Bioactivation Potential of Thiophene-Containing Drugs - American

Review pubs.acs.org/crt

Bioactivation Potential of Thiophene-Containing Drugs Darja Gramec, Lucija Peterlin Mašič, and Marija Sollner Dolenc* Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia ABSTRACT: Thiophene is a five-membered, sulfur-containing heteroaromatic ring commonly used as a building block in drugs. It is considered to be a structural alert, as its metabolism can lead to the formation of reactive metabolites. Thiophene S-oxides and thiophene epoxides are highly reactive electrophilic thiophene metabolites whose formation is cytochrome P450-dependent. These reactive thiophene-based metabolites are quite often responsible for druginduced hepatotoxicity. Tienilic acid is an example of a thiophenebased drug that was withdrawn from the market after only a few months of use, due to severe cases of immune hepatitis. However, inclusion of the thiophene moiety in drugs does not necessarily result in toxic effects. The presence of other, less toxic metabolic pathways, as well as an effective detoxification system in our body, protects us from the bioactivation potential of the thiophene ring. Thus, the presence of a structural alert itself is insufficient to predict a compound’s toxicity. The question therefore arises as to which factors significantly influence the toxicity of thiophene-containing drugs. There is no easy way to answer this question. However, the findings presented here indicate that, for a number of reasons, daily dose and alternative metabolic pathways are important factors when predicting toxicity and will therefore be discussed together with examples.

■

CONTENTS

Introduction Types of Reactive Metabolites of the Thiophene Ring Bioactivation of Thiophene Ring Thiophene S-Oxidation Epoxidation of the Thiophene Ring Thiophene-Based Compounds Forming Reactive Metabolites and Related Toxicities Tienilic Acid (TA) and Tienilic Acid Positional Isomer (TAI) Methapyrilene Tiaprofenic Acid and Suprofen Zileuton OSI-930 Thiophene-Based Compounds without Reactive Metabolites on the Thiophene Ring Duloxetine Eprosartan Rivaroxaban Olanzapine Compounds with Reactive Metabolites Responsible for Their Pharmacologic Action Clopidogrel Ticlopidine Prasugrel Factors Affecting the Toxicity of Thiophene-Containing Drugs Daily Dose and Extent of Covalent Binding Substitution of the Thiophene Ring Competitive Metabolic Pathways Conclusions and Future Trends © 2014 American Chemical Society

Author Information Corresponding Author Funding Notes Acknowledgments Abbreviations References

1344 1346 1346 1346 1346

1356 1356 1356 1356 1356 1356 1356

■

1347

INTRODUCTION One of the main functions of drug metabolism is to remove foreign substances from the body.1 This process involves numerous enzymes that transform a drug into a more hydrophilic metabolite, thus achieving better elimination from the body. Metabolism is considered to be a detoxification process; however, biotransformation can enhance the reactivity of certain drugs. The latter process is called bioactivation and can have detrimental effects.1,2 Chemically, reactive metabolites (RMs) are electrophiles or free radicals and can react readily with proteins, lipids, or nucleic acids, ultimately culminating in a toxic response.1 RMs can be the main reason for an idiosyncratic drug reaction (IDR), an adverse drug reaction unrelated to drug pharmacology which, due to its rare occurrence, often stays undetected during the clinical phases of drug testing but can have life-threatening effects.2−5 Idiosyncratic reactions can affect almost every organ of the body, although drug-induced liver injury is the most frequent effect.5 Formation of RMs, therefore, constitutes a major

1347 1349 1349 1350 1350 1350 1350 1351 1351 1352 1352 1352 1353 1354 1354 1354 1354 1355 1355

Received: April 10, 2014 Published: July 11, 2014 1344

dx.doi.org/10.1021/tx500134g | Chem. Res. Toxicol. 2014, 27, 1344−1358

Chemical Research in Toxicology

Review

Figure 1. In vitro and in vivo biotransformation reactions of the thiophene ring.7,9,14,23,24

related anti-aggregatory drugs.16,17 However, besides the desired anti-aggregatory effect, such drugs show a diverse palette of side effects, among which hepatotoxicity and aplastic anemia are the most serious.17 Side effects are especially common with ticlopidine (65, Figure 16), which is taken at considerably higher daily doses than clopidogrel.17 Thus, daily dose has an important effect on the toxicity caused by the bioactivated thiophene moiety. Drugs that are taken at very low doses are unlikely to result in toxicity derived from RMs.6 In addition to daily dose, numerous other factors influence the toxicity of a thiophene-containing drug. The structural characteristics of the drug as well as substitution of the thiophene ring have an important impact on the metabolic pathways utilized for that drug. Nevertheless, certain substances, or only minor parts of them, are not metabolized on the thiophene ring. Although a particular drug may undergo bioactivation in vivo, it is rarely toxic at effective doses. This is due to an effective detoxification system that can neutralize small quantities of formed RMs. The overall toxicity depends on various factors, among which detoxification route and relevance of the modified protein or proteins are the most significant.3,18 The aim of this review is to present current knowledge about the metabolism of thiophene-containing molecules. We have focused on the metabolic bioactivation of pharmaceutical substances with a thiophene-containing moiety and on the role of their RMs in drug toxicity. The reasons why some drugs containing a thiophene ring cause toxic effects and others do

problem in the pharmaceutical industry and is the leading cause for the withdrawal of a new drug from the market.6 Five-membered aromatic rings are building blocks of many drugs.7 They are used as a bioisosteres for structures like phenyl, which can result in improved pharmacokinetic and pharmacodynamic properties of a drug.7 Among them, thiophene, a five-membered aromatic sulfur-containing heterocycle, has proven to be an attractive isostere, resulting in improved effectiveness of a drug.7 On the other hand, it is very well known that thiophene metabolism can lead to the formation of RMs, which are the main reason for the toxicity of several drugs containing the thiophene moiety. Adverse effects of drugs containing the latter are caused by cytochrome P450 (CYP450) mediated oxidation of the thiophene ring. RMs reported for thiophene bioactivation are thiophene-S oxides,8,9 thiophene epoxides,9 and sulphenic acids.10 They are highly reactive and can rapidly react with small molecule nucleophiles (water, glutathione). A further, less desired event is reaction with nucleophilic residues of proteins, resulting in toxic effects. Suprofen (29, Figure 6) and tienilic acid (13, Figure 2), for example, are thiophene-containing drugs that were, due to serious side effects (e.g, acute renal failure and severe immunoallergic hepatitis), withdrawn from the market.11−14 In contrast, there are certain drugs containing a thiophene moiety that are not subject to bioactivation of the thiophene ring, with related toxicity. An example of this is duloxetine (44, Figure 10), extensively used an antidepressant.15 Furthermore, the thiophene moiety is required for the antiplatelet activity of clopidogrel (60, Figure 15) and some 1345



