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  1. 1. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Metabolic Activation of Natural Products Ling Yang Lab of Pharmaceutical Resource Discovery Dalian Institute of Chemical Physics, the Chinese Academy of Sciences www.pharm.dicp.ac.cn Sep 23rd, 2011
  2. 2. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Natural Products Totally Natural !!! ButNatural products are generally either Safe?of prebiotic origin or originate frommicrobes, plants, or animal sources
  3. 3. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Mechanisms of Toxicity Chemicals Metabolism About 80% Interaction with Metabolites DetoxicificationDose-reaction Receptors relationship About 20% On Target Off Target Reactive Structure- Type A1 Toxicity Type A2 Toxicity Products reaction relationship Protein Adducts GSH… Adducts DNA Adducts Deplete Mutation/Block Oxidative Defences Polymerases Haptenized Trigger High Dose Overwhelm: get oxidative damage Type D ToxicityType B Toxicity Immune Response Type C Toxicity Apoptosis/ Period of Dosing: Exemplified Prime Sites Hypersensitivity Liver, Blood cells, Skin Necrosis Carcinogenicity and Teratology Liebler & Guengerich (2005) Nat Rev Drug Discov 4(5):410-420.
  4. 4. Dalian Institute of Chemical Physics, Chinese Academy of SciencesDrugs Withdrawn Associated With Idiosyncratic Toxicity or Drug Interactions Idiosyncratic Toxicity Drug-Drug interactions Aclcofenac (antiinflammatory) Astemizole Alpidem (anxiolytic) Mibefradil Amineptine (antidepressant) Propulsid Amodiaquine (antimalarial) Posicor Benoxaprofen (antiinflammatory) Seldane Bromfenac (antiinflammatory) Hismanal Carbutamide (antidiabetic) Palladone Ibufenac (antiinflammatory) Iproniazid (antidepressant) Metiamide (antiulcer) Nomifensine (antidepressant) Practolol (antiarrhythmic) Remoxipride (antipsychotic) Sudoxicam (antiinflammatory) Tienilic Acid (diuretic) Tolrestat (antidiabetic) Troglitazone (antidiabetic) Zomepirac (antiinflammatory) Crit. Rev. Toxicol. 35 (2005) 325-361.
  5. 5. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Classification of Reactive MetabolitesElectrophiles (Most): Hard: a localized positive charge Soft (Most): a delocalized charge Free radicals: Free radicals are characterized by containing an unpaired electron and they usually abstract a hydrogen atom from molecules, resulting in a new free radical and thus initiating a chain reaction.
  6. 6. Dalian Institute of Chemical Physics, Chinese Academy of Sciences List of Some Groups liable to Metabolic Activation Structural Alerts Herbal Components Anilines: R1 R2 Quinone-imine R2 + R1 N N R1 R2 CYP1A2, 3A4 N OH O OH O NH2 HN N Rutaecarpine (Rut) Nitroso metaboliteR1, R2=H, alkyl, phenyl acyl, O O HOacyloxy, sulfonyl Benzo-dioxolanes: N N N O O HO O . H H H . H H H O OCH3 OCH3 OH OCH3 O O O H carbene CYP2C9 O O O CYP3A4 O OH O OCH3 O OCH3 H3CO H 3CO OCH3 H3CO catechol OH Quinone O Noscapine ortho-quinone Furans: OH O O O O CYPs O O O O O βа , -unsaturated dicarbonyl N Pyrrolizidine alkaloids (PAs) Nitrobenzenes: R1 R1 R1 N NO2 NH OH O R1=phenyl,acyl or heterocyclic Fang et al. Expert Opin Drug Metab Toxicol. 2011;7(8):989-1007. ...... Zhou et al. Life Sciences (2004) 74: 935–968.
