Increased Drug Safety - avoiding reactive metabolites

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This is an educational presentation directed towards professionals working with drug design. At the end of the presentaion it is demonstrated how the tool SpotRM+ (based on the workbench Bioclipse) provides analysis of planned drug structures regarding structural alerts for formation of reactive metabolites; it is available at http://awametox.com/. The free web tool with a similar name is no longer updated and will not be available after April 2016.

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Increased Drug Safety - avoiding reactive metabolites

  1. 1. Reactive Metabolites - Hints How to Avoid a Drug Safety Hazard Alf Claesson © 2012 Awametox Consulting, Stockholm, Sweden. Contact: +4670 553 7131, info@awametox.se. LinkedIn Reactive Metabolite Reactions with DNA Reactions with proteins Drug The liver is usually the first casualty Problems…problems…problems…problems..problems…problems…
  2. 2. 2 Modified from Gerry Kenna and Roger Bonnert, AstraZeneca Reactive Metabolite (RM) Reactions with DNA Reactions with proteins • An important cause of drug-induced illness and fatality, e.g. DILI= Drug Induced Liver Injury • A major concern for drug industry and regulators • Low-dose drugs cause less/no problems Mutagenicity Carcinogenicity Teratogenicity - Benzidine - Safrole Target organ toxicity (reproducible or idiosyncratic) - Paracetamol - Diclofenac Immune hypersensitivity reactions (idiosyncratic) - Antibacterial sulfon- amides - Halothane Drug The liver is usually the first casualty NH2 N H S OO N N OH Br Cl F F F
  3. 3. 3 Slightly modified from Steve Swallow (AZ Drug Safety) at the SCI Conference ”Designing Safer Medicines in Discovery: Current and Emerging Opportunities to Reduce Attrition”, 25th March 2011 Substructural alerts Genotox/RM Reactive cpd Toxic metabolite (e.g. forms F-acetic acid) logP Light sens. Solubility Stability + ADME person
  4. 4. Substance related failures of drugs are of great concern Drug safety is a major cause of drug attrition.  It will take a very long time before we can predict all hazards  This presentation highlights one factor, i.e. metabolism to reactive species – Indeed, we can hope to master this cause of attrition! By experimentation and increased knowledge Potency in secondary test model Potency in primary test model Practical synthesis Pharmacokinetics Decreased off-target I potency Patent issues Solubility (for example) Lead CD Decreased off- target II potency Not efficient 22% Toxicology 32% Clinical safety 12% DMPK 8% Portfolio 11% Other reasons 15%
  5. 5. 5 The different fates of reactive species formed by enzymatic action In the scheme below “I” represents a drug which acts as an irreversible inhibitor of a P450 enzyme – The general term is Mechanism Based Inhibition (MBI) – also known as suicide inactivation/inhibition. – Indicated by in vitro experiments where enzymes lose activity by time, Time Dependent Inhibition (TDI) P= RM Covalent binding to macromolecules Reaction with water or glutathion (inactivation) Extensive MBI of CYPs by a drug is worrysome since it often leads to interference with metabolism of other drugs (DDI). There is good correlation between covalent binding of a drug to proteins and TDI. Nakayama et al. Drug Met Disp 2011, 54, 1247
  6. 6.  Reactions that do not occur in most patients at any dose.  But please don’t call them dose independent; drugs given at a dose of 10 mg/day or less are relatively safe!  Characteristics suggest immune mechanism  Also known as hypersensitivity reactions, allergic reactions, type B reactions, type II reactions Idiosyncratic Drug Reactions (Jack Uetrecht’s definition) 6 Some toxicity is reproducible and dose-dependent in animals, e.g. that of paracetamol (acetaminophen). This is referred to as Type A toxicity. For some drugs toxicitiy in man is unpredictable from animal research and is relatively infrequent. This is referred to as Type B toxicity (truly idiosyncratic).
