Metabolites of

Xenobiotics : Properties and Significance

1. Introduction: Definitions and Enzymes

2. Physicochemical and Pharmacokinetic Properties

3. Pharmacological Properties

4. Toxicological Relevance

Biological System Drug

PharmacoDynamic effects

PharmacoKinetic effects A, D, E

Act + Tox

Metabolite

Act + Tox

M A, D, M, E

1. Introduction: Definitions and Enzymes

Absorption Distribution Metabolism Excretion

What are Xenobiotics?

Foreign compounds without physiological functions :

• Drugs (therapeutic and diagnostic agents)

• Food constituents devoid of physiological function

• Food additives (preservatives, flavors, coloring agents ...)

• Cosmetics

• Doping agents (EPO, anabolic steroids, growth hormone, ...)

• Hallucinogens (ecstasy, LSD, THC, cocaine,...)

• "Social stimulants" (nicotine, alcohol, caffeine, …)

• Natural toxins (animal venoms, plants and bacterial toxins)

• Innumerable technical and industrial compounds (insecticides, herbicides, fertilizers, plasticizers, ...)

• Environmental natural pollutants produced by vulcanos, etc

• Environmental synthetic pollutants (metals, insecticides ...)

Therapeutic effects

Unwanted effects; Toxicity

Concentration in relevant

compartments

Oral administration

Dissolution

Intestinal excretion

Liver metabolism

Extrahepatic metabolism

Tissue binding

General circulation

Protein binding

Excretion in urine, air, sweat, etc

Biliary excretion

active GI absorption

Passive and

active distribution into tissues

Passive and

What the body does to xenobiotics

GI metabolism

Chemical entities being transferred to or from the substrates Functionalizations

(Phase I) Conjugations (Phase II)

Redox reactions Hydrolyses

O H2O

O2 HO–

e–

2 e–

H– (hydride)

Methyl group

Sulfate and phosphate moieties

Glucuronic acid and some sugars Acetyl and other acyl groups

Following conjugation

with Coenzyme A

Glycine and other amino acids Diglycerides

Cholesterol and other sterols Chiral inversion

β-Oxidation Elongation by two-carbon units

Glutathione

Acetaldehyde, pyruvic acid, other carbonyl compounds, CO2

The Multiplicity of Enzymes and Reactions

Oxidoreductases (EC 1)

playing a role in drug metabolism

• Alcohol dehydrogenases (EC 1.1.1.1, ADH; EC 1.1.1.2, AKR1A1) • Aldehyde dehydrogenases (EC 1.2.1.3, -5, ALDH) • Aldo-keto reductases (EC 1.1.1.50, -213, etc, AKR1) • Dihydrodiol dehydrogenases (EC 1.3.1.20, DHDH; AKR1C)

• CYTOCHROMES P450 (EC 1.14.13 and 1.14.14.1, CYP)

• Flavin-containing monooxygenases (EC 1.14.13.8, FMO) • Dopamine β-monooxygenase (EC 1.14.17.1, DBH)

• Myeloperoxidases (EC 1.11.1.7, MPO) and other peroxidases (EC 1.11.1) • Prostaglandin-endoperoxide synthase (EC 1.14.99.1, PTGS) • Quinone oxidoreductases (EC 1.6.5.2, 1.10.99.2, NQO)

• Monoamine oxidases (EC 1.4.3.4, MAO) • Copper-containing amine oxidases (EC 1.4.3.6, AOC)

• Aldehyde oxidase (EC 1.2.3.1, AOX1) • Xanthine dehydrogenase/oxidase (EC 1.17.1.4, -3.2, XDH)

A phylogenetic tree of

57 human CYP genes

and 3 pseudogenes

2C8 2C19 2E1 2B6 2A7 2G1P 2S1 2D6 2R1 2W1 1A2 17A1 3A4 3A5 5A1 4A22 4Z1/4A20 4F2 4F11 4F8 4V2 26A1 26C1 11B1 27A1 27C1 19 8A1 7B1 39

Family 2

Family 1

Family 3

2C9 2C18 2F1 2A6 2A13 2G2P 2J2 2T2P 2U1 1A1 1B1 21 3A7 3A43 4A11 4B1 4X1 4F3 4F12 4F22 46 26B1 11A1 11B2 27B1 24 20 8B1 7A1 51

Hydrolases (EC 3) playing a role in drug metabolism

Of major importance Amidases Carboxylesterases (CES) Endopeptidases Exopeptidases Of lesser importance Aryl esterases Cholinesterases Phosphoric ester hydrolases Sterol esterases Sulfuric ester hydrolases Triacylglycerol lipases Tropinesterase Thiolester hydrolases

