4.ly korea

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 !!!


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


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Drugs Withdrawn Associated With Idiosyncratic Toxicity or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


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


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


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)


� 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


– 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


�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


�Structural elucidation of GSH conjugate of evodiamine

CYP1A2 or 2C9GSH

Evodiamine

In publication




Our Progress




– noscapine


metabolites

Metabolic Activation of Pyrrolizidine alkaloids (PAs)

P450

N

OO

O

OR

DHP DHR

N

OHOH

High activity and instability


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


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


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


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


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.




Our Progress




– noscapine


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）


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


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


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


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 [email protected] (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


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




Our Progress




– noscapine


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


�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


Time- and NADPH-dependant Inhibition�For CYP3A4: IC50 shift 10-fold

10*decrease10*decrease

�For CYP2C9: IC50 shift 10-fold


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


-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:



Using Cmax,u:

Reversible inhibition: Time-dependent inhibition:

(S)-warfarin: CYP2C9

(R)-warfarin: CYP3A4, CYP1A2


max,u



max,u



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.




Our Progress




– noscapine


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.


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


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


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


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

[email protected]

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