Designing Drugs to Avoid Toxicity

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Designing Drugs to Avoid Toxicity
Progress in Medicinal Chemistry v50 Ch 1
Graham F Smnith

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Designing Drugs to Avoid Toxicity

  1. 1. 1 12 1 Designing Drugs to Avoid Toxicity 23 34 45 GRAHAM F. SMITH 56 67 Central Chemistry Team Lead, Merck Research Laboratories Boston, 78 33 Avenue Louis Pasteur, Boston, MA 02115, USA 89 910 1011 1112 INTRODUCTION 1 1213 13 THE SAFETY WINDOW 214 1415 COMMON SAFETY RISKS AND THEIR SAR 2 1516 Toxicity associated with the liver 2 1617 Cardiovascular toxicity ( h ERG, ETC.) 13 1718 Genotoxicity/mutagenicity 16 1819 Phospholipidosis 39 19 Phototoxicity 4020 20 Idiosyncratic toxicity 4221 2122 CONCLUSIONS 42 2223 2324 REFERENCES 43 2425 2526 2627 27 INTRODUCTION28 2829 29 Two thousand four data from the Centre for Medicines Research show that toxicity is now30 30 the leading cause of failure of compounds in clinical development. With the improved31 31 systemic exposure, which came with better understanding of drug metabolism and phar-32 32 macokinetics (DMPK), came increased observations of dose-limiting toxicity [1]. The33 33 leading causes of drug failure are now tied at 30%, with toxicity as likely to be the demise34 34 of a drug’s development as lack of efficacy, (PK-related attrition now stands at 10%).35 35 Nevertheless, most safety-related attrition (70%) occurs pre-clinically following candidate36 36 selection, suggesting that we are still in need of better predictive models of in vivo toxicity.37 37 Where in vitro assays, or simple in vivo experiments, are predictive of adverse events in38 38 humans, then these are increasingly carried out earlier in the drug discovery cycle.39 39 The structure–toxicity relationships for mutagenicity and hepatotoxicity are already well40 40 established owing to robust in vitro assays which translate well to clinical outcomes. These41 41 assays have frequently been used to implicate common alerting structures or so-called42 42 ‘structure alerts’. Identifying structural alerts for toxicity, and high-throughput assays for43 43 early indicators of toxicity issues in vivo, have become a normal part of early drug discovery.44 44 Regulatory authorities require that these robust assays be run on all new chemical entities45 45 before entering first-in-human trials.46 46 Progress in Medicinal Chemistry – Vol. 50 1 Ó 2011, Elsevier B.V. Edited by G. Lawton and D.R. Witty All rights reserved. DOI: 10.1016/B978-0-12-381290-2.00001-X
  2. 2. 2 DESIGNING DRUGS TO AVOID TOXICITY1 Sometimes, inadvertently, medicinal chemists do introduce toxicophores into drug mole- 12 cules. Most often their reactive nature is produced or enhanced in vivo during normal meta- 23 bolic processes. Wherever possible this review elaborates the biochemical mechanism attrib- 34 uted to this type of toxicity. This allows medicinal chemists to validate the mechanism in their 45 own case and also to contextualize their own molecules in terms of their likelihood to undergo 56 similar biotransformation. Some successfully marketed drugs are positive in glutathione 67 binding assays [2], however, it is well established that the toxicities of known compounds 78 with chemically reactive metabolites can be correlated with the generation of hepatic protein 89 adducts and/or the detection of stable phase II metabolites such as glutathione conjugates. 910 The genotoxic carcinogens have the unifying feature that they are either electrophiles per se 1011 or can be activated to form electrophilic reactive intermediates. Hard electrophiles generally 1112 react with hard nucleophiles such as functional groups in DNA and lysine residues in proteins. 1213 Soft electrophiles react with soft nucleophiles, which include cysteine residues in proteins and 1314 in glutathione. Glutathione has a concentration of approximately 10 mM in the liver. Free 1415 radicals can also react with lipids and initiate lipid peroxidative chain reactions [3]. The 1516 presence of a toxicity risk, or even the confirmation of a metabolic pathway to known toxicity, 1617 does not preclude a molecule from entering development. The risks are evaluated in the 1718 context of the body’s highly developed ability to clear toxic molecules from circulation and to 1819 recover from damage. 1920 2021 2122 THE SAFETY WINDOW 2223 2324 All drugs are toxic at some level and so a major challenge in drug discovery is to find a 2425 margin of efficacy, over adverse events or toxicities, sufficient to provide clinical benefit to 2526 patients whilst avoiding putting them at unnecessary risk. The therapeutic index (TI) is 2627 commonly used in the pharmaceutical industry and is the ratio of the no observable adverse 2728 event level (NOAEL) divided by the human efficacious exposure level (Ceff) or exposure at 2829 the maximum anticipated human dose (Cmax). 