  7. 7. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Techniques available to assess RM formationTrapping and characterizing reactive metabolites Nucleophilic trapping agents: Thiols: GSH; NAC Soft nucleophile Amine: semicarbazide; methoxylamine Hard nucleophileMechanism-based inhibition (Time dependant inhibition)Trapping and characterizing Protein / DNA adducts Immunoassays Proteomics Electrophoresis
  8. 8. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Progress• Metabolic Activation-mediated Toxicity – Rutaecarpine (Rut) and Evodiamine(Ed) – Pyrrolizidine alkaloids (PAs) – Strychnine (Str)• Metabolic Activation-mediated Drug Interactions – noscapine• Ginseng-drug interaction via the inhibition by intestinal metabolites
  9. 9. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Progress• Metabolic Activation-mediated Toxicity – Rutaecarpine (Rut) and Evodiamine(Ed) – Pyrrolizidine alkaloids (PAs) – Strychnine (Str)• Metabolic Activation-mediated Drug Interactions – noscapine• Ginseng-drug interaction via the inhibition by intestinal metabolites
  10. 10. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Metabolic Activation of Rutaecarpine (Rut) Evodia fruit is considered by herbalists to be potential toxic. A wide range of pharmacological activities: vasorelaxation, uterotonic action, anoxia and control of body temperature.Wuzhuyu CYP1A2, 3A4 GSH Rutaecarpine (Rut) Screening CYP isoforms involved in Structural elucidation bioactivation -273 -129 O NH2 -273 H N O O N H OH S O HO HO N O N -129 H N -249 In publication
  11. 11. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Metabolic Activation of Evodiamine (Ed)Five metabolites were detected after evodiamine was incubated with HLMs and PB-induced RLMsMetabolic pathways Metabolic pathwaysStructural elucidation of GSH conjugate of evodiamine CYP1A2 or 2C9 GSH Evodiamine In publication
  12. 12. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Progress• Metabolic Activation-mediated Toxicity – Rutaecarpine (Rut) and Evodiamine(Ed) – Pyrrolizidine alkaloids (PAs) – Strychnine (Str)• Metabolic Activation-mediated Drug Interactions – noscapine• Ginseng-drug interaction via the inhibition by intestinal metabolites
  13. 13. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Metabolic Activation of Pyrrolizidine alkaloids (PAs) R O O OH OH O O P450 N N DHP DHR High activity and instability1988 WHO issue the information about toxicity of PA Acute liver toxicity: liver cell necrosis, liver hemorrhage Chronic liver toxicity: the nucleus increases, giant cell disease of2002 MHRA proposed to prohibit the drug containing PA liver; the liver venous congestion of the lungs2004 WTO, Qianbai Rhinitis Tablet containing toxic plant Senecio, Genotoxicity: genetic combination, DNA cross-linking, DNA- liver toxicity protein cross-linking; carcinogenic, teratogenic2005 China , Qianbai Rhinitis Tablet managed as prescription drug OH OH N-oxide O O Competitive Metabolic Hydrolysis O activation O O O metabolic pathway O O 11 Deacetylation 22 N N Phase II ?
  14. 14. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Metabolic pathway of PAsEsterase, FMO, and UGT may play a detoxicification role via competitiveconsumption of PAs, resulting in a reduction of available PAs for P450 activation.N-glucuronidation is a common metabolic pathway of most of PAs. He et al. (2010) Drug Metabolism and Disposition 38 (4): 626-634.
  15. 15. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Toxicity of Senecionine in Primary Human HepatocytesSenecionine does not show toxicity in human hepatocytes . However, when theUGT1A4 activity was inhibited by inhibitor (hecogenin), potent cytotoxicity exhibited ,indicating that glucuronidation may be an important mechanism against PAs toxicity. He et al. (2010) Chemical Research in Toxicology 23 (3): 591-599.
  16. 16. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Species Differences of Senecionine glucuronidationGlucuronidation is critically important for rabbits, sheep and other species to defendtoxicity of Senecionine. As rats, mice, and dogs are lack of UGT1A4 expression, but alsothe lack of other competition with metabolic activation of metabolic pathways, hastherefore become sensitive to toxic species.