  7. 7. 7 In real life Picture to the right from a presentation by Jack Uetrecht. Skin injuries are a common sign of allergic drug reactions. Etiologies of acute liver failure in the US (n = 1,321). Data from the Acute Liver Failure Study Group registry, 1998–2008 (W. M. Lee). Abbreviations: AIH = autoimmune hepatitis; BCS = Budd–Chiari syndrome; HAV = hepatitis A virus; HBV = hepatitis B virus; IDR = idiosyncratic drug reaction. acetaminophen
  8. 8. Drugs associated with IADRs Drugs Withdrawn Aclcofenac (antiinflammatory) Hepatitis, rash Alpidem (anxiolytic) Hepatitis (fatal) Amodiaquine (antimalarial) Hepatitis, agranulocytosis Amineptine (antidepressant) Hepatitis, cutaneous ADRs Benoxaprofen (antiinflammatory) Hepatitis, cutaneous ADRs Bromfenac (antiinflammatory) Hepatitis (fatal) Carbutamide (antidiabetic) Bone marrow toxicity Ibufenac (antiinflammatory) Hepatitis (fatal) Iproniazid (antidepressant) Hepatitis (fatal) Metiamide (antiulcer) Bone marrow toxicity Nomifensine (antidepressant) Hepatitis (fatal), anaemia Practolol (antiarrhythmic) Severe cutaneous ADRs Remoxipride (antipsychotic) Aplastic anaemia Sudoxicam (antiinflammatory) Hepatitis (fatal) Tienilic Acid (diuretic) Hepatitis (fatal) Tolrestat (antidiabetic) Hepatitis (fatal) Troglitazone (antidiabetic) Hepatitis (fatal) Zomepirac (antiinflammatory) Hepatitis, cutaneous ADRs Marketed Drugs Abacavir (antiretroviral) Cutaneous ADRs Acetaminophen (analgesic) Hepatitis (fatal) Captopril (antihypertensive) Cutaneous ADRs, agranulocytosis Carbamazepine (anticonvulsant) Hepatitis, agranulocytosis Clozapine (antipsychotic) Agranulocytosis Cyclophosphamide (anticancer) Agranulocytosis, cutaneous ADRs Dapsone (antibacterial) Agranulocytosis, cutaneous ADRs, aplastic anaemia Diclofenac (antiinflammatory) Hepatitis Felbamate (anticonvulsant) Hepatitis (fatal), aplastic anaemia (fatal), severe restriction in use Furosemide (diurectic) Agranulocytosis, cutaneous ADRs, aplastic anaemia Halothane (anesthetic) Hepatitis Imipramine (antidepressant) Hepatitis Indomethacin (antiinflammatory) Hepatitis Isoniazid (antibacterial) Hepatitis (can be fatal) Phenytoin (anticonvulsant) Agranulocytosis, cutaneous ADRs Procainamide (antiarrhythmic) Hepatitis, agranulocytosis Sulfamethoxazole (antibacterial) Agranulocytosis, aplastic anaemia Terbinafine (antifungal) Hepatitis, cutaneous ADRs Ticlopidine (antithrombotic) Agranulocytosis, aplastic anaemia Tolcapone (antiparkinsons) Hepatitis (fatal) Trazodone (antidepressant) Hepatitis Trimethoprim (antibacterial) Agranulocytosis, aplastic anaemia, cutaneous ADRs Thalidomide (immunomodulator) Teratogenicity Valproic acid (anticonvulsant) Hepatitis (fatal), teratogenicity Temp. Withdrawn or Withdrawn in other Countries Aminopyrine (analgesic) Agranulocytosis Nefazodone (antidepressant) Hepatitis (> 200 deaths) Trovan (antibacterial) Hepatitis Zileuton (antiasthma) Hepatitis For most of these drugs, bioactivation to reactive metabolites has been demonstrated in vitro or in vivo Kalgutkar AS and Soglia JR (2005). Exp. Opin. Drug Metab. & Toxicol. 1:91-141) Anticancer drugs are visibly absent from the lists.