2930 To determine margin, it is recommended to compare plasma Cmax from animal pharma- 3031 cology and toxicity studies with (predicted) human pharmacokinetic Cmax data using 3132 unbound free fraction [4]. Depending on the disease target and nature of toxicity Ctrough 3233 or area under the curve (AUC) can also be used to determine margins. Ideally these margins 3334 would be around 10-fold or more over a reversible toxicity outcome which is observed in 3435 animal testing, but which can also be clinically monitored easily in humans. 3536 3637 3738 COMMON SAFETY RISKS AND THEIR SAR 3839 3940 TOXICITY ASSOCIATED WITH THE LIVER 4041 4142 CYP inhibition 4243 4344 One of the liver’s main physiological roles is the clearance and metabolism of xenobiotics 4445 into hydrophilic metabolites in order to facilitate their excretion. The liver has an abundance 4546 of xenobiotic metabolizing enzymes and a high capacity for both phase I and phase II 46
  3. 3. GRAHAM F SMITH 31 biotransformation. It receives more than 80% of its blood flow from the portal vein into 12 which drugs are absorbed from the gastrointestinal tract and therefore liver is often a primary 23 target for chemical-induced toxicity. There is the possibility that reactions catalysed by 34 cytochrome p450 (CYP) enzymes may generate metabolites that are not only more toxic but 45 also more reactive than the original xenobiotic. Drug-induced liver injury is the most 56 frequent reason for the withdrawal of an approved drug from the market. Drug-induced 67 liver injury has now become the leading cause of liver failure in the Unites States and results 78 in at least 2700 deaths per annum [5]. 89 Time-dependent inhibition (TDI) of CYPs refers to a change in potency during an in vitro 910 incubation or dosing period in vivo, as opposed to a normal reversible inhibitor dose 1011 response. Inhibition of specific CYP enzymes by a drug can lead to pharmacokinetic 1112 changes in another drug, or so-called drug–drug interactions [6]. When inhibition affects 1213 the major metabolic route of another enzyme, and therefore alters (usually increases) 1314 exposure, this leads to unpredictable exposure levels and often to unacceptable risks to 1415 patients. In common with other proteins, CYPs are eventually metabolized and replaced if 1516 they are irreversibly inhibited. CYP enzymes have a turnover of the order of 1–2 days. 1617 However, TDI is often associated with bioactivation to electrophilic species which have the 1718 potential for a number of toxic pathways beyond the simple inhibition of CYPs. 1819 There are several known mechanisms of CYP inhibition: 1920 2021 * Competing enzyme substrates affecting the turnover of other drugs. 2122 * Competitive inhibitors such as quinidine which are not substrates. 2223 * Haem ligands: non-selective metal chelators such as the imidazole antifungals. 2324 * Metal inhibitor complex forming drugs such as erythromycin. 2425 * Inactivation or suicide inhibitors such as tienilic acid. 2526 2627 There are good methods of in vitro assessment of CYP inhibition and induction. The 2728 outcome of this is that common motifs and SAR for these toxic mechanisms exist. 2829 The following structure classes have well-established mechanisms for CYP inhibition. 2930 3031 Alkynes 3132 Mechanism-based CYP inhibition (MBI) can arise from the covalent attachment of alkyne 3233 metabolites to the CYP protein. The formation of these adducts is described in Scheme 1.1. 3334 [(Schem_1)TD$FIG] 3435 3536 3637 3738 3839 3940 4041 4142 4243 4344 4445 4546 Scheme 1.1 Oxidation of alkynes to electrophilic species 46
  4. 4. 4 DESIGNING DRUGS TO AVOID TOXICITY1 Reactive metabolites can also be generated which may form covalent adducts with CYP 12 proteins or other proteins leading to toxicity [7–10]. Generation of alkyne-CYP intermediate 23 (A) can lead directly to the haem-bound product (B). For example, oxirene (C) (derived 34 from ring closure of A) can react with a CYP haem nitrogen generating B, and can also react 45 with other nucleophilic sites in the CYP protein. Ketene D (formed by the migration of the 56 R2 group in intermediate A) can also react with CYP and other proteins to form potentially 67 toxic conjugates. 78 Gestodene (1) is one of many synthetic steroid drugs, including oral contraceptives, 89 which contain an acetylene moiety. This drug was shown to be a mechanism-based inhibitor 910 of CYP3A4 and 3A5. A variety of other alkyne-containing steroids have also been evaluated 1011 and show differing degrees of activity [11]. 