  17. 17. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Possible Metabolism-mediated Toxicity Mechanism of PAs Bioactivation Toxic adducts Interaction with Macromolecule PAs CYPs Idiosyncratic toxicity Toxic Exposure Liver Effective PAs No Effect Time UGTs Increased local exposure Glucuronized PAs Glucuronyl hydrolaseHe et al. DMD 2010, 38 (4): 626-34. Enterohepatic PAsHe et al. Chem Res Toxicol 2010, 23 (3): 591-9. circulation
  18. 18. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Progress• Metabolic Activation-mediated Toxicity – Rutaecarpine (Rut) and Evodiamine(Ed) – Pyrrolizidine alkaloids (PAs) – Strychnine (Str)• Metabolic Activation-mediated Drug Interactions – noscapine• Ginseng-drug interaction via the inhibition by intestinal metabolites
  19. 19. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Introduction of Strychnos nux-vomica L. Major components:alkaloids, glucosides, organic acids , alcohols… Pharmacological Effects: Treatment of rheumatism and rheumatoid arthritis, analgesic effect, anti-inflammatory Leathal dose: 7g(crude herb)Strychnos nux-vomica L.(Loganiaceae) Toxic target: nervous system, immune system, digestive system, cardiovascular system, urinary system Compatibility : Licorice, red spoon, Datura metel, white peony root, Rehmannia To precipitate strychnine Anti-seizure
  20. 20. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Introduction of strychnine and brucine H N H N H H OCH3 H H N O N OCH3 O O O strychnine brucineClinical Effects: analgesic, anti-inflammatory, anti-tumor, anti-arthritisToxicity:seizure, CNS toxicity, nephrotoxicity… LD50 in mice:strychnine: 3.27mg/kg brucine: 233mg/kg Oral toxic doses of brucine are 71 times as strychnine Injection toxic doses of brucine are 45 times as strychnine
  21. 21. Dalian Institute of Chemical Physics, Chinese Academy of SciencesThe Same Enzymes Catalyzes oxidation and Glucuronidation of both Brucine and Strychnine in HLMs O CYP3A4 [ ] UDP UGT1A4 Brucine [ ] H N H O H H N CYP3A4 H H [ O N O ] O N H N H UDP O UGT1A4 H strychnine [ O N O ]
  22. 22. Dalian Institute of Chemical Physics, Chinese Academy of Sciences The Species Difference of Brucine and Strychnine Glucuronidation in Liver Microsomes LD50 values for strychnine Species LD50 (mg/kg) Animal Cat 0.3-0.5 Dog 0.3-0.8 Rabbit 0.4-0.6 Mouse 0.4-2 Rat 2-16 Human child 15 adult 30-120
  23. 23. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Bioactivation of Strychnine Hyperthermia Strychnine Toxicity Rhabdomyolysis Renal failure +EPI (658.40) Charge (+0) CE (50) CES (25) FT (20): Exp 2, 2.867 to 2.984 min from Sample 1 (STRY-RLM) of STRY@27Apr10.wiff (Turbo Spray) Max. 2.0e5 cps. 658.4 2.0e5 1.9e5 m/z=385 HOOC 1.8e5 1.7e5 1.6e5 1.5e5 351.4 -307 H H N HN 1.4e5 1.3e5 1.2e5 S O m/z=529Intensity, cps 1.1e5 1.0e5 H 9.0e4 8.0e4 -129 O N OH 7.0e4 529.4 O NH 6.0e4 5.0e4 184.2 212.3 333.4 -273 O 4.0e4 385.4 3.0e4 2.0e4 182.0 220.2 1.0e4 194.2 291.4 305.3 640.4 239.2 367.3 0.0 100 150 200 250 300 350 400 450 500 550 600 650 m/z, Da HOOC NH 2 HOOC H N H N H H H N HN H CYP3A4 S O H H O H O N O N O N OH O NH O O O Proposed pathway of strychnine bioactivation NH2 HOOC In publication
  24. 24. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Progress• Metabolic Activation-mediated Toxicity – Rutaecarpine (Rut) and Evodiamine(Ed) – Pyrrolizidine alkaloids (PAs) – Strychnine (Str)• Metabolic Activation-mediated Drug Interactions – noscapine• Ginseng-drug interaction via the inhibition by intestinal metabolites
  25. 25. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Noscapine-warfarin Interactions O N O Antitussive (Clinic) H H OCH3 O Antitumor (phase I,II) O OCH3 H3CO Opium Noscapine12 clinical cases of the interaction between noscapine and warfarin International Normalized Ratio (凝血指数 3.0-7.2 (6 days later) 凝血指数): 凝血指数 11 increased INR+1 bleeding An 82-year-old male Warfarin+Noscapine (50 mg tid) O OWarfarin metabolism by CYPs CYP3A4 OH H OH H CYP2C9 O O O O S-warfarin CYP1A2 R-warfarin Aneja et al. (2007) Cancer Chemoth Pharm 60: 831-39. Rosenborg et al. (2008) Br J of Clin Pharmacol 65: 277-78.