Review

containing CYP2C9, suprofen (29), and NADPH; 5-OH suprofen (31, Figure 6) was the main metabolite detected.14 Because both pathways, S oxidation and epoxidation, can result in formation of 5-OH suprofen, isotope labeling was used to distinguish between the two mechanisms. In the S-oxidation pathway, water is the main source of oxygen (1,4 Michael addition of water on thiophene S-oxide), whereas molecular oxygen is the main source for thiophene epoxidation. Microsomal incubation of substance in the presence of H218O or 18O2 therefore helps to distinguish between the metabolic pathways. O’Donnell et al.14 demonstrated that incorporation of 18O from 18O2 into the suprofen molecule provides direct evidence for epoxidation and excludes an S-oxidation pathway. In the presence of glutathione in the incubation mixture, they detected glutathione conjugates (32), and in the presence of semicarbazide, a pyridazine derivative was detected (35, Figure 6). This indicated spontaneous ring opening and formation of an γ-thioketo-α,β-unsaturated aldehyde (34, Figure 6) as a possible electrophilic intermediate in the metabolism of suprofen (29).14 In addition, thiophene epoxides can react with nucleophiles like water, glutathione, and proteins or can be exposed to oxidative CYP-catalyzed opening of the thiophene ring.7,14 Cytochrome P450 can catalyze the formation of the thiolactone metabolite 2-oxo-thiophene (6, Figure 1), which is an intermediate in the bioactivation pathway of antiplatelet drugs clopidogrel and ticlopidine.17 Further metabolism is the CYP-dependent oxidation of 2-oxothiophene to biologically active thiols (8, Figure 1). This requires oxidative opening of the thiolactone ring, resulting in highly reactive sulphenic acid (7, Figure 1) which is further reduced to the corresponding thiol (8) responsible for the biological activity of antiplatelet drugs.10,17,25 The thiolactone ring of 2-oxo-thiophene could be, to a minor extent, hydrolyzed with paraoxonase-1 (PON-1) to the biologically inactive thiol (8a, Figure 1).23 RMs formed after thiophene bioactivation affects, mainly, the liver. The liver is the main metabolic organ, as it contains the highest concentration of drug-metabolizing enzymes. Additionally, the liver is exposed to high concentration of the drug and, consequently, to drug metabolites, which can result in liver dysfunction or, in the worst case, liver failure.3 Hepatocytes have an effective detoxification system; glutathione, for example, is able to detoxify small amounts of RMs. However, if intermediates are highly reactive, then they can react with numerous other targets before they react with GSH. An additional problem is the large quantity of RMs, as the glutathione detoxification capacity can be exceeded. Thiophene RMs can, due to reactivity, bind covalently to the enzyme, which catalyzes its formation. Second, RMs can escape the active site of the enzyme and bind to other proteins in the surroundings; both of these possibilities can, however, result in hepatotoxicity. The possible occurrence of hepatitis may be due to the direct action of RMs on the liver proteins, but it could also be a consequence of immune system activation. Reactive thiophene metabolites, formed after tienilic acid metabolism, bind to CYP2C9 and act as haptens. The modified protein is recognized by the immune system, which can subsequently lead to an adverse immune reaction.11

not, and whether it is possible to anticipate toxic effects, will be discussed.

■

TYPES OF REACTIVE METABOLITES OF THE THIOPHENE RING Bioactivation of Thiophene Ring. Thiophene (1, Figure 1) and several thiophene-containing drugs are toxic to animals and humans. Dreiem et al.19 performed an in vitro assay of thiophene toxicity on rat cerebellar granule cell cultures (CGC). Exposure of rat CGCs to thiophene alone did not affect their viability, whereas addition of the S9 fraction (a source of metabolic enzymes) and corresponding cofactors (nicotinamide adenine dinucleotide phosphate (NADPH) and glucose-6-phosphate), together with thiophene, extensively reduced cell viability.19 Thiophene caused the death of CGCs only when metabolic enzymes and cofactors were both added. It was therefore suggested that metabolic bioactivation is required for thiophene toxicity.19 They found that the microsomal but not the cytosolic fraction of rat liver was responsible for metabolic bioactivation. Additionally, different CYP inhibitors were added to the incubation mixture with the aim of determining the contribution of various CYP isoenzymes to thiophene biotransformation. It was found that the CYP2C subfamily was responsible for the formation of thiophene RMs and the consequent death of CGC.19 Addition of α-tocopherol, glutathione (GSH), or phenyl-N-tert-butylnitrone to the incubation mixture containing CGC, S9, cofactors, and thiophene resulted in reduced cell death, providing further evidence for the involvement of RMs in thiophene toxicity.19 Because of its high reactivity, the identity of thiophene RMs was unknown for a long time. Nevertheless, by performing in vitro assays of thiophene biotransformation, stable hydroxy− thiophene (5, Figure 1) and mercapturic acid conjugates were detected.7,20 Eventually, two major in vitro metabolic pathways were proposed: thiophene epoxidation and thiophene-Soxidation (Figure 1). Thiophene S-Oxidation. Thiophene S-oxides (2, Figure 1) were the first thiophene RMs to be detected. Mansuy and others21 carried out microsomal incubation of benzothiophene. Incubation of benzothiophene with rat liver microsomes resulted in the formation of thiophene S-oxide, which was stable enough to be detected. Treiber et al.22 also confirmed formation of S-oxides (2, Figure 1) as the main thiophene RMs.22 They performed in vivo experiments in rats in which a conjugate with mercapturic acid was found to be a major urinary metabolite. They proposed Michael addition of glutathione on position 2 of thiophene S-oxide (4, Figure 1) followed by loss of glycine and glutamate of the glutathione moiety. The cysteine moiety is then N-acetylated to the corresponding conjugate with mercapturic acid. In addition to glutathione conjugates, a small amount of S-oxide dimers (3, Figure 1) was found in the urine of rats treated with thiophene. Dimers can be formed by Diels−Alder reaction of S-oxides and were frequently detected in in vitro assays, whereas they were rare in in vivo systems. This is a consequence of the very efficient glutathione system in vivo, where reaction with nucleophiles like glutathione is dominant. In contrast, in an in vitro assay with rat liver microsomes, they showed S-oxide dimers (3) to be the major metabolites.22 Epoxidation of the Thiophene Ring. The second metabolic pathway is epoxidation of the thiophene ring with formation of thiophene-epoxide (9, Figure 1). O’Donnell et al.14 analyzed, by LC−MS/MS, an incubation mixture 1346



Review

Table 1. Drugs Containing a Thiophene Moiety That Form RMs

■

Tienilic Acid (TA) and Tienilic Acid Positional Isomer (TAI). Tienilic acid (13) and tienilic acid positional isomer (18, Figure 2) have similar structures but different mechanisms of toxicity.37 Tienilic acid (2,3-dichloro-4-(2-thienylcarbonyl)phenoxy)acetic acid) is a diuretic with potent uricosuric activity that has been used in the effective treatment of arterial hypertension.27,28,38 It was launched on US market in 1979 but, due to suspected hepatotoxicity and related death cases, was withdrawn from the market after only a few months of use.38 It was supposed that the main reason for tienilic acid toxicity was bioactivation to RMs; however, the detailed mechanism of RM formation still remains to be elucidated. It is still debated whether tienilic acid RMs are thiophene epoxides, thiophene-S oxides, or both. First, TA S-oxide (14, Figure 2) was presented as the main RM, which is due to the keto substituent at position 2, a strong

THIOPHENE-BASED COMPOUNDS FORMING REACTIVE METABOLITES AND RELATED TOXICITIES Numerous drugs containing the thiophene moiety have proven to be toxic for humans and animals. Several such drugs, due to severe toxicity, were withdrawn from the market; some stayed on the market, but their use is, in most cases, limited. Suprofen (29), for example, once used as an oral nonsteroidal antiinflammatory drug (NSAID), is nowadays, due to renal toxicity, used only as 1% eye drops. For all of the drugs listed in Table 1, except for tiaprofenic acid (25, Figure 5), RMs were detected on the thiophene ring. However, in most cases, a direct link between the formation of RMs and the toxicity of the drug has not been found. Nevertheless, for some drugs like tienilic acid (13), strong evidence exists that RMs are responsible for their toxicity. 1347



Review

these mutants, a mechanism-based inactivation of the enzyme was not observed.42 López-Garcia et al.39 showed that GSH decreases TA binding to microsomal proteins but has no protective effect on CYP2C9 inactivation.39 Some new studies suggest that the main TA reactive intermediate is thiophene epoxide (15, Figure 2) and not thiophene-S oxide as previously reported.26 The evidence for tienilic acid epoxidation, in contrast to previously reported Soxidation, is given by 97 ± 2% 18O incorporation from molecular oxygen (18O2).26 TA epoxide undergoes an NIH shift rearrangement to 5-OH tienilic acid (16, Figure 2), which is the main stable TA oxidative metabolite detected and is in equilibrium with its thiolactone tautomer (16a, Figure 2). They proved that epoxidation of the thiophene ring is exclusively responsible for the formation of 5-OH TA, whereas S-oxidation does not lead to the hydroxylated product.26 In the blood of patients suffering from TA-induced hepatitis, specific antibodies, anti-liver−kidney microsomal antibodies 2, anti-LKM2 antibodies), were detected.44 They are highly specific and directed against CYP2C9, an enzyme responsible for oxidation of TA.11,44 They bind to both the modified and native forms of CYP2C9 but not to other CYP2C members.11,24 LKM2 antibodies against CYP2C9 are a sign of immune-mediated hepatitis.11 A possible sequence of events for immunoallergic hepatitis involves (Figure 3)

Figure 3. Mechanism of tienilic acid-induced immune hepatitis.11,39 Figure 2. TA bioactivation.