17a-Ethynylestradiol (2) is a common compo- 1112 nent of oral contraceptives and taken by millions of women worldwide. This steroid has 1213 been shown to be a mechanism-based inhibitor of CYP3A4 in vitro[12]. However, admin- 1314 istration of 17a-ethynylestradiol to women has been shown to have no impact on either 1415 intestinal or hepatic CYP3A4 activity [13]. This is most likely to be due to the very low 1516 doses required to achieve effective contraception, thereby mitigating the potential drug– 1617 drug interaction risk.[(Fig._1)TD$IG] 1718 1819 1920 2021 2122 2223 2324 2425 2526 2627 2728 CYP inhibition is the most common toxicity associated with alkyne-containing 2829 drugs. Therefore, early investigation of metabolic routes (in vitro and in vivo), coupled 2930 with reactive metabolite screening, is warranted for medicinal chemists studying 3031 alkynes. Compounds should be evaluated across a range of CYP enzymes/species 3132 (with and without pre-incubation) to ensure that the potential for inhibition is fully 3233 evaluated. 3334 3435 Thiophenes 3536 The thiophene ring is susceptible to hepatic oxidation by CYP and undergoes epoxida- 3637 tion, followed by epoxide ring opening with nucleophilic biomolecules, to give adducts 3738 [14–22] (Scheme 1.2). Alternatively the epoxide can open to give a (-thionoenal which 3839 can also undergo adduct formation. The thiophene sulphur can also undergo oxidation, 3940 thus activating the ring towards nucleophilic addition of biomolecules. Peroxidase addi- 4041 tion of chlorine to the thiophene sulphur can also activate the ring towards nucleophilic 4142 addition. Both the epoxide and the S-oxides have been postulated as reactive intermedi- 4243 ates. Identification of any of these metabolites therefore implies formation of reactive 4344 intermediates. 4445 Tienilic acid (3), a diuretic, is a mechanism-based inhibitor of CYP2C9 and seems 4546 to inactivate it stoichiometrically. The molecule was launched onto the market and 46
  5. 5. GRAHAM F SMITH 5 [(Schem_2)TD$FIG]1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 Scheme 1.2 Metabolism of thiophenes 1819 1920 2021 2122 2223 then subsequently withdrawn in 1982 due to a link with hepatitis [23–25]. The non- 2324 steroidal anti-inflammatory suprofen (4) showed nephrotoxicity in the clinic and is a 2425 mechanism-based inhibitor of CYP2C9. It was marketed and subsequently withdrawn 2526 due to cases of acute renal failure [26, 27]. The antiplatelet drug panaldine 2627 (Ticlopidine) (5) shows TDI of CYP2B6; because it is linked with increased risk of 2728 agranulocytosis its use has been replaced by clopidogrel (Plavix) (6) [28]. OSI-930 (7) 2829 was being developed for oncology when it was discovered that the molecule reacted 2930 via the sulphoxide to form adducts with CYPs 3A4 and 2D6 [29]. In all of these cases 3031 sulphoxide and glutathione adducts of the thiophene moiety have been detected and 3132 are postulated to cause the time-dependent inhibition of CYP enzymes and further 3233 related toxicities. 3334 One way to reduce or inactivate this pathway is to introduce 2,5-substitution on the 3435 thiophene ring. Alternatively, the ring can be deactivated towards nucleophilic attack 3536 through introduction of adjacent functionality. Introduction of an alternative metabolic 3637 weak point elsewhere in the molecule may also reduce toxic exposure overall. 3738 Examples of these strategies can be seen in Zyprexa (8) and Plavix (6) [31–35], 3839 two commercially successful, widely marketed drugs. It appears that a small structural 3940 change between panaldine (5) and Plavix, which introduces an additional metabolic 4041 route, reduces thiophene-related hepatotoxicity. Panaldine generates about 20 metabo- 4142 lites, some of which covalently bind to proteins, while the primary metabolic fate of 4243 Plavix is hydrolysis of the methyl ester and some glucuronidation of the resulting 4344 acid. Plavix is dosed at 75 mg QD, while panaldine is dosed at 250 mg BID, so the 4445 dose difference between panaldine and Plavix may also be a potential mitigating 4546 factor.[(Fig._1)TD$IG] 46
  6. 6. 6 DESIGNING DRUGS TO AVOID TOXICITY1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 1819 1920 2021 2122 2223 2324 2425 2526 [(Fig._1)TD$IG] 2627 2728 2829 2930 3031 3132 3233 3334 3435 3536 3637 3738 Furans 3839 In a similar manner to that for thiophenes, furan toxicity occurs via furan epoxidation 3940 followed by epoxide ring opening to a g-keto aldehyde which in turn forms adducts with 4041 biomolecules and induces toxicity [36, 37] (Scheme 1.3). Alternatively, the epoxide can 4142 ultimately give rise to a lactone which can also form adducts. The epoxide has been 4243 postulated as the reactive intermediate common to all observed metabolites. Identification 4344 of any of these metabolites therefore implies formation of the epoxide. 4445 The clinical development of the 5-lipoxygenase inhibitor L-739,010 (9) was discontinued 4546 by Merck due to hepatotoxicity; the compound is a mechanism-based inhibitor of CYP3A4 46
