bioactivation of herbal constituents: what is the difference?the case of aristolochic acids • in...

Bioactivation of Herbal Constituents:

What is the Difference?

Bo Wen, PhD

GlaxoSmithKline

Collegeville, Pennsylvania

Email: bo.1.wen@gsk.com

Outline

• Introduction

• Herb-based substructures associated with

bioactivation

• Summary

2

Reasons for Termination of Drug Candidates in Development

Wen B and Zhu M, Drug Metab Rev, 2015, 47:71-87.

The principal barrier to bringing a

continuous stream of innovation to

the market place is that converting a

chemical with interesting biological

properties into a drug involves

solving multiple complex issues

“simultaneously” in a single molecule.

The Challenge for Pharmaceutical Industry:

Solving the Multiple Issues Simultaneously

Non-toxic metabolites

Elimination/Excretion

Therapeuticallyactive metabolite

Drug

detoxification

detoxification

Elimination/Excretion

Excreted/repaireddetoxification

Signaling proteins,enzymes and transporters

Reactive metabolites

MacromolecularAdducts

bioactivation

Phase I

P450, PO (MPO, HRP), FMO, MAO, AO

Phase II

UGT, SULT, NAT, GST

GSH depletion

ROS

DNA, nucleic acid Autologous proteins

Teratogenicity Carcinogenicity Necrosis Apoptosis Hypersensitivity

Oxidative Stress

covalent binding

Circumstantial Evidence Links Reactive Metabolites to Adverse

Drug Reactions (ADRs)

Reactive Metabolites in Adverse Outcome Pathways

Mathieu V, Chem. Res. Toxicol. 2015, 28:1391-1397.

• Reactive metabolite-mediated protein alkylation and covalent protein binding in the liver are typically considered as

the molecular initiating event (MIE, blue) for liver fibrosis AOP. This serves as a trigger to provoke hepatocyte

injury, including apoptosis, which in turn activates Kupffer cells.

• As a result, transforming growth factor β1 (TGF-β1) expression is induced, which is a key factor for stellate cell

activation. The latter goes hand in hand with the occurrence of inflammation and oxidative stress.

• The overall end result is accumulation of collagen and changes in the extracellular matrix composition in the liver

(orange), which becomes clinically manifested as the adverse outcome, namely liver fibrosis (red).

Name Substructure Reactive species

Hydrazines, hydrazides R1

HN

HN R2 R1, R2 = alkyl or aromatic groups

diazonium ions, free radicals

Thiophenes, furans, pyrroles X = S, O, NH

X

epoxides, S-oxides (thiophenes),α, β-

unsaturated dicarbonyl (furans)

Anilines, anilides and precursors NH2

nitrosos, quinoneimines, free radicals

Quinones and precursors HO X X = NH2, OH, etc

quinones, quinoneimines, free radicals

Nitroaromatics NO2

nitroso intermediates, free radicals

Halogenated aromatics (Br > Cl > F) X X = Br, Cl, F

arene oxides

Thiazolidinediones

NH

SR

O

OR = alkyl or aromatic groups

isocyanates, isothiocyanates

3-Alkylindoles

NH

3-methyleneindolenines

Alkynes, acetylenes R1 C C R2 R1, R2 = H or alkyl groups oxirenes, ketenes

Arylacetic acids, arylpropionic acids COOH

n = 1, 2n

acyl glucuronides

Structural alerts: a starting point for testing and beyond

Wen B & Fitch W, Expert Opin Drug Metab Toxicol. 5:39-55 (2009)

Does “Natural” mean Safe?

1 Chalasani N et al., Gastroenterology. 2015, 148:1340-1352.2 Navarro VJ et al., Hepatology. 2017, :363-373.

Data from the US DILI Network

indicates HDS are the second

leading cause of DILI1

Use of herbal supplements is

increasing as is their percentage

of DILI cases2

Outline

• Introduction

• Herb-based substructures associated with

bioactivation

- aristolochic acids

- alkenylbenzenes

- indoloquinazoline alkaloids

- furano diterpenoids

• Summary

9

Aromatic amines and nitroaromatics

The case of aristolochic acids

10

• Balkan endemic nephropathy (BEN) is a unique, chronic renal disease often

associated with tubulointerstitial fibrosis and upper urothelial cancer (UUC) which was

first recognized in the late 1950s.

• Occurs exclusively in residents of specific farming villages in the Danube river basin

(Bosnia-Herzegovina, Croatia, Serbia, Macedonia, Bulgaria and Romania) where

~25,000 have the disease.