6,24,26,43

(1) CYP2C9 mediated oxidation to thiophene S-oxide;

electrophile.21,39 Valadon et al.8 carried out in vitro and in vivo studies of TA metabolism. After incubation of TA with rat liver microsomes, NADPH and O2 adducts with microsomal proteins were detected (21% of the parent compound). Addition of mercaptoethanol significantly decreased the covalent binding, and a mercaptoethanol conjugate was the main metabolite detected. The results suggested that microsomal incubation of tienilic acid led to highly electrophilic Soxides (14, Figure 2) that can react easily with diverse nucleophiles. They confirmed that reactions similar to those detected in vitro also occur in vivo in rats.8 Oxidative metabolism of TA is performed by the CYP2C family, especially by CYP2C9.11 RMs formed can, due to their high reactivity, react covalently with the nucleophilic group of the amino acid residue of the active site of CYP2C9 (17, Figure 2), resulting in autoinactivation of the enzyme.11,29,40,41 Melet et al.42 suggested that S-oxide binds to the nucleophilic OH group of serine 365 in the CYP2C9 active site. The role of Ser365 was investigated with two mutants, S365A and S365G. Both catalyzed the formation of 5-OH TA, with Km values comparable to that of the wild-type enzyme. However, with

(2) Alkylation of the CYP2C9 active site; (3) Formation of antibodies LKM2 against the modified and the native form of the protein; (4) Destruction of hepatocytes by the immune system.11,39,44 The hepatotoxic effects of TA and its positional isomer (18, Figure 2, TAI) have been compared.12,39,45,37,46 Despite their being only small structural differences between the isomers (Figure 2), differences in reactivity and toxicity were observed.39 While TA hepatotoxicity is immune-mediated, TAI works as a direct hepatotoxin. Both compounds undergo oxidative metabolism with CYP2C9, resulting in the formation of RMs. TAI, unlike TA, does not act as a mechanism-based inhibitor or CYP2C9.39 This could be due to different reactive sites in the thiophene rings of TAI and TA S-oxides (positions 2 and 5 of the thiophene ring).39 The nucleophilic group in the active site of CYP2C9 may not be in position to attack the TAI metabolite on site 2 of the thiophene ring, and TAI sulfoxide can therefore diffuse out from active site.39 They can react with GSH or a nucleophilic group in proteins, resulting in direct hepatotox1348



Review

icity.46 The other explanation was that the TAI metabolite is less reactive than the TA metabolite and therefore is able to diffuse out of the active site of the enzyme and function as a direct hepatotoxin.43 Rademacher et al.26 suggested that TAI appears to be capable of forming epoxides (19, Figure 2) as well as thiophene-S oxides (20, Figure 2). Thiophene epoxide is further converted to two thiolactone tautomers (21 and 21a, Figure 2), whereas S-oxide can form S-oxide dimers. According to this, the intrinsic hepatotoxicity of TAI may be due to thiophene-S oxides.26 Methapyrilene. Methapyrilene (22, Figure 4) is an H1 antagonist that was used as a sleep aid and an active ingredient

Figure 5. Tiaprofenic acid metabolism.

Figure 4. Methapyrilene bioactivation.

Figure 5). The second pathway is the hydroxylation of the benzene ring in the para position, resulting in 2-(5-(4hydroxybenzoyl)thiophen-2-yl)propanoic acid (28, Figure 5). Both metabolites are excreted in urine in the form of glucuronid (Surgam Data Sheet). Because no RMs on the thiophene ring have been detected, it is possible that reactive acyl glucuronides are involved in tiaprofenic acid toxicity. In contrast, suprofen RMs have been detected. When O’Donnell et al.14 incubated suprofen with CYP2C9 and NADPH, 5-OH suprofen (31, Figure 6) was the main metabolite detected. The source of oxygen for 5-OH suprofen was 18O2, which means that thiophene epoxide was formed (30, Figure 6). Additionally, incubation with glutathione resulted in

30

in formulations for colds and allergies. After more than 20 years of use, it was withdrawn from the market due to proven hepatotoxicity in rats.33 The hepatotoxicity was associated with oxidative stress, mitochondrial dysfunction, and alterations in liver glutathione concentrations.31,32 Ratra and co-workers provided strong evidence that methapyrilene hepatotoxicity depends mainly on CYP metabolism because in the presence of CYP inhibitor, metyrapone toxicity was reduced.31 They found that CYP2C11 is the main enzyme responsible for methapyrilene bioactivation in rats.31 Graham et al. again confirmed cytotoxicity and glutathione depletion after incubating different concentrations of methapyrilene with rat hepatocytes.30 They confirmed irreversible binding of tritium-labeled methapyrilene to rat hepatocytes, which was time- and concentrationdependent. Incubation of methapyrilene with rat liver microsomes resulted in several metabolites, whereas addition of glutathione to the incubation mixture resulted in one major metabolite (glutathione conjugate of mono-oxygenated methapyrilene 24, Figure 4). The reactive precursor for the glutathione conjugate could be either methapyrilene epoxide or methapyrilene S-oxide; however, deuterium exchange confirmed the formation of S-oxides (23, Figure 4).30 Tiaprofenic Acid and Suprofen. Tiaprofenic acid (25, Figure 5) [(RS)-2-(5-benzoyl-2-thienyl)propanoic acid] and suprofen (29) [(RS)-2[4-(2-thienylcarbonyl)phenyl)propanoic acid] are nonsteroidal anti-inflammatory drugs, prescribed mostly for the treatment of musculoskeletal pain, including back pain, osteoarthritis, and rheumatoid arthritis. Both drugs have been reported to cause renal toxicity.34,35 Numerous cases of severe cystitis associated with tiaprofenic acid have been reported.34 Suprofen was, due to cases of flank pain and acute renal failure, withdrawn from the market. Eye drops with 1% suprofen are, however, still used. Suprofen and tiaprofenic acid contain a thiophene moiety, so their toxicity may be due to bioactivation. However, no RMs of tiaprofenic acid have been observed. The metabolism of tiaprofenic acid is shown in Figure 5. Most of it is excreted in the form of acyl glucuronide (26, Figure 5), only 5% is metabolized to two other metabolites (Surgam Data Sheet). The first pathway is the reduction of the keto group to an alcohol, resulting in the formation of 2-(5(hydroxy(phenyl)methyl)thiophen-2-yl)propanoic acid (27,

Figure 6. Suprofen bioactivation.14 1349



Review

treating asthma. One pathway of zileuton metabolism is the formation of 2-acetylbenzothiophene (38, Figure 8), which can be further transformed to reactive metabolites responsible for the zileuton hepatotoxicity.36 Joshi et al.36 proved that zileuton is toxic to MCL-5 cells, which was triggered by CYP-dependent bioactivation. Microsomal incubation of 38 resulted in two metabolites, M + 16 (singly oxidized) and M + 32 (doubly oxidized). M + 16 was identified as 2-acetylbenzothiophene Soxide (39, Figure 8), which, due to its reactivity, reacts rapidly with trapping agents like GSH or NAC. The mercapturic acid derivative 2-acetylamino-3-(2-acetyl-benzo[b]thiophen-3-ylsulfanyl) propionic acid (40, Figure 8) was the main stable metabolite formed in vitro. In addition, this was also main metabolite detected in rat urine after treatment with zileuton. CYP1A2 and CYP2E1 were shown to be very important for bioactivation to S-oxide.36 OSI-930. OSI-930 (41, Figure 9) [3-[(quinolin-4-ylmethyl)amino]-thiophene-2-carboxylic acid (4-trifluoromethoxy-phenyl)-amide] is a potential anti-cancer drug that contains a thiophene moiety.18 Its metabolism is presented in Figure 9. Metabolic reactions studied with human liver microsomes, with the addition of NADPH and GSH as trapping agents, resulted in a glutathione conjugate of OSI-930 (43, Figure 9). The metabolic pathway (S-oxidation or epoxidation) was determined from a stable deuterium isotope retention experiment. On the basis of incorporation of deuterium from water (D2O), they proposed S-oxidation as the major metabolic pathway. Various CYPs were further tested for their capacity to form RMs. CYP3A4 and CYP2D6 were found to be the main enzymes involved in OSI-930 bioactivation. Although the drug is capable of forming RMs in vitro and in vivo, no toxicity has been detected in preclinical phases. The latter may, however, be due to early testing, as some RM effects are rare and can be manifested only after a drug has been tested on a large population.18