  7. 7. GRAHAM F SMITH 71 [38–40]. Upon incubation with recombinant CYP3A4, a covalently bound adduct of the 12 compound was formed, which was identified using mass spectrometry. 23 The HIV protease inhibitor L-754,394 (10) showed hepatotoxicity via potent mechanism- 34 based inhibition of CYP3A4 and its clinical development was discontinued. It also has been 45 shown subsequently for L-756,423 (11) that attachment of benzofuran through the 2- 56 position, potentially blocking epoxidation, results in the removal of the furan-associated 67 toxicity [41–43]. The fungal pneumotoxin Ipomeanol (12) was also developed for oncology 78 and then halted due to hepatotoxicity. Upon activation of Ipomeanol with rabbit CYP4B1 in 89 the presence of N-acetyl cysteine and N-acetyl leucine a major product (13) consistent with 910 furan epoxide formation was observed and characterized [44–46]. 1011 It is interesting to note that there are examples of 2,5-disubstituted benzofurans such as 1112 ranitidine (14) [47] which do not undergo typical furan metabolism. This is probably due to 1213 their low lipophilicity, low dose and additional substitution. Substituted benzofurans have 1314 been observed to undergo metabolism. Benzofuran itself undergoes the typical furan 1415 hydroxylation at the 2-position, possibly through direct hydroxylation and also potentially 1516 through epoxidation, followed by ring opening to generate 2-hydroxyphenylacetic acid [48]. 1617 [(Fig._1)TD$IG] 1718 1819 1920 2021 2122 2223 2324 2425 2526 2627 2728 2829 2930 3031 3132 3233 3334 3435 3536 3637 3738 3839 3940 4041 4142 4243 4344 4445 4546 46
  8. 8. 8 DESIGNING DRUGS TO AVOID TOXICITY [(Schem_3)TD$FIG]1 12 23 34 45 56 67 78 89 910 1011 1112 Scheme 1.3 Furan oxidative metabolism 1213 1314 Benzodioxolanes 1415 The benzodioxolane moiety is associated with mechanism-based irreversible inhibition 1516 and/or induction of CYPs. In addition some compounds that contain the benzodioxolane 1617 moiety are associated with hepatotoxicity. CYP-dependent oxidation of the methylene 1718 leads to both a reactive carbene intermediate (A) which can form irreversible adducts with 1819 the haem of CYPs (metal inhibitor complex) or the catechol (B) which is an ortho- 1920 quinone precursor and known toxin through redox chemistry. The mechanism of toxicity 2021 and CYP inhibition of benzodioxolane compounds has been discussed in detail [49–51] 2122 (Scheme 1.4). 2223 Paroxetine (15) is a marketed selective seratonin reuptake inhibitor (SSRI) with a 2324 known CYP2D6 inhibition profile; it is both a reversible and a time-dependent 2425 CYP2D6 inhibitor. This results in significantly increased exposure to co-medications 2526 that are metabolized by CYP2D6. Metabolism of the benzodioxolane group has been 2627 strongly implicated in the CYP2D6 inhibition shown by paroxetine [52–56] and recent 2728 studies have shown that the potency of paroxetine as a CYP2D6 inhibitor in vitro 2829 increases eightfold following pre-incubation [57]. The increase in potency was asso- 2930 ciated with the formation of a CYP mechanism-based inhibitor complex. 3031 Administration of paroxetine has been shown to convert some volunteers who are 3132 extensive CYP2D6 metabolisers to a poor metaboliser phenotype [58]. In addition 3233 paroxetine inhibits its own metabolism leading to non-linear time-dependent pharma- 3334 cokinetics. The half-life of paroxetine after single doses of 20 mg/day is 10 h but after 3435 3536 3637 [(Schem_4)TD$FIG] 3738 3839 3940 4041 4142 4243 4344 4445 4546 Scheme 1.4 Metabolism of benzodioxolane 46
  9. 9. GRAHAM F SMITH 91 multiple doses of 20 mg/day this increases to 24 h [59]. While, the fate of the benzo- 12 dioxolane is well established in vitro, it is of note that most SSRIs are relatively potent, 23 reversible inhibitors of CYP2D6. 34 Niperotidine (16) is an H2 antagonist structurally related to ranitidine. Twenty-five cases 45 of acute hepatitis (including one death from fulminant hepatitis) associated with niperotidine 56 use were reported in Italy between March and August 1995 and the drug was withdrawn 67 from the market. The methylenedioxy group of niperotidine (absent in ranitidine) is known 78 to undergo metabolism to catechol and quinone metabolites [60, 61]. 89 Methylenedioxymethamphetamine (MDMA) (17) has been shown to inhibit CYP2D in a 910 time-dependent manor through a mechanism producing a UV absorption spectrum consis- 1011 tent with a carbene formation [6]. MDMA causes liver damage in humans.