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