glutathione adducts on the thiophene ring (32, Figure 6), and incubation with semicarbazide resulted in formation of a pyridazine adduct, α-methyl-4-(3-pyridazinylcarbonyl)phenyl acetic acid (35, Figure 6). This may be due to spontaneous ring opening of thiophene-4,5-epoxide and formation of a reactive γ-thioketo-α,β-unsaturated aldehyde intermediate (34, Figure 6). γ-Thioketo-α,β-unsaturated aldehyde and thiophene 4,5-epoxide may be responsible for the mechanism-based inactivation of CYP2C9.14 Suprofen inactivates CYP2C9 in an NADPH-, time-, and concentration-dependent manner. Irreversible inhibition of CYP2C9 may be the leading cause for suprofen-related renal toxicity.14,47 CYP2C9 is highly expressed in the kidney and can play an important role in the epoxidation of arachidonic acid to the epoxyeicosatreonic acid metabolites (EETs). EETs have diverse physiological roles, including vasodilatory and vasoconstrictive activity. Modulation of arachidonic acid metabolism because of the inhibition of CYP2C9 by suprofen can, therefore, result in renal toxicity.14,48 Comparison with the related analogue ketoprofen (36, Figure 7) provided additional evidence that the thiophene ring

Figure 7. Structures of NSAIDs suprofen and ketoprofen.

is responsible for suprofen toxicity. Ketoprofen is a NSAID in which the thiophene moiety is replaced by benzene. In contrast to suprofen, ketoprofen is a widely prescribed medicine, with mild side effects. Zileuton. Zileuton (37, Figure 8) (N-(1-benzo[b]thien-2ylethyl)-N-hydroxyurea) is a 5-lipoxygenase inhibitor used for

■

THIOPHENE-BASED COMPOUNDS WITHOUT REACTIVE METABOLITES ON THE THIOPHENE RING Despite its potential to form RMs, thiophene has been integrated into numerous drugs without causing harmful effects. In certain cases, no thiophene RMs were detected. Some molecules contain structural fragments that are easier to metabolize than thiophene, in which case metabolism takes place along other, more favorable pathways. Drugs that contain the thiophene ring but exhibit no proven bioactivation liability are presented in Table 2. Duloxetine. Duloxetine (44, Figure 10) [(+)-N-methyl-3(1-naphthalenyloxy)-2-thiophenepropanamine] is an antidepressant used for the treatment of generalized anxiety disorder,49 major depressive disorder,50 diabetic neuropathic

Figure 8. Zileuton biotransformation.36

Figure 9. OSI-930 metabolism.18 1350



Review

Table 2. Drugs Containing a Thiophene Moiety That Do Not Form RMs from the Thiophene Ring

the CYP 450 system, and no oxidative metabolites have been detected.55 Eprosartan is excreted in the urine and feces, mainly as the unchanged drug. Therefore, fluconazole (inhibitor of CYP2C9) had no effect on its pharmakokinetics.54 Eposartan appears to be a safe drug without significant interactions with other coadministered drugs.54 Rivaroxaban. Rivaroxaban (45, Figure 11) is an oral anticoagulant indicated for prevention and treatment of thrombo-embolic disorders like deep vein thrombosis and

Figure 10. Duloxetine.15

pain,51 and other diseases. Duloxetine functions in the central nervous system as a selective serotonin and norepinephrine reuptake inhibitor.52 It entered the market in 2004 and was regarded as a safe drug with mild side effects.53 The usual daily dose was 60−120 mg. In the postmarketing phase, effects on the liver were observed. There have been concerns about its safety, especially with patients with preexisting liver disease.53 It was supposed that the hepatotoxic effect could be due to its bioactivation to RMs.15 Duloxetine is characterized by the presence of two toxicoforic groups, the naphthalene and thiophene rings. However, no RMs on thiophene ring were detected.15 Its main route of metabolism is oxidation of the naphthol ring followed by glucuronidation.52 Eprosartan. Eprosartan is a selective angiotensin II receptor blocker used for the treatment of esential hypertension.54 The daily dose is relatively high (Table 2); however, its absorption is very poor (less than 15%).55 Eposartan is not metabolized by

Figure 11. Rivaroxaban metabolism.57 1351



Review

pulmonary embolism (Xarelto Data Sheet). Weinz et al.57 examined the in vivo metabolism of rivaroxaban in rats, dogs, and humans. The most important metabolic pathways observed were cleavage of the amide bond and oxidative degradation of the morpholine group. Elimination of rivaroxaban from the body was rapid, with most of the dose being excreted within 24 h. Despite having a thiophene moiety, RMs were not detected.57 Figure 11 lists the major metabolites of rivaroxaban. 47 was the main metabolite detected in urine (22% of the dose was excreted in that form). Hydrolysis of the amide bond resulted in formation of 48 and 49. 49 was further conjugated with glycine (50) and excreted with urine.57 Olanzapine. Olanzapine (51, Figure 12) is an atypical antipsychotic, widely used for the treatment of shizophrenia

and bipolar disorder.58 Olanzapine is extensively metabolized and excreted in urine (60% of the dose) and in the feces (30% of the dose). Its major metabolites are 10-N-glucuronide (54, Figure 12; 44% of the dose) and N-desmethyl olanzapine (53, Figure 12; 31% of the dose), whereas 2-hydroxymethyl olanzapine (52, Figure 12) is a minor metabolite.58 Olanzapine is not subjected to metabolism on the thiophene ring.

■

COMPOUNDS WITH REACTIVE METABOLITES RESPONSIBLE FOR THEIR PHARMACOLOGIC ACTION Formation of RMs is not always undesirable. In some cases, RMs can be pharmacologically active and thereby contribute to the desired effect of the drug. Clopidogrel (60, Figure 15), ticlopidine (65, Figure 16), and prasugrel (73, Figure 17) are thiophene-containing antiplatelet drugs used to prevent thrombotic events such as myocardial infarction, stroke, or vascular death (Table 3).16,17 They work as irreversible inhibitors of the P2Y12 subtype of the adenosine diphospate (ADP) receptor, thereby preventing ADP-stimulated platelet aggregation.61 They are not active in vitro, and hepatic activation of the thiophene ring to 4mercapto-3-piperidinyliden acetic acid derivatives is required for their anti-aggregating activity.63 Although the mechanism of action of antiplatelet drugs is the same, their in vivo metabolism is slightly different. Clopidogrel. Clopidogrel (60), marketed under trade name Plavix, is a widely used antithrombotic drug. It is extensively metabolized, mainly in the liver. Pereilo and co-workers16 discovered that its stereochemical properties play a crucial role in its biological activity. Incubation with microsomes gave eight stereoisomers, of which only one showed biological activity. They found that the S configuration on carbon 7 is obligatory for antiplatelet activity. The structure of the active metabolite

Figure 12. Olanzapine metabolism.58

Table 3. Drugs in Which RM Are Responsible for Their Pharmacologic Action

1352



Review

was studied with MS and NMR, and 2-(1-[1-(2-chlorophenyl)2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene) acetic acid was proposed to be the active metabolite, which, due to its highly reactive thiol group, is very labile.16 Production of its active metabolite consists of the following two steps (Figure 15). The first is oxidation of the thiophene ring and formation of 2-oxo clopidogrel (61, Figure 15), which, in the second step, is converted to the active metabolite (62, Figure 15).10,17 Formation of 2-oxo-clopidogrel is catalyzed by CYP450 enzymes, in particular CYP1A2, CYP2B6, and CYP2C19. The second step, formation of active metabolite from 2-oxo clopidogrel, is catalyzed mainly by CYP3A4, CYP2C9, CYP2B6, and CYP2C19 isoforms. CYP2C19 makes important contributions to both oxidative steps and therefore has a major effect on the formation of active metabolites.10,17,59 It is unclear why CYP-dependent activation is required for hydrolysis of the thioester bond of 2-oxoclopidogrel. Dansette et al.25 suggested CYP-mediated oxidation of 2-oxoclopidogrel to the highly reactive S-oxide (56, Figure 13), which reacts Figure 14. Covalent binding of clopidogrel to its receptor.