[(Fig._1)TD$IG] 1112 1213 1314 1415 1516 1617 1718 1819 1920 2021 2122 2223 2324 2425 2526 2627 2728 2829 Fortunately, several viable isosteric replacements are available for the benzodioxo- 2930 lane structure. Replacement of one of the oxygen atoms with a methylene results in a 3031 dihydrobenzofuran moiety, which may often show similar pharmacology to a benzo- 3132 dioxolane. The dihydrobenzofuran system can be rather susceptible to oxidative metab- 3233 olism, and this should be checked promptly when this group is employed (Scheme 1.5). 3334 The difluorobenzodioxolane group is a metabolically blocked at the ‘methylene’ car- 3435 bon, and this does not undergo the same metabolic reactions as the methylenedioxy 3536 group. The difluorobenzodioxolane group is rather unusual as it is considerably more 3637 lipophilic than the methylenedioxy group. There are no drugs in the MDDR (molecular 3738 detection of drug resistance) drug database containing this moiety. The methylene 3839 carbon may also be blocked with other groups, for example as a dimethylketal, although 3940 the stability of such groups towards acid-catalysed hydrolysis needs to be carefully 4041 assessed. 4142 There are many additional groups that have the potential to mimic a benzodioxolane. 4243 Owing to their instability towards hydrolysis in dilute aqueous acid, benzoxazoles should 4344 also be employed with caution, if at all. The benzodioxane ring-expanded system appears 4445 not to be implicated in the same kinds of toxicity/mechanism-based CYP inhibition as the 4546 benzodioxolane group. 46
  10. 10. 10 DESIGNING DRUGS TO AVOID TOXICITY [(Schem_5)TD$FIG]1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 1819 1920 20 Scheme 1.5 Some potential benzodioxolane isosteres21 2122 2223 2324 2425 Haem Ligands 25 The previous examples of potential liver toxins all form covalent inhibitor complexes with26 26 CYPs and other proteins. Another commonly encountered class of inhibitors is the haem27 27 ligands which offer lone pair donation, usually from nitrogen, to stabilize the iron in the28 28 haem complex. These molecules have an affinity for the active site of CYPs in both the29 29 oxidized and reduced forms but are reversible inhibitors (Scheme 1.6). Many heterocycles30 3031 3132 [(Schem_6)TD$FIG] 3233 3334 3435 3536 3637 3738 3839 3940 4041 4142 4243 4344 4445 4546 Scheme 1.6 Haem ligands 46
  11. 11. GRAHAM F SMITH 111 frequently used in drug-like molecules are capable of performing this role, for example 12 pyridines, azines and azoles [62]. 23 The 11-b-hydroxylase inhibitor metyrapone (18) is an inhibitor of cortisol synthesis and 34 of CYP3A4 [63, 64]. Metyrapone also causes induction of CYP3A4 synthesis in hepato- 45 cytes. The HIV protease inhibitor ritonavir (19) [65, 66] contains two 5-substituted thia- 56 zoles. Ritonavir is a potent inhibitor of CYP3A-mediated biotransformations (e.g. nifedi- 67 pine oxidation and terfenadine hydroxylation). Ketoconazole (20) is a member of the 78 antifungal imidazole drugs. Ketoconazole strongly inhibits CYP3A4 selectively [67]. 89 Sulconazole (21), another member of the antifungal imidazole derivatives, strongly inhibits 910 most CYPs (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4).[(Fig._1)TD$IG] 1011 1112 1213 1314 1415 1516 1617 1718 1819 1920 2021 2122 2223 2324 2425 2526 2627 2728 2829 2930 CYP induction 3031 3132 CYP induction occurs when a drug or chemical causes an increase in enzyme activity, 3233 usually via increased gene transcription [68–70]. In many cases, inducers are also hepato- 3334 toxic. CYP induction can lead to a reduction in efficacy of co-medications and also to an 3435 increase in reactive metabolite-induced toxicity. CYP induction is therefore a metabolic 3536 liability in drug therapy and it is highly desirable to develop new drug candidates that are not 3637 potent CYP inducers. 3738 Most commonly, ligand activation of key receptor transcription factors leads to 3839 increased transcription. In the human liver, some of these enzymes, but not all, are 3940 inducible. Human CYP1A, CYP2A, CYP2B, CYP2C, CYP2E and CYP3A enzymes 4041 are currently known to be inducible. CYP gene families 2 and 3 have a similar 4142 mechanism of gene activation through a ligand-activated nuclear receptor constitutive 4243 androstane receptor or constitutively active receptor CAR and/or pregnane X receptor 4344 (PXR). CYP3A4 is the most highly expressed CYP enzyme representing up to 28% of 4445 all CYPs and is highly inducible by a wide variety of xenobiotics. CYP3A4 has been 4546 implicated in the metabolism of more than 50% of prescribed pharmaceuticals [71]. 46