Aromatic amines and nitroaromatics

The case of aristolochic acids

11

• The plant Aristolochia clematitis (Birthwort) grew in local wheat fields as a weed and led to

contamination of the wheat grain during harvesting process.

• Similar nephropathy developed in rabbits after administration of Aristolochia seeds or fed with flour

containing Aristolochia seeds.

• It was proposed that contamination of the baking flour in endemic areas by seeds of the plant

Aristolochia clematitis was the cause of BEN (Ivic and Lovrie, 1967)

• However, Ivic’s work was neglected for many decades till 1992.

Ivic and Lovrie. Acta Medica Med 5:1-3, 1967

Aromatic amines and nitroaromatics

The case of aristolochic acids

• In 1992, an epidemic of Chinese herbs nephropathy (CHN), lately known as aristolochic

acid nephropathy (AAN), was first reported in Belgium in a cohort of young female

patients who take slimming pills containing Chinese herbs Aristolochia fangchi rich in AAs.

• High similarity of renal histopathology noticed between CHN/AAN and BEN.

Debelle FD et al, Kidney Int. 74:158-169, 2008

Association of AT→TA transversion mutations with

AA-DNA adducts and upper urothelial cancer (UUC)

• A specific AT→TA transversion mutation in codon 139 of exon 5 in the tumor suppressor gene TP53 is

associated with urothelial tumors and formation of the AA-DNA adducts in CHN/AAN patients (both

serve as diagnostic biomarkers of UUC).

• Mutation pattern in TP53 of UUC in the IARC database. A) AAN patients in United Kingdom; B) BEN

patients in Croatia, Bosnia and Serbia; C) AAN patients in Taiwan; D) UUC patients not associated with

AA exposure.

Stiborová M et al, Arch Toxicol. 90:2595-2615, 2016

Aristolochic acids in BEN, CHN, AAN

Debelle FD et al, Kidney Int. 74:158-169, 2008

Bioactivation and detoxication pathways of aristolochic

acid I (AA-I)

TP53 AT→TA

transversion mutations

Chinese herbs nephropathy (CHN)

Aristolochic acid nephropathy (AAN)

Balkan endemic nephropathy (BEN)

• Mice treated with dicoumarol,

an inhibitor of NQO1, exhibited

attenuated nephrotoxicity and

higher levels of the non-toxic

demethylated metabolite AA-Ia.

• Pretreatment of CYP1A

inducers in rodents suppressed

formation of DNA adducts and

protected animals from AA-

induced nephrotoxicity.

Chen M et al, Toxicol Sci 122:288-296, 2011.

Feng C et al, Environ Toxicol Pharmacol 36:850-857, 2013.

Mechanisms of aristolochic acid nephrotoxicity

Chang SY et al, JCI Insight. 2017

• AA is converted to AL-NOSO3H in human liver via nitroreduction by NQO1 and sulfation by SULTs,

followed by efflux into blood by MRP3/4 and active uptake into kidney cells by OAT transporters, where

AL-NOSO3H forms DNA and protein adducts leading to nephrotoxicity.

• Interplay of NQO1, CYP1A1/2, SULTs in AA metabolism and MRP3/4, OATs in transport of AL-NOSO3H

may explain the mechanism of AA-induced nephrotoxicity, and genetic polymorphisms in these enzymes

and transporters may be critical determinants on individuals’ susceptibility to AAN and BEN.

NO2

CF3

HN

O

NO2

CF3

HN

O

HO

NO2

CF3

N

O

OGSH

FLU-G3

GSH

FLU-G2NO2

CF3

N

O

Flutamide (FLU)

NO2

CF3

NH

O

NO2

CF3

HN

O

HO

NO2

CF3

N

O

GS

GS

GSH

NO2

CF3

NH2

HO

GS

FLU-G4

NO2

CF3

HN

O

OHGSH

NO2

CF3

HN

O

OH

SG

FLU-G1

NH2

CF3

HN

O

NH

CF3

N

O

FLU-G6

FLU-G5

NH

CF3

NH

FLU-G7

NH2

CF3

HN

O

NH

CF3

N

O

GSH

OH OH

GSH

NH2

CF3

NH2

GS

NH2

CF3

HN

O

GS

NH2

CF3

HN

O

OH

GS

FLU-6

GSH

Oxidative activation

Reductive activation

NO2

CF3

HN

O

2e

N

CF3

HN

O

O

2e

HN

CF3

HN

O

OHNH2

CF3

HN

O

CY P/ RedNADPH

CY P/ RedNADPH

NO2

CF3

HN

O

ee

O2

O2

2e

CYP/ RedNADPH

FLU FLU-6

Free radicals,redox cycling

Example of anilines/

masked anilines:

Flutamide

Wen B, et al. Chem. Res. Toxicol. 21:2393-2406, 2008

❖ a nonsteroidal

antiandrogen drug that is

widely used for the

treatment of prostate cancer

❖ Nitroaromatics associated

with idiosyncratic

hepatotoxicity in patients

* Rare but severe liver injury caused by flutamide in patients

Outline

• Introduction

• Herb-based substructures associated with

bioactivation

- aristolochic acids

- alkenylbenzenes

- indoloquinazoline alkaloids

- furano diterpenoids

• Summary

18

Alkenylbenzenes

A case of food-borne compounds

• A class of food-borne compounds naturally occurring in spices and herb medicines, including sassafras, nutmeg,

cinnamon, anise, fennel, basil, and tarragon.

• Increased incidents of hepatocellular adenoma and carcinoma in rodents; safrole, methyleugenol and estragole were

categorized as “possible human carcinogens” by IARC and “reasonably anticipated to be a human carcinogen” by NTP.

• Flavor and Extract Manufactures’ Association (FEMA) of the United States concluded that the present exposure to

methyleugenol and estragole from spices and other food additives does not pose a significant cancer risk.

Bioactivation of alkenylbenzenes

Bioactivation of benzylic and allylic alcohols

via sulfo-conjugation

• Benzylic alcohols of polyaromatic hydrocarbons (PAHs) undergo SULT-mediated sulfonation

to form the extremely reactive sulfuric acid esters and carbonium ions that can play a role as

ultimate carcinogenic and mutagenic metabolites.

Surh YJ, Chem Biol Interact 109:221-235, 1998

SAR of alkenylbenzenes bioactivation

• Methyl isoeugenol and isoeugenol possess the alkenyl double bond at the 1ʹ, 2ʹ- position, instead of the terminal

2ʹ, 3ʹ- position, and metabolism of these structural analogs leads to formation of noncarcinogenic 3ʹ-hydroxy

metabolites instead of the proximate carcinogenic 1ʹ-hydroxy metabolites.

• Eugenol possess a free phenolic hydroxyl group at the para-position to the side chain (instead of a methoxyl group

in methyleugenol), which is readily subject to rapid and efficient glucuronidation/sulfation and subsequently

excretion in urine.

nevadensin

Bioactivation and detoxification of alkenylbenzenes

The protective role of nevadensin in methyleugenol-

induced hepatocarcinogenicity in rats

• The basil flavonoid nevadensin, a SULT inhibitor, inhibited SULT-mediated bioactivation of methyleugenol reflected in

a reduction of DNA adduct formation and inhibition of hepatocellular altered foci (HAF) induction, an early marker of

hepatocarcinogenicity in rats.

Alhusainy et al, Food Chem Toxicol. 74:28-34, 2014

Indoloquinazoline alkaloids

The case of evodiamine and rutaecarpine

❖ Main active alkaloids of the herbal

medicine Evodia rutaecarpa (Wu-zhu-yu

in Chinese) for the treatment of

gastrointestinal disorders, headache, and

hypertension.

❖ However, immune-mediated toxicity

and acute toxicity were reported in mice.

❖ Mechanism-base inactivation of

CYP3A4 and CYP1A2.

Jeon TW, et al, Toxicol Lett 164:155-166 (2016)

CYP-dependent bioactivation of rutaecarpine

• A cell counting kit-8 cytotoxicity assay indicated that rutaecarpine (Rut) can decrease the primary rat hepatocyte viability,

increase lactate dehydrogenase and reactive oxygen species, reduce JC-1 (an indicator of mitochondrial membrane

potential, an early sign of apoptosis), and cause cell stress and membrane damage

Zhang FL, et al, Xenobiotica 45(11):978-89, 2015

Bioactivation of 3-alkylindole-containing evodiamine

Wen B, et al, Drug Metab. Dispos. 42:1044-1054, 2014.

Bioactivation of 3-alkylindole-containing evodiamine

Wen B, et al, Drug Metab. Dispos. 42:1044-1054, 2014.

❖ The 3-alkylindole moiety in evodiamine and rutaecarpine undergoes CYP–catalyzed dehydrogenation to form an

electrophile 3-methyleneindolenine.

❖ Evodiamine was a mechanism-based inactivator of CYP3A4, with KI = 29 μM and kinact = 0.029 min-1, respectively.