  26. 26. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Effects of Noscapine on Human CYPs, % Control Activity Positive % Control ActivityCYP Isoforms Probe reactions Remaining IC50 (µM) Control remaining (Positive Control) CYP1A2 Phenacetin O-deethylation 58.9±0.1 >100 Furafylline 15.6±0.8 CYP2A6 Coumarin 7-hydroxylation 69.2±2.0 >100 8-methoxypsoralen 11.5±0.5 CYP2C8 Paclitaxel 6α-hydroxylation 51.2±0.8 >100 Montelukast 11.2±0.1 CYP2C9 Diclofenac 4-hydroxylation 16.3±0.1 13.3±1.2 Sulfaphenazole 6.0±0.1 CYP2D6 Dextromethorphan O-demethylation 75.1±1.8 >100 Quinidine 8.6±0.4 CYP2E1 Chlorzoxazone 6-hydroxylation 99.8±3.9 >100 Clomethiazole 18.9±0.4 CYP3A4 Testosterone 6β-hydroxylation 12.3±0.7 10.8±2.5 Ketoconazole 5.0±0.3 For CYP3A4: Competitive, Ki=5.2µM For CYP2C9: Noncompetitive, Ki= 8.8µM
  27. 27. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Time- and NADPH-dependant InhibitionFor CYP2C9: IC50 shift 10-fold For CYP3A4: IC50 shift 10-fold 10*decrease 10*decrease KI= 8.9µM KI=9.3µM kinact= 0.014 min-1 kinact=0.06 min-1
  28. 28. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Metabolic-activation of NoscapineA GSH conjugate was detected in HLMs Structural elucidation Conjugate HLMs -GSH -NADPH Bioactivation process Fang et al. (2010) British Journal of Clinical Pharmacology 69(2): 193-199.
  29. 29. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Prediction of noscapine and Warfarin Drug Interactions (S)-warfarin: CYP2C9 (R)-warfarin: CYP3A4, CYP1A2Reversible inhibition: Time-dependent inhibition:Using Cmax: Using Cmax:AUC of (S)-warfarin: 1.5% AUC of (S)-warfarin: 110.9%AUC of (R)-warfarin: 1.1% AUC of (R)-warfarin: 48.9%Using Cmax,u: Using Cmax,u:AUC of (S)-warfarin: 0.5% AUC of (S)-warfarin: 41.8%AUC of (R)-warfarin: 0.4% AUC of (R)-warfarin: 32.7%Warfarin Concentration Toxic in Plasma Effective TimeNoscapine-Warfarin Interactions Fang et al. (2010) Br J of Clin Pharmacol 69(2):193-199. Rosenborg et al. (2010) Clin Pharmacol Ther 88(3):343-346.