  12. 12. 12 DESIGNING DRUGS TO AVOID TOXICITY1 CYP1A genes belong to the Per-Arnt-Sim (PAS) family of transcription factors and 12 require the aliphatic hydrocarbon receptor (AhR). CYP1A2 is also one of the major 23 CYPs in human liver, accounting for approximately 10% of total amount of hepatic 34 CYPs. 45 There are four main mechanisms of CYP induction [72]: 56 67 1. PXR upregulates the important CYP3A and 2C enzymes. PXR is referred to as the 78 master regulator of CYP enzymes. The classic substrate for PXR is the antibiotic 89 rifampicin (22). Similarly, the glucocorticoid anti-inflammatory and immunosuppres- 910 sant dexamethasone (23) has been reported to be a substrate [73]. It has been hypoth- 1011 esized that unwanted activation of the PXR is responsible for approximately 60% of all 1112 observed drug–drug interactions [74]. Today, many drug companies routinely include 1213 the PXR reporter gene assay at the drug discovery stage as part of the selection processes 1314 of drug candidates for clinical development. 1415 2. Aliphatic hydrocarbon (Ah) or aryl hydrocarbon receptor (AhR) induces CYP1A 1516 enzymes 1 and 2; certain polycyclic aromatic hydrocarbons in the diet and environ- 1617 ment induce their own metabolism, for example hydrocarbons in cigarette smoke, 1718 charbroiled meats and cruciferous vegetables. 2,3,7,8-Tetrachlorodibenzo-p-dioxin 1819 (TCDD) (24) and the related TCDF (25) are the prototypical CYP1A inducers. 1920 Tryptophan derivatives, caffeine, eicosanoids and some prostaglandins are also 2021 AhR substrates. 2122 3. CAR induces CYP2B and CYP3A enzymes. Typical substrates are barbiturates such as 2223 phenobarbital (26). 2324 4. Peroxisome proliferator-activated receptors (PPARs) upregulate CYP4A. Typical exam- 2425 ples include the fibrates, PPAR alpha receptor agonists such as clofibrate (27). The 2526 thiazolidinedione antidiabetic agents such as rosiglitazone (28) act as PPAR gamma 2627 agonists. 2728 2829 Transcription factors such as HNF4a are also involved and there is also significant post- 2930 translational regulation of protein half-life, especially of CYP2E1. The glucocorticoid 3031 receptor (GR) and estrogen receptor (ER) may also be involved. Two other nuclear 3132 receptors, designated LXR and FXR, which are respectively activated by oxysterols 3233 and bile acids, also play a role in liver CYP7A1 induction [75]. Together all of these 3334 receptors are able to sense a great variety of xenobiotics and consequently regulate 3435 numerous phase I and phase II drug-metabolizing enzymes and drug transporters. In this 3536 way they attempt to adjust the body’s metabolic response to the challenges of the 3637 chemical environment. 3738 To avoid toxicity associated with potential CYP induction, it is important to divert 3839 the structure–activity relationship of interest from that of the nuclear receptor which is 3940 also being activated. The screening approaches to avoiding CYP induction are 4041 reviewed by Pelkonen et al.[75]. It is possible to establish in vitro assays for AhR, 4142 CAR, PPAR gamma and PXR, and SAR from these assays may be used to refine a 4243 QSAR model. In this way in silico models have been developed for all of these 4344 receptors using QSAR and docking approaches, some of which reach up to 80% 4445 successful prediction. 4546 [(Fig._1)TD$IG] 46
  13. 13. GRAHAM F SMITH 131 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 1819 1920 2021 2122 2223 2324 2425 2526 2627 2728 2829 2930 3031 3132 3233 3334 CARDIOVASCULAR TOXICITY (hERG, ETC.) 3435 3536 Virtually all cases of extended QT interval are traced to the inward rectifying potassium 3637 ion channel (IKr) related gene known as hERG (human ether-a-go-go-related gene), 3738 which encodes the protein Kv 11.1. Inhibition of the cardiac IKr current leads to pro- 3839 longation of the QT interval and to a risk of lethal ventricular arrhythmia (torsade de 3940 pointes (TdP)) [76–78]. The electrocardiogram (ECG) traces in Figure 1.1 show the 4041 prolongation of QT leading to TdP. Once hERG involvement in inherited long QT was 4142 established, QT-prolonging TdP-prone drugs began to be tested on hERG. This showed 4243 hERG to be a major contributor to drug-acquired QT prolongation. This phenomenon 4344 was once considered a trivial finding, in fact IKr was a valid drug target for the class III 4445 arrhythmic drugs, but more recently QT prolongation has become a major regulatory 4546 46
