine interactions with inducing anticonvulsants.

UGT1A6

UGT1A6 is the most important enzyme for the conjugation of planar phenols and amines.

It displays high activity for a variety of aromatic alcohols, including 1-naphthol, 4-nitrophenol, 4-methylumbelliferone, and APAP. However, these planar phenols are substrates for several other UGT enzymes. Immunoinhibition studies with an antibody raised against the 120 amino acid N-terminal region UGT1A6 peptide fused to Staphylococcus aureus protein A revealed that approximately 50% of the 1-naphthol glucuronidation activity in human liver microsomes (HLMs) could be inhibited (52). Cats are highly susceptible to APAP liver toxicity because UGT1A6 is a pseudogene in this species (6). Serotonin appears to be a highly selective endogenous substrate for this enzyme (53). The first exon sequence of UGT1A6 is divergent from other UGT1A sequences, being most similar to UGT1A9 with only a 54% homology. In rats, UGT1A6 is inducible by polycyclic aromatic hydrocarbons (PAH). UGT1A6 was also induced in human hepatocytes byb-naphthoflavone and in some, but not all, hepatocyte preparations by rifampin. APAP glucuronidation appears to be increased in smokers, perhaps due to PAH-mediated induction of UGT1A6. Serotonin glucuronidation was doubled in microsomes from persons with moderate-to-heavy alcohol use (54).

Krishnaswamy discovered several variants in the UGT1A6 gene (55). The UGT1A6*2 variant (S7A/T181A/R184S) showed a twofold higher activity (lower K_m) for several substrates (serotonin 4-nitrophenol, APAP, valproic acid) when cloned and expressed in HEK-293 cells compared with wild-type enzyme; however, the K_m was higher than wild type in (*2/*2) HLMs (54). Allele frequencies in Caucasians for the S7A, T181A, and R184S variants were 0.32 to 0.37. In Japanese, the frequency of these mutations is somewhat lower (0.22) (56). Response elements for HNF1-a, Nrf-2, AhR, PXR/CAR have been identified in the regulatory region of this gene (55). In a small study of 15 b-thalassemia/hemoglobin E patients, those subjects with a UGT1A6*2 variant without UGT1A1*28 showed a significant, lower AUC of APAP, APAP-glucuronide, and APAP-sulfate than those of the patients with wild-type UGT1A1 and UGT1A6 (57).

Interactions involving APAP and its glucuronidation are listed in Table 1.

Approximately 50% of a typical dose of APAP is glucuronidated (58). UGT1A1, UGT1A6, and UGT1A9 are the principal UGTs involved in glucuronidation. UGT1A6 is a high-affinity (K_m¼ 2.2 mM), low-capacity enzyme. UGT1A1 has intermediate affinity

(9 mM) with high capacity, and UGT1A9 is a low-affinity, high-capacity enzyme (21 mM) (58). With a kinetic model, Court et al. estimated that at typical therapeutic concentrations (0.05 5 mM), UGT1A9 was the most important enzyme (>55% of total activity). Consequently, the mechanism of induction of APAP glucuronidation by oral contraceptives, phenytoin, and rifampin is unclear and may involve multiple enzymes.

UGT1A7, UGT1A8, UGT1A9, AND UGT1A10

There is a 93 94% sequence homology in the first exon of UGT1A7 to UGT1A10;

however, these enzymes show great variation in the level of tissue expression. This group of UGT1A enzymes is highly divergent from UGT1A3 to UGT1A5 with approximately 50% identity in the first exon compared with UGT1A9. UGT1A9 is expressed in human hepatic and kidney tissues, whereas UGT1A7, UGT1A8, and UGT1A10 are expressed extrahepatically. Liver expression appears to be controlled by the presence of an HNF4-a response element at 372 to 360, that is present only in UGT1A9 and a distal response element to HNF-1 (65,66). UGT1A8 and UGT1A10 are intestinal forms (and UGT1A7 is expressed in esophagus and gastric epithelium). In both rat and rabbit, UGT1A7 is expressed in liver. The rabbit (legomorph) enzyme (UGT1A7l) displays high activity for a variety of small phenolic compounds such as 4-methylumbelliferone, p-nitrophenol, vanillin, 4-tert-butylphenol, and octylgallate. In addition, the rabbit enzyme is capable of catalyzing the N-glucronidation of imipramine to a quaternary ammonium glucuronide, similar to UGT1A4 (67). Rat UGT1A7 catalyzes the glucuronidation of benzo(a)pyrene phenols and is inducible by both 3-methylcholanthrene (3-MC) and oltipraz. Ciotti demonstrated that human UGT1A7 has very high activity for the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, and therefore may play a role in the gastrointestinal first-pass metabolism of this drug

however, these enzymes show great variation in the level of tissue expression. This group of UGT1A enzymes is highly divergent from UGT1A3 to UGT1A5 with approximately 50% identity in the first exon compared with UGT1A9. UGT1A9 is expressed in human hepatic and kidney tissues, whereas UGT1A7, UGT1A8, and UGT1A10 are expressed extrahepatically. Liver expression appears to be controlled by the presence of an HNF4-a response element at 372 to 360, that is present only in UGT1A9 and a distal response element to HNF-1 (65,66). UGT1A8 and UGT1A10 are intestinal forms (and UGT1A7 is expressed in esophagus and gastric epithelium). In both rat and rabbit, UGT1A7 is expressed in liver. The rabbit (legomorph) enzyme (UGT1A7l) displays high activity for a variety of small phenolic compounds such as 4-methylumbelliferone, p-nitrophenol, vanillin, 4-tert-butylphenol, and octylgallate. In addition, the rabbit enzyme is capable of catalyzing the N-glucronidation of imipramine to a quaternary ammonium glucuronide, similar to UGT1A4 (67). Rat UGT1A7 catalyzes the glucuronidation of benzo(a)pyrene phenols and is inducible by both 3-methylcholanthrene (3-MC) and oltipraz. Ciotti demonstrated that human UGT1A7 has very high activity for the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, and therefore may play a role in the gastrointestinal first-pass metabolism of this drug