  30. 30. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Progress• Metabolic Activation-mediated Toxicity – Rutaecarpine (Rut) and Evodiamine(Ed) – Pyrrolizidine alkaloids (PAs) – Strychnine (Str)• Metabolic Activation-mediated Drug Interactions – noscapine• Ginseng-drug interaction via the inhibition by intestinal metabolites
  31. 31. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Ginsenosides Biotransformation by Intestinal Bacteria 20(S)-protopanaxadiol type 20(S)-protopanaxatriol type Glc Glc Glc-Xyl Glc6-1X Rb3 Rb1,Rb2,Rc Re HO Rg1 Glc2-1Glc Glc2-1Glc HO Glc2-1Rha Glc-O Naturally abundant Glc OH Glc Glc2-1Glc Rd Rg2 HO F1 Glc2-1Rha HO HO OH OH F2 Rh1 Glc Glc-Xyl HO Glc Mx Glc HO HO C-K Ppd OH HO Ppt OH Gastro- HO HO intestine .942–542 ,12 .lluB .mrahP .loiB .)8991( .la te oakA .5841–1841 ,32 .lluB .mrahP .loiB .)0002( .la te eaB .668–168 ,52 .lluB .mrahP .loiB .)2002( .la te awagesaH 044–634:36 deM atnalP )7991( .la te awagesaH Blood Effect .751–351 ,59 .icS .locamrahP .J .)4002( awagesaH .1701–5601 ,13 .sopsiD .bateM gurD .la te bawaT 50 46 Positive 79% 45 40 Negative 21% N o. of specimens Glc-Glc6-O Glc-O 35 OH OH 30 25 20 15 12 Glc-Glc6-O HO 10 5 Rb1 C-K 0individual differences of hydrolysis 1 in ability of Rb1 Hasegawa et al. (1997) Planta Med 63:436–440.
  32. 32. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Natural Occurring Ingredients of Natural Products How about Metabolites by Intestinal Bacteria? Example: Ginsenosides
  33. 33. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Possible Ginseng-drug Interactions via CYPs inhibition by Ginsenoside Intestinal metabolites Intestinal Naturally rare Naturally abundant Bacterial Metabolites S- Ppt Ppd CK Rg2 Rg3 Rg3 Rh1 Rh2 F1 Rb1 Rb2 Rc Rd Re Rg1 CYP1A2 CYP2A6 CYP2C9 CYP2D6 CYP2E1 CYP3A4 IC50 ( μ μ μ μ μ μ μ μ M) 100 50 10 1 μ μ μ μ μ μ μ μ μ μ μ μ M Prediction of potential for DDI from In Vitro Data CB,max CL,max CYP2C9 CYP3A4Ginsenosides (µM ) (µM ) Cmax/Ki Prediction Cmax/Ki Prediction >0.59~0.83 Ppt 1.9 < 0.1 Remote Possible (0.47, rat) Ppd >0.12 >1.3 < 0.1 Remote >0.1 Possible C-K >0.75 >7.8 > 0.1 Possible - - Liu et al. Toxicological Sciences 92: 356-364. Liu et al., Planta Medica 72:126-131 Liu et al. Biological & Pharmaceutical Bulletin 27:1555-1560
  34. 34. Dalian Institute of Chemical Physics, Chinese Academy of SciencesRole of Cytochrome P450 in Drug Metabolism Elimination pathways of top 200 drugs Cytochrome P450 involved in ------70 % CYP3A4 involved in ------ 45 % CYP2C9 involved in ------ 16 % Bjornsson TD, et al. J Clin Pharmacol 2003; 43 (5): 443-69 Wienkers LC, Heath TG. Nat Rev Drug Discov. 2005 Oct;4(10):825-33.
  35. 35. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Acknowledgement The National Basic Research Program (also called 973 Program) of China National High-tech R&D Program (863 Program) of China National Natural Science Foundation of China National Key Technology R&D Program in the 11th Five year Plan of China Outstanding Overseas Chinese Scientists (One Hundred Talent Program) from the Chinese Academy of Sciences Innovation Fund of Chinese Academy of Sciences
  36. 36. Dalian Institute of Chemical Physics, Chinese Academy of Sciences Our Group
  37. 37. Thanks for your attention! yling@dicp.ac.cn www.pharm.dicp.ac.cn

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