  14. 14. 14 DESIGNING DRUGS TO AVOID TOXICITY [(Fig._1)TD$IG]1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 1819 1920 2021 Fig. 1.1 A: Normal ECG. B: Long QT syndrome. C: Ventricular arrhythmia (torsade de pointes). 2122 2223 2324 2425 issue. Since 2005, the FDA has required that all new drug candidates are evaluated to 2526 determine the drug’s effect on the QT interval. Other channels which may play a more 2627 minor role include Nav1.5 and Ca2+. 2728 QT prolongation can routinely lead to a drug being withdrawn from the market or from 2829 development as happened in the cases of the antihistamine terfenadine (29) and the gastric 2930 prokinetic cisapride (30). Astemizole (31) a long duration antihistamine drug, the anti- 3031 psychotic sertindole (32) and the quinolone antibacterial grepafloxacin (33) were also all 3132 withdrawn post-launch over concerns about life threatening TdP. 3233 Today nearly all drug discovery programmes include an early assessment of hERG 3334 liabilities, including an in vitro primary radioligand binding assay in the IKr ion channel 3435 [79]. In addition to IKr, early assessment of Nav1.5 and Cav1.2 channels is also being 3536 conducted earlier. Functional alternatives to these binding assays are patch clamp and patch 3637 express. Apart from an earlier and cheaper alert to hERG toxicity these high-throughput 3738 assay data provide excellent data for validating structure–activity relationships and building 3839 computational models. 3940 Cavalli et al.[80] was able to build a 3D QSAR model (Figure 1.2) based on 4041 known drugs. This model is often used as a first pass design tool to avoid hERG 4142 activity. The empirically based model has been validated and enhanced by homology 4243 models related to known crystal structures of four other bacterial potassium channels 4344 [81–83]. These models have been used successfully in the development of drugs such 4445 as maraviroc (34) to overcome hERG binding issues encountered in the discovery 4546 phase [84]. 46
  15. 15. GRAHAM F SMITH 15 [(Fig._2)TD$IG]1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 1819 Fig. 1.2 The Cavalli hERG pharmacophore model. 1920 2021 Workers from Merck showed that bio-isosteres which might improve IKr profile based on 2122 previous pairwise analysis of molecules assayed can be used to computationally point the 2223 way towards reduced hERG affinity [85]. Bell and Bilodeau [86] recently gave a good 2324 overview of medicinal chemistry tricks to avoid hERG SAR. Techniques usually involve 2425 reducing basicity and lipophilicity (Scheme 1.7). The IKr channel seems to have high affinity 2526 for many types of lipophilic bases, therefore adding polar groups, for example alcohols or 2627 ethers, removing hydrophobic groups, reducing Pi-stacking interactions and removing or 2728 modifying aryl rings are all good approaches chemically.[(Fig._1)TD$IG] 2829 2930 3031 [(Schem_7)TD$FIG] 3132 3233 3334 3435 3536 3637 3738 3839 3940 4041 4142 4243 4344 4445 4546 Scheme 1.7 Simple modifications which often reduce the risk of hERG activity 46
  16. 16. 16 DESIGNING DRUGS TO AVOID TOXICITY1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 1415 1516 1617 1718 1819 1920 2021 2122 2223 2324 2425 2526 2627 2728 GENOTOXICITY/MUTAGENICITY 2829 2930 Genotoxicity describes a deleterious action on a cell’s genetic material affecting its 3031 integrity. The term genotoxicity includes DNA reactivity, resulting in mutation, and also 3132 interaction with various protein targets, for example spindle microtubules, leading to 3233 numerical chromosome changes or aneuploidy. It is regulatory practice to view DNA- 3334 reactive effects as having no acceptable threshold (or no-effect level), whereas reaction 3435 with protein targets might have an acceptable threshold and potential to establish a safety 3536 margin as is the case for other toxicities. Genotoxic substances are potentially mutagenic 3637 or carcinogenic. This definition includes both some classes of chemical compounds and 3738 certain types of radiation. 3839 Typical genotoxins such as aromatic amines are believed to cause mutations because they 3940 are nucleophilic and form strong covalent bonds with DNA, resulting in the formation of 4041 aromatic amine-DNA adducts and preventing accurate replication. Genotoxins affecting 4142 sperm and eggs can pass genetic changes to descendants who have never been exposed to 4243 the genotoxin. As many mutations can contribute to the development of cancer, many 4344 mutagens are carcinogens. So-called spontaneous mutations are also known to occur due 4445 to errors in DNA replication, repair and recombination, and the many endogenous products 4546 of cellular metabolism such as oxygen radicals. 46