Figure 13. Bioactivation of antiplatelet drugs (ticlopidine and clopidogrel).10,25

rapidly with water, resulting in formation of the corresponding sulfenic acid (57, Figure 13). Sulfenic acid intermediates have been trapped with dimedone and analyzed by MS and 1H NMR. Sulfenic acid intermediates can react with thiol nucleophiles like glutathione to form mixed dithioethers (58, Figure 13) and are then converted to the pharmacologically active thiols (59, Figure 13).25 Dansette et al.,23 in newer study, described two possible pathways for the hydrolysis of the thioester bond of 2oxoclopidogrel. The major path is P450- and NADPHdependent, as described previously, and leads to the active thiol (62, Figure 15) with an exocyclic double bond. The second pathway is paraoxonase-1 (PON-1) catalyzed hydrolysis of the thiolactone ring of 2-oxoclopidogrel, which leads to a pharmacologically inactive thiol with an endocyclic double bond (63, Figure 15).23 The thiophene moiety is therefore essential for the activity of clopidogrel. The active metabolite with a thiol group can bind covalently to P2Y12 by forming a disulfide bond with cysteine residues on the receptor (Figure 14), thus preventing ADPmediated platelet activation and aggregation.17,61 Nevertheless, only a minor proportion of the clopidogrel undergoes metabolic conversion to active metabolite. The majority (90%) is converted to a carbocyclic acid derivate (64, Figure 15), which is formed by hydrolysis of the methyl ester and shows no pharmacological activity.17 Ticlopidine. Similar to that of clopidogrel, several antiplatelet drugs need metabolic transformation to become pharmacologically active. Ticlopidine (65, Figure 16) exhibits complex metabolism. One metabolic pathway is N-dealkylation

Figure 15. Clopidogrel metabolism.10,17,61

Figure 16. Ticlopidine metabolism.10,17,61,62

and formation of metabolites 66 and 67, whereas N-oxidation of ticlopidine results in formation of 68 (Figure 16).62 The metabolic conversion to a pharmacologically active metabolite is similar to that described for clopidogrel.10,17 First, the thiophene ring of ticlopidine is oxidized, forming 2-oxo ticlopidine (71, Figure 16), which is then metabolized to the 1353



Review

electrophiles like iminoquinones or Michael acceptors.1 The importance of daily dose is evident in the case of the antiplatelet drugs ticlopidine and clopidogrel. They share a similar structure, but ticlopidine is taken at considerably higher doses than clopidogrel. Ticlopidine treatment is associated with severe side effects, like neutropenia and thrombotic thrombocytopenic purpura, whereas clopidogrel appears to be much safer.17 The daily dose of eprosartan is high (600−800 mg); however, this is safe drug, with mild side effects. Its absorption is very low, so the available dose of eprosartan is much lower.55 An important factor in addition to the daily dose is the dosing regimen. Drugs intended for chronic treatment are more prone to IDRs from drugs for acute treatment. Substitution of the Thiophene Ring. Hu et al.66 pointed out that the pattern of substitution in the thiophene ring exerts a major influence on RM formation. They investigated in vivo and in vitro metabolic activation for a series of monoamino- and diamino-substituted thiophenes. HPLC/UV/MS analysis of incubation mixtures containing a substance with a 2,5diaminothiophene structure (77, Figure 18), pooled rat or

active metabolite (72, Figure 16) in the CYP-dependent pathway.17 Shimizu et al.62 identified novel ticlopidine metabolites. They proposed that ticlopidine undergoes Soxidation and epoxidation on its thiophene ring. Both pathways can lead to the formation of 2-oxoticlopidine, a precursor for the pharmacologically active metabolite. RMs of ticlopidine Soxides (69, Figure 16) and epoxides (70, Figure 16) can cause rare, but potentially fatal, side effects. Among them, agranulocytosis, aplastic anemia, and hepatic injury are the most serious. Prasugrel. The metabolism of prasugrel (73, Figure 17) differs from that described for ticlopidine and clopidogrel.17

Figure 17. Prasugrel metabolism.10,17,61

The first step is hCE2-mediated ester bond hydrolysis to the corresponding thiolactone ring (75, Figure 17).10 The following step is metabolic transformation to an active metabolite (76, Figure 17). The major enzymes involved in its formation are CYP3A4 and CYP2B6, whereas CYP2C9 and CYP2C19 play only a minor role in its bioactivation.10,17

■

Figure 18. Reactivity of monoamino- and diamino-substituted thiophenes.66

FACTORS AFFECTING THE TOXICITY OF THIOPHENE-CONTAINING DRUGS Daily Dose and Extent of Covalent Binding. Numerous factors affect the toxicity of drugs containing a structural alert. Among the most important appear to be the daily dose and extent of covalent binding.6 Although IDR is considered to be dose-independent, Lammert et al.64 observed a relationship between daily dose and hepatotoxicity: drugs used at higher daily doses (above 50 mg) exhibited a higher incidence of IDR.64 Nakayama et al.65 established a zone classification system to access the impact of dose and covalent binding on the risk of idiosyncratic toxicity. According to a zone classification system, drugs with considerable protein binding should be taken in low doses, for example, a drug covalently binding 10 pmol/mg protein is relatively safe at doses below 100 mg, whereas a drug covalently binding 50 pmol/mg protein is, due to zone classification, safe only at doses below 25 mg.65 Metabolites that are highly reactive (hard electrophiles) will react with hard nucleophiles in the surroundings, R−OH and R−NH2 groups of the proteins, and will therefore bind to proteins to a greater extent.1 Consequently, those drugs should be taken at very low doses. Drugs containing a thiophene moiety that have been withdrawn from the market were usually taken at very high daily doses (for example, the daily dose of suprofen was 400 mg) (Table 1). In contrast, no drugs taken at low daily doses exert serious side effects. Although the drug is capable of RM formation, the total body burden stays low. Furthermore, a small amount of RMs can be effectively removed with small molecular nucleophiles like water or GSH and therefore do not present a health hazard. GSH, which is a soft nucleophile, is especially effective at removing soft

human liver microsomes, and cysteine nucleophiles (GSH or NAC) resulted in formation of glutathione or NAC conjugates (79). GSH was shown to form adducts with thiophene on position 3 of the parent compound. Conjugates detected in vitro were further confirmed in in vivo experiments with rats. CYP 3A4, 1A2, 2C, and 2D6 are the enzymes responsible for thiophene metabolism. On the contrary, no glutathione conjugates of 2,4-diamino (81) or 2-monoamino (80) thiophene derivatives were detected, so it was assumed that these groups of compounds do not form RMs. On the basis of these results and the fact that no metabolites were formed from S-oxidation or epoxidation pathways, a novel 2,5-diiminethiophene (78) bioactivation pathway was proposed. This is consistent with the fact that amino substitution adjacent to the sulfur is obligatory for bioactivation. Structural modifications of the thiophene ring can therefore eliminate bioactivation, and these results can be used to create new drugs.66 As described above, substitution of the thiophene ring can alter thiophene metabolism. Properties of the substituent and its position on the thiophene ring are important variables.45 Chen et al. determined a structure−bioactivation potential relationship for C4- and C5-substituted 2-acetylthiophenes.45 C5 substitution reduced formation of RMs to a greater extent than did C4 substitution. Additionally, methyl substitution on C5 completely prevents formation of RMs.45 Furthermore, they incubated microsomes with five 2-chloro-5-substituted thiophenes (82, Figure 19). Although 2-chloro substitution is common with substances containing a thiophene moiety, for 1354