  9. 9. 9 Recent failures of drugs that were on the market Lumiracoxib (Prexige® from Novartis) launched 2003- 2004, withdrawn autumn 2007 due to reports of serious liver adverse events N H O Cl F OH Sitaxentan (Encysive Pharma) was withdrawn in 2010 after having been on the European market for only four years. This compound has obvious liabilities regarding hazard for RM formation, yet no proven link between ADRs and RMs. Cl ON NH S O O S O O O
  10. 10. S O O O Alkyl Alkyl halides and sulfonates [Br,I,Cl] Electrophilic esters SNAr electrophiles N A [F,Cl,Br] N A OSO2R Wide variety of structures! (EWG= electronwithdrawing group) [F,Cl] EWG O O Ar O O O N H Oxiranes and aziridines O SO2 Michael acceptors Awareness/avoidance of intrinsic reactivity [F ,C l] E W G Very useful presentation at SCI Conference, March 25, 2011, on ”Designing Safer Medicines in Discovery” Title: ChEMBL & Structural Alerts By Francis Atkinson Chemogenomics Group EMBL – EBI, Hinxton http://www.soci.org/News/Fine-Safer-Medicines-2011-Papers.aspx 10
  11. 11. 11  Shortlived electrophilic intermediates are formed from most drugs during ‘detoxification’ - Imines and iminium ions from alkylamines - Epoxides from double bonds - Unsaturated carbonyls  Do not cause a safety problem unless - Defence mechanisms are overwhelmed - Key macromolecules are altered - manifested by organ and/or immune toxicity.  RMs can have selective affinity to certain macromolecules - Genotoxicity vs. other organ tox? - Structure Reactivity Relationships poorly understood Reactive metabolites (RMs) from xenobiotics N + O from paracetamol (acetaminophen); not extremely shortlived. Quinoid species very common as RMs. Most RM- forming reactions are Phase 1 reactions Most common RMs listed at StopRM.org where references to reviews are also listed.
  12. 12. 12  Phase 1 reactions, i.e. oxidative (cytochromes P-450), reductive (-NO2), and hydrolytic pathways. These are behind most of the RM generation.  Phase 2 reactions, i.e. conjugation reactions like sulfatation and glucuronidation, in general less prone to cause problems by RM generation. - Important exceptions are formations of nitrene, quinoid and carbenium ion species which are initialized by acetylation, sulfatation, and more - Formation of acylglucuronides and acyl-CoA thioesters as acylating agents Metabolic reactions that can generate RMs All dependent on context - just like organic synthesis… OO O O OH OH OH O R R= rest of the drug R S O CoA Skonberg et al. Exp Op DM Tox 4 (2008) 425 Review
  13. 13. The molecular mechanism of genotoxicity by aromatic amines Persistent mutations caused by intercalated adducts in hotspots of DNA lead to cancer Repairs Primary target atom Nitrenium ion or nitrene NH N NH N N O NH2 H N dR N NH N N O NH2 dR N + H+ P450 + + H+ + NH2 NH N NH N N O NH2 H N dR N NH N N O NH2 dR N Guanine-rich DNA motif CYP1A2 Acetyltransferases H+ + • Only 30% of arylamines follow this path • Rate of HO-N formation and nitrenium ion stability determine mutagenicity. Sulfotransferases N..N + H N Reactive nitroso compounds can also be formed from anilines and nitro compounds DNA products, CRT 2003 See Shamowsky et al. JACS 2011, 133, 16168, and McCarren et al. J Cheminfo 2011, 3:51
  14. 14. 14 Precursors of RMs  Phenyls/benzene – Can form arene oxides and quinoid species – Problem substituents: nitro, amino (anilines and masked anilines) – Halo substituents – influence fate of arene oxides – Alkoxy groups (facilitate hydoxylation and also undergo dealkylation) – Alkyl groups on aromatics  HO-alkyl  eliminations to reactive quinomethanes (benzoquinone methides)  Heteroaryls – Thiophenes – Thiazoles – Furans, and more…  Other groups – Many, e.g. alkenes ( allylic alcohols), alkynes, alkyl halides. Also carboxylic acids (form acylglucuronides and thioesters), and more… Aromatics Heteroaromatics Aliphatics Oxidations by cytochromes P- 450, FMNs, peroxidases Reduction