  17. 17. GRAHAM F SMITH 171 The international test guidelines require a bacterial mutagenicity test (the Ames test) 12 and an in vitro test for chromosome aberrations or for mutation in a mouse lymphoma cell 23 line, before the first human clinical trials. An in vivo test for chromosome damage 34 (typically a micronucleus test) must be done before phase II clinical trials [87]. Many 45 companies also use early versions of these regulatory assays for screening, or some of the 56 wide range of relatively high-throughput screening assays available for early detection of 67 genotoxicity. The Ames test is a bacterial assay that allows the detection of strong early 78 signals of mutagenicity [88–90]. Ames tests use a histidine-free medium with a genet- 89 ically engineered strain of bacteria that can only proliferate into colonies after certain 910 mutations restore their ability to synthesize histidine. It has been established that the 1011 predictive power of positive Ames test results for rodent carcinogenicity is high, ranging 1112 from 60 to 90% depending on the compound set examined. An assay is also used that 1213 identifies chromosomal damage, either visible as chromosome breaks at metaphase, or as 1314 micronuclei (chromatin that is left outside the main nucleus and comprises either frag- 1415 ments of broken chromosomes (clastogenicity), or whole chromosomes, indicating 1516 potential for aneuploidy). An in vitro and in vivo chromosomal aberration assay is 1617 required before first-in-human studies; these studies are often conducted in the presence 1718 of metabolic activation in order to assess the toxicity of any metabolites which may be 1819 formed. 1920 Following extensive testing, the validation of structural types leading to mutagenicity is 2021 well established. The development of the so-called ‘structure alerts’ related to mutage- 2122 nicity from the 1950s to the current day is well reviewed by Benigni and Bossa [91]. 2223 In general the alkylation of DNA by electrophilic chemicals leads to mutagenicity. The 2324 other mechanism is via molecules which intercalate with DNA, changing its tertiary 2425 structure, and interfering with normal DNA function and replication. In this section 2526 biochemical pathways which explain the reactivity of these groups are elaborated, so 2627 that they might be more appropriately used and modified by medicinal chemists to reduce 2728 the risk of mutagenicity. 2829 2930 Electrophiles not requiring metabolic activation 3031 3132 32 During the course of in vitro testing in research programmes, certain chemical inter-33 33 mediates and mild electrophiles find their way into the screening cascade by design or34 34 by accident. Despite some of these being perfectly stable chemicals in buffered35 35 solution, it must be noted that the body is perfectly able to find nucleophiles with36 36 sufficient potency such as amines and thiols which will unselectively react with37 37 these electrophiles. The toxicity of these functional groups will in general be related38 38 to their chemical reactivity. Figure 1.3 shows a set of common electrophiles which39 39 should be avoided unless targeting a specific drug–protein covalent interaction is the40 40 desired goal.41 4142 4243 Alkyl halides and sulphonates 4344 4445 Alkyl halides and sulphonates are susceptible to nucleophilic attack by a cysteine-SH or 4546 other bio-molecule nucleophiles to form adducts [92]. Their toxicity is directly related to 46
  18. 18. 18 DESIGNING DRUGS TO AVOID TOXICITY [(Fig._3)TD$IG]1 12 23 34 45 56 67 78 89 910 1011 1112 1213 1314 Fig. 1.3 Some common electrophiles encountered in medicinal chemistry. 1415 1516 1617 their chemical reactivity. Leaving groups beta to an electron-withdrawing group (EWG 1718 such as carbonyls, aryl groups, nitriles, etc.) are also of concern due to possible elimi- 1819 nation to form a Michael acceptor molecule. Mammalian response to such agents in- 1920 volves elevation of activity of phase II detoxifying enzymes [93, 94]. An SN1 mechanism 2021 for the substitution is also possible. For example mono alkyl fluorides are less susceptible 2122 to nucleophilic attack, but are likely to be converted via cationic (SN1-like) mechanisms 2223 where possible. 2324 Toremifene (Fareston) (35) is an oral anti-estrogen drug for the treatment of metastatic 2425 breast cancer. There are numerous adverse events and toxicities reported with the use of 2526 toremifene as described in the pharmaceutical documentation ring (PDR) entry. However, 2627 many of these may be due to its estrogenic activity rather than the presence of an alkyl 2728 halide. Many alkyl halides and sulphates have been reported as anticancer agents. Their 2829 designed mode of action is alkylation of DNA, and hence they are cytotoxic with many side 2930 effects. In these cases, the genetic toxicity is incorporated by design and part of the risk 3031 analysis for development and usage. 3132 [(Fig._1)TD$IG] 3233 3334 3435 3536 3637 3738 3839 3940 4041 4142 4243 4344 4445 In addition to mechanisms seen for the other halides, organic iodides can cause hypo- 4546 thyroidism, hyperthyroidism, phototoxicity, photosensitivity and skin sensitization. 46

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