Review

Figure 19. Bioactivation of 2-chloro-5-acetylthiophene.45

four of them, glutathione adducts were detected (84, Figure 19). An NMR study revealed that glutathione replaced the chlorine atom at position 2 of the thiophene ring. 2-Chloro substitution can reduce the formation of RMs but cannot prevent it completely.45 Competitive Metabolic Pathways. Involvement of other metabolic pathways can also affect toxicity. Duloxetine, which is metabolized on a naphthalene ring rather than on thiophene, is an example of a competitive metabolic pathway that results in safe metabolites (Figure 10). In contrast, some substances contain additional reactive sites that can be responsible for toxicity. An example of this is sitaxentan (85, Figure 20), a

Figure 21. Raloxifene metabolism.69

Some minor structural modifications in a molecule can eliminate the bioactivational potential of the drug.66

■

CONCLUSIONS AND FUTURE TRENDS The thiophene moiety has proven to be the leading cause for the toxicity of numerous drugs. Moreover, many drugs have been withdrawn from the market due to thiophene-associated toxicity. However, avoiding incorporation of a thiophene moiety in new drug candidates can prevent the development of potentially valuable drugs. Several aspects of thiophene toxicity have been described in this review. The toxicity of thiophene-containing compounds depends on oxidative metabolism of the thiophene ring. Thiophene metabolism is CYPmediated and can lead to highly reactive thiophene epoxides and thiophene S-oxides. However, inclusion of a thiophene ring in the structure of new drug does not necessarily result in a toxic outcome. Duloxetine (Cymbalta) is a blockbuster antidepressant without an adverse effect associated with formation of RMs. The questions therefore arise as to the reasons for such diverse outcomes and how they can be avoided. A number of factors are responsible for the fate of thiophene-containing drugs. Daily dose is of great importance.6 Most of the drugs withdrawn from the market were used at high daily doses, more than 200 mg. Low-dose drugs are considered to be safe, as it is not likely that a drug will cause toxicity related to RMs when the daily dose is below 20 mg. An additional factor is the reactivity of oxidative metabolites, which is determined mainly by the nature of the substitution of the thiophene ring. Highly reactive metabolites will result in autoinactivation of the enzyme, whereas less reactive metabolites can migrate out of the active site and affect vicinal protein targets. Different targets can lead to different toxic effects; for instance, tienilic acid is an immune-mediated hepatotoxin, whereas its positional isomer is a direct hepatotoxin. The metabolism of drugs is usually extremely complex. Thiophene-containing compounds can undergo metabolism through other competitive metabolic pathways, leading to less toxic products. Drug metabolism and bioactivation have to be taken into account in drug design. Every drug entering the market must be safe and effective. Even drugs containing a structural alert can meet these requirements. When dealing with the thiophene moiety, as a known structural alert, targets should therefore include production of low-dose drugs, blocking thiophene

Figure 20. Sitaxentan metabolism.68

selective endothelin A receptor antagonist used for treating pulmonary arterial hypertension.67,68 It was withdrawn from the market in 2010 because of fatal cases associated with hepatic injury.67 Although preclinical studies revealed no hepatotoxic effects of sitaxentan, a few cases of severe liver injury were reported when the drug was on the market. Idiosyncratic hepatotoxicity can be explained by formation of RMs. Although sitaxentan contains the potentially dangerous thiophene moiety, some newer studies suggest that formation of quinone metabolites is responsible for sitaxentan’s hepatotoxic effects.68 Metabolic changes on the 1,3-benzodioxole moiety of sitaxentan have been observed.68 On incubation with microsomes, o-catechol (86, Figure 20) was formed as the main metabolite. Glutathione conjugates of o-catechol were also detected, leading to the proposed oxidation of o-catechol to reactive o-quinone (87, Figure 20). The proposed metabolic pathway is presented in Figure 20. However, no metabolic changes on thiophene moiety were observed.68 An additional example that clearly displays the importance of competitive metabolic pathways is that of raloxifene (88, Figure 21), a selective estrogen receptor modulator. Raloxifene contains benzothiophene moiety, but the main oxidative metabolite formed is an extended diquinone (89, Figure 21).69 Nevertheless, the primary pathway of raloxifene metabolism is glucuronidation, resulting in two glucuronides, 90 and 91 (Figure 21). The intestinal UGT enzymes UGT1A8 and UGT1A10 are the main isoforms involved in raloxifene glucuronidation.70 Because of extensive intestinal glucuronidation, oxidative metabolism of raloxifene is limited.69 Despite its potential to form RMs, raloxifene remains a relatively safe drug. These examples have demonstrated that the chemical structure of a drug, together with the pattern of substitution on the thiophene ring, can exert a major influence on the potential for bioactivation of a drug and its related toxicity. 1355



Review

epoxidation of thiophene derivatives. Biochem. Biophys. Res. Commun. 338, 450−455. (10) Mansuy, D., and Dansette, P. M. (2011) Sulfenic acids as reactive intermediates in xenobiotic metabolism. Arch. Biochem. Biophys. 507, 174−185. (11) Lecoeur, S., Bonierbale, E., Challine, D., Gautier, J. C., Valadon, P., Dansette, P. M., Catinot, R., Ballet, F., Mansuy, D., and Beaune, P. H. (1994) Specificity of in vitro covalent binding of tienilic acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity: comparison with two directly hepatotoxic drugs. Chem. Res. Toxicol. 7, 434−442. (12) Bonierbale, E., Valadon, P., Pons, C., Desfosses, B., Dansette, P. M., and Mansuy, D. (1999) Opposite behaviors of reactive metabolites of tienilic acid and its isomer toward liver proteins: use of specific antitienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds. Chem. Res. Toxicol. 12, 286−296. (13) Nishiya, T., Kataoka, H., Mori, K., Goto, M., Sugawara, T., and Furuhama, K. (2006) Tienilic acid enhances hyperbilirubinemia in Eisai hyperbilirubinuria rats through hepatic multidrug resistanceassociated protein 3 and heme oxygenase-1 induction. Toxicol. Sci. 91, 651−659. (14) O’Donnell, J. P., Dalvie, D. K., Kalgutkar, A. S., and Obach, R. S. (2003) Mechanism-based inactivation of human recombinant P450 2C9 by the nonsteroidal anti-inflammatory drug suprofen. Drug Metab. Dispos. 31, 1369−1377. (15) Chan, C. Y., New, L. S., Ho, H. K., and Chan, E. C. (2011) Reversible time-dependent inhibition of cytochrome P450 enzymes by duloxetine and inertness of its thiophene ring towards bioactivation. Toxicol. Lett. 206, 314−324. (16) Pereillo, J. M., Maftouh, M., Andrieu, A., Uzabiaga, M. F., Fedeli, O., Savi, P., Pascal, M., Herbert, J. M., Maffrand, J. P., and Picard, C. (2002) Structure and stereochemistry of the active metabolite of clopidogrel. Drug Metab. Dispos. 30, 1288−1295. (17) Farid, N. A., Kurihara, A., and Wrighton, S. A. (2010) Metabolism and disposition of the thienopyridine antiplatelet drugs ticlopidine, clopidogrel, and prasugrel in humans. J. Clin. Pharmacol. 50, 126−142. (18) Medower, C., Wen, L., and Johnson, W. W. (2008) Cytochrome P450 oxidation of the thiophene-containing anticancer drug 3[(quinolin-4-ylmethyl)-amino]-thiophene-2-carboxylic acid (4-trifluoromethoxy-phenyl)-amide to an electrophilic intermediate. Chem. Res. Toxicol. 21, 1570−1577. (19) Dreiem, A., and Fonnum, F. (2004) Thiophene is toxic to cerebellar granule cells in culture after bioactivation by rat liver enzymes. Neurotoxicology 25, 959−966. (20) Bray, H. G., Carpanini, F. M., and Waters, B. D. (1971) The metabolism of thiophen in the rabbit and the rat. Xenobiotica 1, 157− 168. (21) Mansuy, D., Valadon, P., Erdelmeier, I., Lopez-Garcia, P., Amar, C., Girault, J. P., and Dansette, P. M. (1991) Thiophene S-Oxides as new RM: formation by Cytochrome P450 dependent oxidation and reaction with nucleophiles. J. Am. Chem. Soc. 113, 7825−7826. (22) Treiber, A., Dansette, P., Amri, H., Girault, J., Ginderow, D., Mornon, J. P., and Mansuy, D. (1997) Chemical and biological oxidation of thiophene: preparation and complete characterization of thiophene S-oxide dimers and evidence for thiophene S-oxide as an intermediate in thiophene metabolism in vivo and in vitro. J. Am. Chem. Soc. 119, 1565−1571. (23) Dansette, P. M., Rosi, J., Bertho, G., and Mansuy, D. (2012) Cytochromes P450 catalyze both steps of the major pathway of clopidogrel bioactivation, whereas paraoxonase catalyzes the formation of a minor thiol metabolite isomer. Chem. Res. Toxicol. 25, 348−356. (24) (2008) The Practice of Medicinal Chemistry (Wermuth, C. G., Ed.) 3rd ed, Elsevier, Amsterdam, The Netherlands. (25) Dansette, P. M., Libraire, J., Bertho, G., and Mansuy, D. (2009) 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. 22, 369−373.

