Post on 24-Jun-2015
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Metabolic Activation of Natural
Products
Lab of Pharmaceutical Resource Discovery
Dalian Institute of Chemical Physics, the Chinese Academy of Sciences
www.pharm.dicp.ac.cn
Ling Yang
Sep 23rd, 2011
Natural Products
Totally Natural !!!
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
But
Safe?Natural products are generally either
of prebiotic origin or originate from
microbes, plants, or animal sources
Mechanisms of Toxicity
Chemicals
Metabolites
Metabolism
Interaction with
Receptors
On Target Off Target
Type A1 Toxicity Type A2 Toxicity
Detoxicification
Reactive
Products
About 80%
Dose-reaction
relationship
About 20%
Structure-
reaction
relationship
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Trigger
Immune Response
Protein Adducts GSH… Adducts DNA Adducts
Haptenized
Deplete
Oxidative Defences
Type B Toxicity
Hypersensitivity Apoptosis/
NecrosisPrime Sites
Liver, Blood cells, Skin
High Dose Overwhelm:
get oxidative damage
Type C Toxicity
Mutation/Block
Polymerases
Period of Dosing: Exemplified
Carcinogenicity and Teratology
Type D Toxicity
Liebler & Guengerich (2005) Nat Rev Drug Discov 4(5):410-420.
relationship
Drugs Withdrawn Associated With Idiosyncratic Toxicity or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
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Crit. Rev. Toxicol. 35 (2005) 325-361.
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)
Classification of Reactive Metabolites
� Electrophiles (Most):
Hard: a localized positive charge
Soft (Most): a delocalized charge
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
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.
List of Some Groups liable to Metabolic Activation
N
R1 R2
N
R1 R2
OH
NH2
R1, R2=H, alkyl, phenyl acyl,
acyloxy, sulfonyl
HN
OH
N
O
N
R2
O
R1+
Nitroso metabolite
Quinone-imineAnilines:
O
H
OH
O
O
.
.
carbene
Benzo-dioxolanes:
Structural Alerts Herbal Components
O
O
OCH3
N
O
H H
O
O
OCH3
N
O
H H
HO
HO
OCH3
N
O
H H
CYP2C9
Rutaecarpine (Rut)
CYP1A2, 3A4
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
O
carbene
OH
OH
catechol
O
O
Quinone
O O
OO Oа,β-unsaturated dicarbonyl
Furans:
......
O
H3COOCH3
Noscapine
O
H3COOCH3
O
H3COOCH3
CYP2C9
CYP3A4
ortho-quinone
Nitrobenzenes:R1
NO2
R1
N
O
R1
NHOH
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.
N
OO
O
OOH
CYPs
Pyrrolizidine alkaloids (PAs)
Techniques available to assess RM formation
� Trapping and characterizing reactive metabolites
Nucleophilic trapping agents:
� Thiols: GSH; NAC Soft nucleophile
� Amine: semicarbazide; methoxylamine Hard nucleophile
� Mechanism-based inhibition (Time dependant inhibition)
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
� Mechanism-based inhibition (Time dependant inhibition)
� Trapping and characterizing Protein / DNA adducts
� Immunoassays
� Proteomics
� Electrophoresis
• Metabolic Activation-mediated Toxicity
– Rutaecarpine (Rut) and Evodiamine(Ed)
– Pyrrolizidine alkaloids (PAs)
Our Progress
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
– Strychnine (Str)
• Metabolic Activation-mediated Drug Interactions
– noscapine
• Ginseng-drug interaction via the inhibition by intestinal
metabolites
• Metabolic Activation-mediated Toxicity
– Rutaecarpine (Rut) and Evodiamine(Ed)
– Pyrrolizidine alkaloids (PAs)
Our Progress
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
– Strychnine (Str)
• Metabolic Activation-mediated Drug Interactions
– noscapine
• Ginseng-drug interaction via the inhibition by intestinal
metabolites
Metabolic Activation of Rutaecarpine (Rut)
Wuzhuyu
� 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.