  15. 15. 15 Epoxides, especially on aromatic rings (‘arene oxides’)  Benzene very frequent group in drug candidates – Lots of varied substituents and fusions – Indispensible to medicinal chemists?  RMs formed by epoxidation of the ring – Labile arene oxides are formed – As an example, 1,2-naphtalene oxide was isolated in 1968 by famous NIH scientists (Daly, Jerina, Witkop et al. JACS 50 6525) – Enzymatic inactivation/detoxification by epoxide hydrolases and/or GSH S-transferases (also by direct reaction with GSH) GSH is used in vitro to trap RMs and thus indicate/measure their presence ‘NIH shift’ O More easily formed than benzene oxide, E = 70.1 and 59.8 kcal/mol, respectively (Mats Svensson, AZ). Glutathione (GSH) mM conc. in hepato- cytes CYP O R OH S G R OH OH R OH R GSH H2O Enzyme Rearr.
  16. 16. 16 Characteristics of dangerous epoxides  Not inactivated fast enough (in relation to amounts formed)  Toxic quantities able to reach and modify macromolecules  Learning from experience is possible: – Polyaromatic oxides are known carcinogens, being stabilized arene oxides, e.g from benzopyrene – Other epoxides also have insidious behaviour - balanced and targeted reactivity towards sensitive proteins/DNA O O O ‘Stable’ epoxide from toxic - naphtoflavone. Aflatoxins easily form epoxides on their fused dihydrofurans (lead to 1,4-dioxo compounds) O O O O O O H H NO NH2 O ‘Stable’ epoxide from carbamazepine
  17. 17. Lamotrigine is known to form epoxide(s) NH2 N NH2 N N Cl Cl NH2 N NH2 N N Cl Cl O Cl N N NH2 N NH2 Cl GS NH2 N NH2 N N Cl Cl OH GS Human P450 2A6. Rat P450 2C11 GSH - H2O M-I M-II Maggs et al. (2000) and Chen et al. (2009). Both research groups conclude that a reactive, but somewhat stabilized, epoxide is formed. Formed in minor amounts in vivo (reaction products isolated from bile only). Notably, it is also formed in keratinocytes. The isomeric analogue irsogladine, a PDE4 inhibitor, is metabolized to an epoxide via a major pathway. This isomerizes to phenols (Sugiyama et al. Arzneimittelforschung 1986, 36, 1229) N N N Cl Cl NH2 NH2 N N N Cl Cl NH2 NH2 O N N N Cl Cl NH2 NH2 OH
  18. 18. Quinoids comprise a major category of RMs O [C,N,O] R OH [C,N,O] R OH [C,N,O] Nu R NuP450 or other enzyme In this large group the electrophilic system consists of a quinone, a quinone- imine, a quinone-diimine, or the corresponding methides, the quinone methides (quinomethanes) and quinone-imine methides. Only the para isomers are depicted below. Testa et al. (Drug Disc Today 2012, 17, 549) have analysed the literature and conclude: “A markedly greater source of worry and potential toxicity is seen with redox reactions, most significantly with the formation of quinones, quinonimines, quinonimides and quinone-diimines, which accounted for 40% of all toxic and/or reactive metabolites identified in this work.” In addition, from the abstract of a review (Monks et al. Current Drug Metab 2002): Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi and bacteria… For example, the quinones of polycyclic aromatic hydrocarbons are prevalent as environmental contaminants and provide a major source of current human exposure to quinones. ... . Quinones are oxidants and electrophiles, and the relative contribution of these properties to quinone toxicity is influenced by chemical structure, in particular substituent effects.