oxidative metabolism with substituents on the thiophene ring and accelerating metabolism through other metabolic pathways. However, with all such changes, drug effectiveness must not be affected.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +386-1-476-9572. Fax: +386-1-425-8031. E-mail: marija.sollner@ffa.uni-lj.si. Funding

This work was supported partially by a young researcher grant to D.G. and by program group “Medicinal Chemistry: Drug Design, Synthesis and Evaluation of the Drugs” (program code P1-0208). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The authors thank Dr. Roger Pain for his critical reading of the manuscript. ABBREVIATIONS ALT, alanine aminotransferase; AST, aspartate aminotransferase; CGC, cerebellar granule cell cultures; CYP450, cytochrome P450; GSH, glutathione; IDR, idiosyncratic drug reaction; LKM2 antibodies, liver−kidney microsomal antibodies; NAC, N-acetylcysteine; NADPH, nicotinamide adenine dinucleotide phosphate; NSAID, nonsteroidal anti-inflammatory drug; RM, reactive metabolite; TA, tienilic acid; TAI, tienilic acid isomer; UGT, uridinediphosphate glucuronosyltransferase

■

REFERENCES

(1) Attia, S. M. (2010) Deleterious effects of reactive metabolites. Oxid. Med. Cell. Longevity 3, 238−253. (2) Park, B. K., Laverty, H., Srivastava, A., Antoine, D. J., Naisbitt, D., and Williams, D. P. (2011) Drug bioactivation and protein adduct formation in the pathogenesis of drug-induced toxicity. Chem.−Biol. Interact. 192, 30−36. (3) Kalgutkar, A. S. (2009) Role of bioactivation in idiosyncratic drug toxicity: structure−toxicity relationships, in Advances in Bioactivation Research (Elfarra, A., Ed.) Springer, New York. (4) Uetrecht, J. (2008) Idiosyncratic drug reactions: past, present, and future. Chem. Res. Toxicol. 21, 84−92. (5) Li, A. P. (2002) A review of the common properties of drugs with idiosyncratic hepatotoxicity and the “multiple determinant hypothesis” for the manifestation of idiosyncratic drug toxicity. Chem.−Biol. Interact. 142, 7−23. (6) Stepan, A. F., Walker, D. P., Bauman, J., Price, D. A., Baillie, T. A., Kalgutkar, A. S., and Aleo, M. D. (2011) Structural alert/reactive metabolite concept as applied in medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a perspective based on the critical examination of trends in the top 200 drugs marketed in the United States. Chem. Res. Toxicol. 24, 1345−1410. (7) Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R. S., and O’Donnell, J. P. (2002) Biotransformation reactions of fivemembered aromatic heterocyclic rings. Chem. Res. Toxicol. 15, 269− 299. (8) Valadon, P., Dansette, P. M., Girault, J. P., Amar, C., and Mansuy, D. (1996) Thiophene sulfoxides as reactive metabolites: formation upon microsomal oxidation of a 3-aroylthiophene and fate in the presence of nucleophiles in vitro and in vivo. Chem. Res. Toxicol. 9, 1403−1413. (9) Dansette, P. M., Bertho, G., and Mansuy, D. (2005) First evidence that cytochrome P450 may catalyze both S-oxidation and 1356



Review

(26) Rademacher, P. M., Woods, C. M., Huang, Q., Szklarz, G. D., and Nelson, S. D. (2012) Differential oxidation of two thiophenecontaining regioisomers to reactive metabolites by cytochrome P450 2C9. Chem. Res. Toxicol. 25, 895−903. (27) Lemieux, G., Beauchemin, M., Gougoux, A., and Vinay, P. (1978) Treatment of arterial hypertension with tienilic acid, a new diuretic with uricosuric properties. Can. Med. Assoc. J. 118, 1074−1078. (28) Lau, K., Stote, R. M., Goldberg, M., and Agus, Z. S. (1977) Mechanisms of the uricosuric effect of the diuretic tienilic acid (Ticrynafen) in man. Clin. Sci. Mol. Med. 53, 379−386. (29) López-García, M. P., Dansette, P. M., and Coloma, J. (2005) Kinetics of tienilic acid bioactivation and functional generation of drug-protein adducts in intact rat hepatocytes. Biochem. Pharmacol. 70, 1870−1882. (30) Graham, E. E., Walsh, R. J., Hirst, C. M., Maggs, J. L., Martin, S., Wild, M. J., Wilson, I. D., Harding, J. R., Kenna, J. G., Peter, R. M., Williams, D. P., and Park, B. K. (2008) Identification of the thiophene ring of methapyrilene as a novel bioactivation-dependent hepatic toxicophore. J. Pharmacol. Exp. Ther. 326, 657−671. (31) Ratra, G. S., Morgan, W. A., Mullervy, J., Powell, C. J., and Wright, M. C. (1998) Methapyrilene hepatotoxicity is associated with oxidative stress, mitochondrial disfunction and is prevented by the Ca2+ channel blocker verapamil. Toxicology 130, 79−93. (32) Ratra, G. S., Powell, C. J., Park, B. K., Maggs, J. L., and Cottrell, S. (2000) Methapyrilene hepatotoxicity is associated with increased hepatic glutathione, the formation of glucuronide conjugates, and enterohepatic recirculation. Chem.−Biol. Interact. 129, 279−295. (33) Ratra, G. S., Cottrell, S., and Powell, C. J. (1998) Effects of induction and inhibition of cytochromes P450 on the hepatotoxicity of methapyrilene. Toxicol. Sci. 46, 185−196. (34) Crawford, M. L., Waller, P. C., and Wood, S. M. (1997) Severe cystitis associated with tiaprofenic acid. Br. J. Urol. 79, 578−584. (35) Hart, D., Ward, M., and Lifschitz, M. D. (1987) Suprofenrelated nephrotoxicity. A distinct clinical syndrome. Ann. Intern. Med. 106, 235−238. (36) Joshi, E. M., Heasley, B. H., Chordia, M. D., and Macdonald, T. L. (2004) In vitro metabolism of 2-acetylbenzothiophene: relevance to zileuton hepatotoxicity. Chem. Res. Toxicol. 17, 137−143. (37) Coen, M., Rademacher, P. M., Zou, W., Scott, M., Ganey, P. E., Roth, R., and Nelson, S. D. (2012) Comparative NMR-based metabonomic investigation of the metabolic phenotype associated with tienilic acid and tienilic acid isomer. Chem. Res. Toxicol. 25, 2412− 2422. (38) New Zealand Hypertension Study Group (1983) Multicentre controlled trial of tienilic acid in hyperuricaemic, hypertensive subjects, with intensive monitoring of liver function. Br. J. Clin. Pharmacol. 15, 513−518. (39) López-Garcia, M. P., Dansette, P. M., and Mansuy, D. (1994) Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid. Biochemistry 33, 166−175. (40) Koenigs, L. L., Peter, R. M., Hunter, A. P., Haining, R. L., Rettie, A. E., Friedberg, T., Pritchard, M. P., Shou, M., Rushmore, T. H., and Trager, W. F. (1999) Electrospray ionization mass spectrometric analysis of intact cytochrome P450: identification of tienilic acid adducts to P450 2C9. Biochemistry 38, 2312−2319. (41) Mancy, A., Broto, P., Dijols, S., Dansette, P. M., and Mansuy, D. (1995) The substrate binding site of human liver cytochrome P450 2C9: an approach using designed tienilic acid derivatives and molecular modeling. Biochemistry 34, 10365−10375. (42) Melet, A., Assrir, N., Jean, P., Pilar Lopez-Garcia, M., MarquesSoares, C., Jaouen, M., Dansette, P. M., Sari, M. A., and Mansuy, D. (2003) Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid. Arch. Biochem. Biophys. 409, 80−91. (43) Smith, D. A., van de Waterbeemd, H., and Walker, D. K. (2001) Pharmacokinetics and Metabolism in Drug Design, Wiley, New York.