Pharmaceutical drugs containing 3-alkylindoles

Outline

• Introduction

• Herb-based substructures associated with

bioactivation

- aristolochic acids

- alkenylbenzenes

- indoloquinazoline alkaloids

- furano diterpenoids

• Summary

30

Germander-induced human liver injury

• Germander (Teucrium chamaedrys L.), a furano diterpenoid-containing herbal plant, has been used

as a folk medicine as a diuretic and antiseptic agent.

• In the early 1990s, promotion of slimming capsules/tablets containing germander powder led to an

epidemic of liver injury with over 30 cases of cytolytic hepatitis in France, including several fatalities

due to fulminant hepatic necrosis.

• Signs of immunoallergic type of hepatitis since a rechallenge caused early recurrence of the toxicity.

Castot and Larrey D, Gastroenterol Clin Biol. 16:916-22, 1992

Lancet, 1992, 340:674

Germander (Teucrium chamaedrys)

Reproducing hepatotoxicity of Germander and its major

furano diterpenoid teucrin A in mouse

Kouzi SA, et al, Chem Res Toxicol. 7:850-856, 1994

(Top) Hepatic midzonal necrosis in mice @ 24 h after the oral administration of teucrin A alone; (middle) almost no necrosis

after teucrin A and pretreatment with the P450 inhibitor PBO (piperonyl butoxide); (bottom) modestly enhanced hepatic

necrosis after pretreatment of PB (sodium phenobarbital).

Bioactivation of the furano diterpenoid teucrin A

NAC: N-acetyl cysteine; NAL: N-acetyl lysine

Non-toxic metabolites

Elimination/Excretion

Therapeuticallyactive metabolite

Drug

detoxification

detoxification

Elimination/Excretion

Excreted/repaireddetoxification

Signaling proteins,enzymes and transporters

Reactive metabolites

MacromolecularAdducts

bioactivation

Phase I

P450, PO (MPO, HRP), FMO, MAO, AO

Phase II

UGT, SULT, NAT, GST

GSH depletion

ROS

DNA, nucleic acid Autologous proteins

Teratogenicity Carcinogenicity Necrosis Apoptosis Hypersensitivity

Oxidative Stress

covalent binding

What about the signs of immunological type of hepatitis?

Laliberté L and Villeneuve JP. CMAJ. 1996, 154:1689-1692

Autoimmune hepatitis by pharmaceutical drugs

Cho and Uetrecht, Chem. Res. Toxicol. 2017, 30, 295−314

• Drug-induced autoimmune hepatitis: 1) formation of reactive metabolites by CYP enzyme; 2) covalent binding of reactive

metabolites on the CYP enzyme leading to formation of a neoantigen, a metabolite-modified self-protein; 3)

immunological response including production of antiCYP autoantibodies; 4) specific recognition of the CYP enzyme by

autoantibodies leading to immune destruction of hepatocytes and subsequent autoimmune hepatitis.

Teucrin A-induced autoimmune hepatitis in patients

• Microsomal samples were analyzed by SDS-PAGE and immunoblotting for expression of neoantigen (the teucrin A-

adducted hmEH) specifically recognized by the anti-hmEH autoantibodies in patient’s sera.

• Among the broad range of targets affected most by teucrin A treatment in rat liver were mitochondrial and endoplasmic

reticulum (ER) proteins which included several cytochrome P450 enzymes, UGTs and notably epoxide hydrolase which

converts the putative epoxide to non-toxic dihydrodiols.

Druckova A et al, Chem. Res. Toxicol. 2007, 20, 1393–1408.

De Berardinis V et al, Mol Pharmacol 2000, 58:542–551.

Teucrin A-induced autoimmune hepatitis in patients

• hmEH is the target of germander- or teucrin A-induced autoantibodies on the surface of human hepatocytes,

leading to immune destruction of hepatocytes and subsequent autoimmune hepatitis.

• No anti-CYP3A4 autoantibodies identified in patients although CYP-teucrin A adducts were formed in vitro

(challenge in immune-mediated IDR prediction?)

TA RM

De Berardinis V et al, Mol Pharmacol 2000, 58:542–551.

Summary

• The globally widespread use of herbal medicines comes with a substantial

increase in case reports of herbal-induced toxicity nowadays -- “Natural does not

mean Safe”.

• Like synthetic drugs, bioactivation of herbal constituents often represents a

molecular initiating event for herb-induced toxicity, including immune-mediated

toxicity.

• It is important to understand complexity of herbal constituents, daily exposure,

disposition, therapeutic regimen (acute vs chronic) and intended target

population. i.e. the methyleugenol/nevadensin case.

• It is imperative to use comprehensive in vitro and in vivo systems in the hazard

identification and risk assessment of herbal constituents. i.e. microphysiological

systems, untargeted metabolite profiling, and systems pharmacology.