CYP1A2, 3A4GSH
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
�Structural elucidation
-273
-129
-249
N
H
N
N
O
HO
S
HN
O
NH
O
O
HO
NH2
O
OH
-129-273
Rutaecarpine (Rut)
�Screening CYP isoforms involved in
bioactivation
In publication
Metabolic Activation of Evodiamine (Ed)�Five metabolites were detected after evodiamine was incubated with HLMs and PB-induced RLMs
�Metabolic pathways�Metabolic pathways
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
�Structural elucidation of GSH conjugate of evodiamine
CYP1A2 or 2C9GSH
Evodiamine
In publication
• Metabolic Activation-mediated Toxicity
– Rutaecarpine (Rut) and Evodiamine(Ed)
– Pyrrolizidine alkaloids (PAs)
Our Progress
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
– Strychnine (Str)
• Metabolic Activation-mediated Drug Interactions
– noscapine
• Ginseng-drug interaction via the inhibition by intestinal
metabolites
Metabolic Activation of Pyrrolizidine alkaloids (PAs)
P450
N
OO
O
OR
DHP DHR
N
OHOH
High activity and instability
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
1988 WHO issue the information about toxicity of PA
2002 MHRA proposed to prohibit the drug containing PA
2004 WTO, Qianbai Rhinitis Tablet containing toxic plant Senecio,
liver toxicity
2005 China , Qianbai Rhinitis Tablet managed as prescription drug
Acute liver toxicity: liver cell necrosis, liver hemorrhage
Chronic liver toxicity: the nucleus increases, giant cell disease of
liver; the liver venous congestion of the lungs
Genotoxicity: genetic combination, DNA cross-linking, DNA-
protein cross-linking; carcinogenic, teratogenic
1
2N
OO
O
OOH
N
OO
O
OOH
Metabolicactivation
Competitivemetabolic pathway
N-oxide
Hydrolysis
Deacetylation
Phase II
1
2
?
Metabolic pathway of PAs
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
N-glucuronidation is a common metabolic pathway of most of PAs.He et al. (2010) Drug Metabolism and Disposition 38 (4): 626-634.
Esterase, FMO, and UGT may play a detoxicification role via competitive
consumption of PAs, resulting in a reduction of available PAs for P450 activation.
Toxicity of Senecionine in Primary Human Hepatocytes
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
He et al. (2010) Chemical Research in Toxicology 23 (3): 591-599.
Senecionine does not show toxicity in human hepatocytes . However, when the
UGT1A4 activity was inhibited by inhibitor (hecogenin), potent cytotoxicity exhibited ,
indicating that glucuronidation may be an important mechanism against PAs toxicity.
Species Differences of Senecionine glucuronidation
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Glucuronidation is critically important for rabbits, sheep and other species to defend
toxicity of Senecionine. As rats, mice, and dogs are lack of UGT1A4 expression, but also
the lack of other competition with metabolic activation of metabolic pathways, has
therefore become sensitive to toxic species.
Possible Metabolism-mediated Toxicity Mechanism of PAs
Toxic
Exposure
PAs
Liver
CYPs
Bioactivation Toxic adducts
Idiosyncratic
toxicity
Interaction with Macromolecule
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Increased local
exposure
Effective
No Effect
Time
ExposureLiver
PAs
UGTs
Glucuronized PAs
Enterohepatic
circulation
PAs Glucuronyl hydrolase
He et al. DMD 2010, 38 (4): 626-34.
He et al. Chem Res Toxicol 2010, 23 (3): 591-9.
• Metabolic Activation-mediated Toxicity
– Rutaecarpine (Rut) and Evodiamine(Ed)
– Pyrrolizidine alkaloids (PAs)
Our Progress
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
– Strychnine (Str)
• Metabolic Activation-mediated Drug Interactions
– noscapine
• Ginseng-drug interaction via the inhibition by intestinal
metabolites
Introduction of Strychnos nux-vomica L.
Pharmacological Effects:
Treatment of rheumatism and rheumatoid arthritis, analgesic effect,
anti-inflammatory
Major components:alkaloids, glucosides, organic acids , alcohols…
Leathal dose: 7g(crude herb)
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
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
ON
N
O
H
HH
ON
N
O
H
HH
OCH3
OCH3
strychnine brucine
Clinical Effects: analgesic, anti-inflammatory, anti-tumor, anti-arthritis
Introduction of strychnine and brucine
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Toxicity:seizure, CNS toxicity, nephrotoxicity…
Oral toxic doses of brucine are 71 times as strychnine
Injection toxic doses of brucine are 45 times as strychnine
LD50 in mice:strychnine: 3.27mg/kg
brucine: 233mg/kg
The Same Enzymes Catalyzes oxidation and Glucuronidation
of both Brucine and Strychnine in HLMs
Brucine
[ ]
O
UDP
[ ]
CYP3A4
UGT1A4
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Brucine
strychnine
ON
N
O
H
HH
ON
N
O
H
HH
[ ]
ON
N
O
H
HH
[ ]
O
[ ]
UDP
CYP3A4
UGT1A4
The Species Difference of Brucine and Strychnine
Glucuronidation in Liver Microsomes
Species LD50 (mg/kg)
Animal
Cat 0.3-0.5
Dog 0.3-0.8
LD50 LD50 LD50 LD50 values for strychnine values for strychnine values for strychnine values for strychnine
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Dog 0.3-0.8
Rabbit 0.4-0.6
Mouse 0.4-2
Rat 2-16
Human
child 15
adult 30-120
Bioactivation of Strychnine
NH
H HN
HOOC
m/z=529
m/z=385
+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.