  19. 19. All roads lead to Rome…or to quinoids (1) From phenols  diphenols  quinones When R= X-H the formation of a phenol or aminophenol is facilitated. Even more facilitation…. Real drug examples existN H F R O N H F R N O R - HF O X H OH X O X OH X H X = O,N 1,4-elimi- nation Oxid.Rearr. O R OH RO ALK R OH OH R O O R OH OH Nu R O OH Me R Nu Oxid. Oxid. COMT 19
  20. 20. All roads lead to Rome…or to quinoids (2) Quinone methides (quinomethanes) and quinone-imine methides. Thompson et al. studied the phenol below (Toxicology 2001, 160, 197). OH O S O O O [O,N] H H [N,O][O,N] OH H - H2OOxid. OH OOH OH When the benzylic alcohol forms a sulfate (via SULT enzymes) the elimination is even faster The sulfates of the corresponding 4- alkoxy-benzylic alcohols would also be quite reactive. O O S O O O R 20
  21. 21. All roads lead to Rome…or to quinoids (3) Spontaneous loss of HF gives rise to a reactive, toxic species (Thompson et al. 2000) Merck cpd published in 2005. Decomposes to a carboxylic acid on standing in a water solution Order of events can be different (Kalgutkar et al. DMD 2007) The example also shows that the leaving group can be a ”phenol” Lefluonomide is a licensed rheumatoid arthritis drug. Instability due to isoxazole ring opening dominates. No reports of imine-methide formation. OH F FF O FF - HF N H O N O OH F F F F N N H F O O F NH N N N O Cl N N O Cl OH R Cl O Cyp N N OR 21
  22. 22. 22 Paracetamol can be directly oxidised to a reactive acetylated quinoneimine Massive amounts of NAPQI will exhaust GSH reserves N O O N O O S R NH O OH S R NH O OH R-SHOx. Proton shifts NAPQ Well-known quinoid-forming motifs of real drugs Kassahun, K. Studies on the Metabolism of Troglitazone to Reactive Intermediates in Vitro and in Vivo. Chem Res Toxicol 2001, 14, 62 O OH O S N H O O O O O S N H O O O OH GS R The thiazolidine ring might also entail RM problems. But these are not significant in low-dose rosiglitazone (Avandia®, GSK) and pioglitazone (Actos®, Lilly). Troglitazone - withdrawn from the market.
  23. 23. 23  Oxidation of heteroaromatics  Acyl glucuronides (AGs) and acyl-CoA as RMs  Special case – the antiepileptic felbamate  Formation of acyl halides (halothane) A few other mechanisms of RM formation are shown on the following four slides
  24. 24. 24 Heteroaromatics can also cause problems  Thiophenes form epoxide and/or S-oxides (tienilic acid, a diuretic drug, is a classic example; withdrawn 1982) – The epoxide can also hydrolyse and ring-open – Simple thiophenes largely abandoned within AZ  Thiazoles can also cause problems S O O COOH Cl Cl S + R O S R Nu S R O S R Nu S R Nu(p450) OH NuH Add.-Elim. NuH Tienilic acid + O S N H Duloxetine (Cymbalta®) does not appear to show RM tox problems. Daily dose is 60 mg. Thiabendazole (an anthelmintic), given 1-3 g/day shortterm N H N S N N H N O O S NH2 H+ P450
  25. 25.  Invoked mechanisms: – Direct acylation (of amino groups) – In theory, the acyl glycoside can isomerize by acyl migration to expose a free aldehyde (semiacetal) which can react with amines and rearrange further.  Rare proven cases. Benzoic acids don’t have AG problems whereas aryl acetic acids (many NSAIDs) might have – Many NSAIDs withdrawn from the market because of hepatotoxicity. Mechanism? Most stuctures have other potential liabilities stemming from aromatic substructures, e.g. zomepirac 25 Reviews: Skonberg et al. Exp Op DM Tox 4 (2008) 425. Bailey & Dickinson. Chemico-Biological Interactions 145 (2003) 117/137 Acyl glucuronides (AGs) and acyl-CoA as RMs From AZ workshop 2007: “The poorly defined link between acyl glucuronides and toxicity was considered not to reflect evidence of absence (of such a link), but rather absence of evidence (of such a link).” Zomepirac, withdrawn 1984 or O OH OH OH O O O O R R O N H R1R1-NH2 R S O CoA O R O O N H OH OH O O Prot O N OH OCl