(44) Beaune, P., Dansette, P. M., Mansuy, D., Kiffel, L., Finck, M., Amar, C., Leroux, J. P., and Homberg, J. C. (1987) Human antiendoplasmic reticulum autoantibodies appearing in a drug-induced hepatitis are directed against a human liver cytochrome P-450 that hydroxylates the drug. Proc. Natl. Acad. Sci. U.S.A. 84, 551−555. (45) Chen, W., Caceres-Cortes, J., Zhang, H., Zhang, D., Humphreys, W. G., and Gan, J. (2011) Bioactivation of substituted thiophenes including α-chlorothiophene-containing compounds in human liver microsomes. Chem. Res. Toxicol. 24, 663−669. (46) Mansuy, D. (1997) Molecular structure and hepatotoxicity: compared data about two closely related thiophene compounds. J. Hepatol. 26, 22−25. (47) Hutzler, J. M., Balogh, L. M., Zientek, M., Kumar, V., and Tracy, T. S. (2009) Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acid and (±)-suprofen: a comparison of kinetics and probe substrate selection. Drug Metab. Dispos. 37, 59−65. (48) Wang, M. H., Brand-Schieber, E., Zand, B. A., Nguyen, X., Falck, J. R., Balu, N., and Schwartzman, M. L. (1998) Cytochrome P450derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors. J. Pharmacol. Exp. Ther. 284, 966−973. (49) Wu, W. Y., Wang, G., Ball, S. G., Desaiah, D., and Ang, Q. Q. (2011) Duloxetine versus placebo in the treatment of patients with generalized anxiety disorder in China. Chin. Med. J. 124, 3260−3268. (50) Dhillon, S. (2013) Duloxetine: a review of its use in the management of major depressive disorder in older adults. Drugs Aging 30, 59−79. (51) Pergolizzi, J. V., Raffa, R. B., Taylor, R., Rodriguez, G., Nalamachu, S., and Langley, P. (2013) A review of duloxetine 60 mg once-daily dosing for the management of diabetic peripheral neuropathic pain, fibromyalgia, and chronic musculoskeletal pain due to chronic osteoarthritis pain and low back pain. Pain Pract. 13, 239− 252. (52) Lantz, R. J., Gillespie, T. A., Rash, T. J., Kuo, F., Skinner, M., Kuan, H. Y., and Knadler, M. P. (2003) Metabolism, excretion, and pharmacokinetics of duloxetine in healthy human subjects. Drug Metab. Dispos. 31, 1142−1150. (53) Carter, N. J., and McCormack, P. L. (2009) Duloxetine: a review of its use in the treatment of generalized anxiety disorder. CNS Drugs 23, 523−541. (54) Schmidt, B., and Schieffer, B. (2003) Angiotensin II AT1 receptor antagonists. Clinical implications of active metabolites. J. Med. Chem. 46, 2261−2270. (55) McClellan, K. J., and Balfour, J. A. (1998) Eprosartan. Drugs 55, 713−718. (56) Wu, G., Vashishtha, S. C., and Erve, J. C. (2010) Characterization of glutathione conjugates of duloxetine by mass spectrometry and evaluation of in silico approaches to rationalize the site of conjugation for thiophene containing drugs. Chem. Res. Toxicol. 23, 1393−1404. (57) Weinz, C., Schwarz, T., Kubitza, D., Mueck, W., and Lang, D. (2009) Metabolism and excretion of rivaroxaban, an oral, direct factor Xa inhibitor, in rats, dogs, and humans. Drug Metab. Dispos. 37, 1056− 1064. (58) Callaghan, J. T., Bergstrom, R. F., Ptak, L. R., and Beasley, C. M. (1999) Olanzapine. Pharmacokinetic and pharmacodynamic profile. Clin. Pharmacokinet. 37, 177−193. (59) Kazui, M., Nishiya, Y., Ishizuka, T., Hagihara, K., Farid, N. A., Okazaki, O., Ikeda, T., and Kurihara, A. (2010) Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metab. Dispos. 38, 92−99. (60) Dansette, P. M., Levent, D., Hessani, A., Bertho, G., and Mansuy, D. (2013) Thiolactone sulfoxides as new reactive metabolites acting as bis-electrophiles: implication in clopidogrel and prasugrel bioactivation. Chem. Res. Toxicol. 26, 794−802. (61) Wallentin, L. (2009) P2Y(12) inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use. Eur. Heart J. 30, 1964−1977. 1357



Review

(62) Shimizu, S., Atsumi, R., Nakazawa, T., Fujimaki, Y., Sudo, K., and Okazaki, O. (2009) Metabolism of ticlopidine in rats: identification of the main biliary metabolite as a glutathione conjugate of ticlopidine S-oxide. Drug Metab. Dispos. 37, 1904−1915. (63) Richter, T., Mürdter, T. E., Heinkele, G., Pleiss, J., Tatzel, S., Schwab, M., Eichelbaum, M., and Zanger, U. M. (2004) Potent mechanism-based inhibition of human CYP2B6 by clopidogrel and ticlopidine. J. Pharmacol. Exp. Ther. 308, 189−197. (64) Lammert, C., Einarsson, S., Saha, C., Niklasson, A., Bjornsson, E., and Chalasani, N. (2008) Relationship between daily dose of oral medications and idiosyncratic drug-induced liver injury: search for signals. Hepatology 47, 2003−2009. (65) Nakayama, S., Atsumi, R., Takakusa, H., Kobayashi, Y., Kurihara, A., Nagai, Y., Nakai, D., and Okazaki, O. (2009) A zone classification system for risk assessment of idiosyncratic drug toxicity using daily dose and covalent binding. Drug Metab. Dispos. 37, 1970−1977. (66) Hu, Y., Yang, S., Shilliday, F. B., Heyde, B. R., Mandrell, K. M., Robins, R. H., Xie, J., Reding, M. T., Lai, Y., and Thompson, D. C. (2010) Novel metabolic bioactivation mechanism for a series of antiinflammatory agents (2,5-diaminothiophene derivatives) mediated by cytochrome p450 enzymes. Drug Metab. Dispos. 38, 1522−1531. (67) (2010) Thelin (sitaxentan) to be withdrawn due to cases of unpredictable serious liver injury, European Medicines Agency Press Release, London. (68) Erve, J. C., Gauby, S., Maynard, J. W., Svensson, M. A., Tonn, G., and Quinn, K. P. (2013) Bioactivation of sitaxentan in liver microsomes, hepatocytes, and expressed human P450s with characterization of the glutathione conjugate by liquid chromatography tandem mass spectrometry. Chem. Res. Toxicol. 26, 926−936. (69) Dalvie, D., Kang, P., Zientek, M., Xiang, C., Zhou, S., and Obach, R. S. (2008) Effect of intestinal glucuronidation in limiting hepatic exposure and bioactivation of raloxifene in humans and rats. Chem. Res. Toxicol. 21, 2260−2271. (70) Kemp, D. C., Fan, P. W., and Stevens, J. C. (2002) Characterization of raloxifene glucuronidation in vitro: contribution of intestinal metabolism to presystemic clearance. Drug Metab. Dispos. 30, 694−700.

1358


Bioactivation Potential of Thiophene-Containing Drugs - American

Recommend Documents