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5658.4
351.4
-307
Hyperthermia
Renal failure
RhabdomyolysisToxicityStrychnine
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
O N
N
O
H
HH
HOOC
NH
HN
HOOC
NH2
O
S O
OHO N
N
O
H
HH
O N
N
O
H
HH
O
CYP3A4
Proposed pathway of strychnine bioactivation
O N
O
H
HOOC
NH
NH2
O
S O
OH
m/z=529
100 150 200 250 300 350 400 450 500 550 600 650m/z, Da
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
Intensity, cps
529.4
333.4212.3
184.2385.4
220.2182.0305.3291.4 640.4194.2 367.3239.2
-129
-273
In publication
• Metabolic Activation-mediated Toxicity
– Rutaecarpine (Rut) and Evodiamine(Ed)
– Pyrrolizidine alkaloids (PAs)
Our Progress
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
– Strychnine (Str)
• Metabolic Activation-mediated Drug Interactions
– noscapine
• Ginseng-drug interaction via the inhibition by intestinal
metabolites
Opium
O
O
OCH3
N
O
O
H3CO
OCH3
HH
Noscapine
Antitussive (Clinic)
Antitumor (phase I,II)
Noscapine-warfarin Interactions
�12 clinical cases of the interaction between noscapine and warfarin
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
�12 clinical cases of the interaction between noscapine and warfarin
International Normalized Ratio (凝血指数凝血指数凝血指数凝血指数): 3.0-7.2 (6 days later)
�11 increased INR+1 bleeding
�An 82-year-old male Warfarin+Noscapine (50 mg tid)
O O
OH
O
H
O O
OH
O
H
S-warfarin R-warfarin
CYP2C9
CYP1A2
CYP3A4
Aneja et al. (2007) Cancer Chemoth Pharm 60: 831-39.
Rosenborg et al. (2008) Br J of Clin Pharmacol 65: 277-78.
�Warfarin metabolism by CYPs
Effects of Noscapine on Human CYPs,
% Control Activity Positive % Control Activity
Remaining 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
CYP Isoforms Probe reactionsIC50 (µM)
�For CYP3A4: Competitive, Ki=5.2µM �For CYP2C9: Noncompetitive, Ki= 8.8µM
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Time- and NADPH-dependant Inhibition�For CYP3A4: IC50 shift 10-fold
10*decrease10*decrease
�For CYP2C9: IC50 shift 10-fold
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
KI=9.3µM
kinact=0.06 min-1
KI= 8.9µM
kinact= 0.014 min-1
Metabolic-activation of Noscapine
�A GSH conjugate was detected in HLMs
HLMs
-GSH
Conjugate
�Structural elucidation
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
-NADPH
�Bioactivation process
Fang et al. (2010) British Journal of Clinical Pharmacology 69(2): 193-199.
Prediction of noscapine and Warfarin Drug Interactions
Using Cmax:
AUC of (S)-warfarin: 1.5%
AUC of (R)-warfarin: 1.1%
Using Cmax,u:
Using Cmax:
AUC of (S)-warfarin: 110.9%
AUC of (R)-warfarin: 48.9%
Using Cmax,u:
Reversible inhibition: Time-dependent inhibition:
(S)-warfarin: CYP2C9
(R)-warfarin: CYP3A4, CYP1A2
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
max,u
AUC of (S)-warfarin: 0.5%
AUC of (R)-warfarin: 0.4%
max,u
AUC of (S)-warfarin: 41.8%
AUC of (R)-warfarin: 32.7%
Toxic
Effective
Time
Warfarin Concentration
in Plasm
a
Noscapine-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.