  26. 26. O O NH2 O NH2 O O H O NH2 O O O NH2 O H O H O NH O O H N H N Alb O N N Alb H Esterases Alcohol dehydro- genase Form ed via spon- taneous loss of carbam ic acid 26 Special case – the antiepileptic felbamate Mechanism Reaction of the isolated hemiaminal 1 with albumin (Alb) was studied by Roller et al. in Chem Res Tox 2005, 15, 815. They concluded that conjugate addition mainly goes via a histidine residue. 1 Within a year of its release in 1993 • 34 cases of aplastic anemia resulting in 13 deaths (Incidence rate 1:4800 – 1:37000) • 23 cases of hepatotoxicity resulting in 5 deaths (Incidence rate 1:18000 – 1:25000 Black box warning (severe restriction in use) • thousands of patients estimated to be on drug Note No glutathione conjugates in liver microsomes and human hepatocytes. No covalent binding to liver microsomes and human hepatocytes.
  27. 27. 27 Volatile anaesthetics and hepatotoxicity Halothane – introduced1956, “Delayed” liver injury in ~1: 3000 patients who receive multiple exposures, and liver failure in ~1: 30,000 Enflurane - introduced in UK in 1981 “Delayed” liver injury in ~1: 100,000 patients who receive multiple exposures Isoflurane - introduced in UK in 1984 Very rare case reports of “Delayed” liver injury, ~ <1: 100,000 patients who receive multiple exposures Sevoflurane - introduced in 1990, initially in Japan Currently one of the most widely prescribed volatile anaesthetics in developed nations. Rare case reports of “delayed” liver injury, from Japanese literature; Desflurane - introduced 1956 Currently one of the most widely prescribed volatile anaesthetics in developed nations. A few isolated case reports of “delayed” liver injury F C C F F Br H Cl F C C F F O Cl F C C F F O Protein CYP 2E1 Halothane TFA-Cl TFA-protein Mechanism of halothane activation
  28. 28. 28 Resources on reactive metabolites  Literature reviews (see comprehensive list on stoprm.org), for example – Kalgutkar et al. (Pfizer), have written many reviews; large one from 2005* – Uetrecht – perspectives.. – Baillie – 20 years experience…  Presentations – Some available on the Internet. Also search for presentations at http://www.slideshare.net. – A Claesson has collated several presentations at stoprm.org  Databases etc. – SpotRM+ is a new application (2016) available at awametox.com – ’MDL METAB’ from Accelrys; many industrial chemists and ADMET pros use this. – Mechanism Based Toxicity Database (MBT), from GVK Biosciences – Lhasa Limited and MultiCASE are providers of computational tools on software and knowledge – Chemistry databases containing drugs & data: ChEMBLdb, Chemspider, and ChemPub  Other resources – Drug metabolism predictive tools (MetaSite, MetaPrint2D, Meta-PC, and more): see ‘Directory of computer-aided Drug Design tools’ (ADME Toxicity) * A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups. 65 pages See next slides
  29. 29. SpotRM+ is a new (Jan 2016) tool that helps you avoid the test substances that have issues regarding reactive metabolites © 2012 Awametox Consulting, Sweden. Contact: info@awametox.se Most likely, the freely accessible web application SpotRM (since 2012) will not be available after March 2016. “SpotRM+ is a new analytical expert tool that confronts your ideas of new drug structures with the real world of drugs that have serious adverse effects in clinical use, due to reactive metabolites (RM). SpotRM+ highlights potential hazards in your structure by matching it with a set of well thought-out structural alerts that have proven or likely associations with adverse clinical events.” There is also a DEMO version available.

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