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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


Natural Products

Totally Natural !!!

But
Natural products are generally either
Safe?
of prebiotic origin or originate from
microbes, plants, or animal sources


Mechanisms of Toxicity
Chemicals
Metabolism
About 80% Interaction with Metabolites Detoxicification
Dose-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 Toxicity
Type 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.


Drugs Withdrawn Associated With Idiosyncratic Toxicity or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
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.


Classification of Reactive Metabolites
Electrophiles (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.


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 metabolite

R1, R2=H, alkyl, phenyl acyl, O O
HO
acyloxy, 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.


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)

Trapping and characterizing Protein / DNA adducts
Immunoassays
Proteomics
Electrophoresis


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


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


Metabolic Activation of Evodiamine (Ed)
Five metabolites were detected after evodiamine was incubated with HLMs and PB-induced RLMs
Metabolic pathways Metabolic pathways

Structural elucidation of GSH conjugate of evodiamine

CYP1A2 or 2C9 GSH

Evodiamine

In publication


Metabolic Activation of Pyrrolizidine alkaloids (PAs)

R O
O OH OH
O O
P450
N N
DHP DHR
High activity and instability

1988 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 of
2002 MHRA proposed to prohibit the drug containing PA
liver; the liver venous congestion of the lungs
2004 WTO, Qianbai Rhinitis Tablet containing toxic plant Senecio, Genotoxicity: genetic combination, DNA cross-linking, DNA-
liver toxicity
protein cross-linking; carcinogenic, teratogenic
2005 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 ？


Metabolic pathway of PAs

Esterase, FMO, and UGT may play a detoxicification role via competitive
consumption 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.


Toxicity of Senecionine in Primary Human Hepatocytes

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.
He et al. (2010) Chemical Research in Toxicology 23 (3): 591-599.


Species Differences of Senecionine glucuronidation

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
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 hydrolase
He et al. DMD 2010, 38 (4): 626-34. Enterohepatic PAs
He et al. Chem Res Toxicol 2010, 23 (3): 591-9. circulation


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


Introduction of strychnine and brucine
H N H N
H H
OCH3

H H

N O N OCH3
O

O O

strychnine brucine

Clinical Effects: analgesic, anti-inﬂammatory, anti-tumor, anti-arthritis

Toxicity：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


The 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
]


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


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=529
Intensity, 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


Noscapine-warfarin Interactions
O

N
O Antitussive (Clinic)
H
H
OCH3
O
Antitumor (phase I,II)
O
OCH3
H3CO

Opium Noscapine
12 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 O

Warfarin 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.


Effects of Noscapine on Human CYPs,
% Control Activity Positive % Control Activity
CYP 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


Time- and NADPH-dependant Inhibition
For 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


Metabolic-activation of Noscapine

A 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.


Prediction of noscapine and Warfarin Drug Interactions
(S)-warfarin: CYP2C9
(R)-warfarin: CYP3A4, CYP1A2

Reversible 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

Time
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.


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
0
individual differences of hydrolysis
1

in ability of Rb1 Hasegawa et al. (1997) Planta Med 63:436–440.


Natural Occurring Ingredients of Natural Products

How about
Metabolites by
Intestinal Bacteria?

Example: Ginsenosides

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 CYP3A4
Ginsenosides
(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


Role 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.


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


Our Group

Thanks for your attention!
yling@dicp.ac.cn
www.pharm.dicp.ac.cn

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