• Metabolic Activation-mediated Toxicity
– Rutaecarpine (Rut) and Evodiamine(Ed)
– Pyrrolizidine alkaloids (PAs)
Our Progress
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
– Strychnine (Str)
• Metabolic Activation-mediated Drug Interactions
– noscapine
• Ginseng-drug interaction via the inhibition by intestinal
metabolites
Ginsenosides Biotransformation by Intestinal Bacteria20(S)-protopanaxadiol type 20(S)-protopanaxatriol type
Rb1,Rb2,Rc Re
Rd
F2
C-K Ppd
Rh1
Ppt
Naturally
abundant
Gastro-
intestine
F1
Glc2-1Glc
Glc2-1Glc
Glc
HO
Glc6-1X
Glc
OH
OHGlc
HO
Glc
OH
Glc
HO
OH
HO
GlcRg2
OH
Glc2-1Rha
Rg1
Glc2-1Rha Glc-O
Glc
Rb3Glc2-1Glc
Glc-Xyl
MxGlc-Xyl
HO
HO
HO
HO
HO
HO
HO
20(S)-protopanaxadiol type 20(S)-protopanaxatriol type
Rb1,Rb2,Rc Re
Rd
F2
C-K Ppd
Rh1
Ppt
Naturally
abundant
Gastro-
intestine
F1
Glc2-1Glc
Glc2-1Glc
Glc
HO
Glc6-1X
Glc
OH
OHGlc
HO
Glc
OH
Glc
HO
OH
HO
GlcRg2
OH
Glc2-1Rha
Rg1
Glc2-1Rha Glc-O
Glc
Rb3Glc2-1Glc
Glc-Xyl
MxGlc-Xyl
HO
HO
HO
HO
HO
HO
HO
Akao et al. (1998). Biol. Pharm. Bull. 21, 245–249.Bae et al. (2000). Biol. Pharm. Bull. 23, 1481–1485.Hasegawa et al. (2002). Biol. Pharm. Bull. 25, 861–866.
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
46
12
0
5
10
15
20
25
30
35
40
45
50
1
No. of specim
ens
Positive 79%
Negative 21%
HO
OH
Glc-O
Glc-Glc6-O
OH
Glc-Glc6-O
Rb1 C-K
Hasegawa et al. (1997) Planta Med 63:436–440.
individual differences of hydrolysis in ability of Rb1
BloodEffect
BloodEffect
Hasegawa et al. (2002). Biol. Pharm. Bull. 25, 861–866.Hasegawa et al. (1997) Planta Med 63:436–440Hasegawa (2004). J. Pharmacol. Sci. 95, 153–157.Tawab et al. Drug Metab. Dispos. 31, 1065–1071.
Natural Occurring Ingredients of Natural Products
How about
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
Example: Ginsenosides
How about
Metabolites by
Intestinal Bacteria?
Possible Ginseng-drug Interactions via CYPs inhibition by
Ginsenoside Intestinal metabolites
Rb1 Rb2 Rc Rd Re Rg1Rg2 Rg3
S-
Rg3 Rh1 Rh2 F1
Naturally rare Naturally abundant
CKPpdPpt
Intestinal Bacterial Metabolites
CYP1A2
CYP2A6
CYP2C9
CYP2D6
CYP2E1
CYP3A4
Rb1 Rb2 Rc Rd Re Rg1Rg2 Rg3
S-
Rg3 Rh1 Rh2 F1
Naturally rare Naturally abundant
CKPpdPpt
Intestinal Bacterial Metabolites
CYP1A2
CYP2A6
CYP2C9
CYP2D6
CYP2E1
CYP3A4
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
100 50 10 1 μμμμMIC50 (μμμμM) 100 50 10 1 μμμμM100 50 10 1 μμμμMIC50 (μμμμM)
�Prediction of potential for DDI from In Vitro Data
CYP2C9 CYP3A4 Ginsenosides
CB,max
(µM )
CL,max
(µM ) Cmax/Ki Prediction
Cmax/Ki Prediction
Ppt 1.9 < 0.1 Remote >0.59~0.83
(0.47, rat) Possible
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
Role of Cytochrome P450 in Drug Metabolism
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
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.
�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
AcknowledgementDalian Institute of Chemical Physics, Chinese Academy of Sciences
China
�Outstanding Overseas Chinese Scientists (One Hundred Talent
Program) from the Chinese Academy of Sciences
�Innovation Fund of Chinese Academy of Sciences
Our GroupDalian Institute of Chemical Physics, Chinese Academy of Sciences
Thanks for your attention!
www.pharm.dicp.ac.cn
yling@dicp.ac.cn