CES1 CES2 CES3 CES5 CES6 CES7 CES8

Enzyme nomenclature: www.chem.qmul.ac.uk/iubmb/

Aminopeptidases Dipeptidases

Serine-type carboxypeptidases Metallocarboxypeptidases

Cysteine-type carboxypeptidases Omega peptidases

Serine endopeptidases Cysteine endopeptidases Aspartic endopeptidases Metalloendopeptidases

Transferases (EC 2) and Ligases (EC 6)

playing a role in drug metabolism

• UDP-GLUCURONOSYLTRANSFERASES (EC 2.4.1.17, UGT)

• Glutathione S-transferases (EC 2.5.1.18, GST)

• Sulfotransferases: Alcohol SULT (EC 2.8.2.2, SULT2B1; -14, SULT2A1)

Steroid SULT (EC 2.8.2.15)

Phenol SULT (EC 2.8.2.1, SULT1A1, 1A2, 1A3)

Estrogen SULT (EC 2.8.2.4, SULT1E1)

Amine SULT (EC 2.8.2.3, SULT3A1)

• N-Acetyltransferases (EC 2.3.1.5, NAT1, NAT2)

• Methyltransferases: N-MT (EC 2.1.1.1, NNMT; -8, HNMT; -28, PNMT)

O-MT (EC 2.11.1.6, COMT)

S-MT (EC 2.1.1.9, TMT)

• Acyl-CoA synthetases (EC 6.2.1.1, ACSS; -2, ACSM; -3, ACSL)

• Phosphotransferases (EC 2.7)

A phylogenetic tree of human UGT families 1 and 2

UGT1A1

UGT1A3

UGT1A5

UGT1A4

UGT1A6

UGT1A7

UGT1A8

UGT1A9

UGT2A1

UGT1A10

UGT2A2

UGT2B4

UGT2B7

UGT2B11

UGT2B28

UGT2B10

UGT2B15

UGT2B17

Family 1

Family 2

The structure of human UGT2B7 with bound morphine and UDPGA

O NH2

OH

O NH

OH

OH

O NH2

OH

OH

O

OH

CHO

O OH

OH

O

OH

COOH

O

OH

COOH

OH

O OH

OH

OH

O COOH

O NH

OH

HO

NH2

O NH

OH

GLUC

SULF

GLUC

GLUC

GLUC

GLUC

SULFGLUC

SULF

GLUC

GLUC

SULF

OCH3

A metabolism scheme: The example of propranolol

Distribution of metabolites according to generations (Testa et al., Drug Discovery Today 17, 549, 2012)

Distribution of metabolites according to enzyme (super)families

CYPs (40.0%)

Dehydrogenases (7.98%)

UGTs (13.6%)

Non-enz. hydrol (2.73%)

Hydrolases (7.02%)

Peroxidases (2.05%) FMOs (2.21%)

Other reductases (3.69%)

SULTs (4.68%)

GSTs (5.54%)

NATs (1.46%) Other transferases or non-enz. conj. (2.99%)

Acyl-CoA ligases (1.75%) MeTs (2.14%)

2. Physicochemical and Pharmacokinetic

Properties

Examples of decreased lipophilicity due to Phase I metabolism

∆logP metab – xeno

∆logP metab – xeno = ?

2. Physicochemical and Pharmacokinetic

Properties

Examples of decreased lipophilicity due to Phase I metabolism

(∆logP metab – xeno)

R CH3 R CH2OH R CHO

CYP

R COO¯ (ca. -1.7)

(ca. -1.6)

R COOH

(ca. -5.5)

ADH ALDH

R N

CH3

CH3

R N

H

CH3

R N

H

H

H2C=O

CYP

H2C=O

CYP

(ca. -1.9) (ca. -1.6)

NR

NR

OFMO

CYP

(ca. -1.7)

Caron et al., Pharm Res 1996

R R OH R OH

OH

(ca. -0.5)

CYP CYP

(ca. -0.4)

R CH2 N

CH3

CH3

R CH2 N

CH3

CH3

OFMO

CYP Caron et al., Helv Chim Acta 1999

(ca. -2.6)

SR'R

SR'R

O

SR'R

OO

FMO

CYP CYP

(ca. -2.2)

Caron et al., Helv Chim Acta 1999

(ca. -2.2)

What about Phase II metabolites?

R OH

OH

R OH

O CH3

COMT (ca. +0.6)

R OO

HOH

OH OH

COOH

R OHUGT

(ca. -2.0)

Giroud et al., Helv Chim Acta 1998

SO

O

O

R OR OH ¯

(ca. -5 ??)

SULT

CoA- Ligases

O

CH3

H

H

H

CH3

CH3

CH3

CH3

R

O

R COOH

CH2O

C HO

CH2 O P

O

O

O (CH2)2N(CH3)3

RO

CH3(CH2)14

O

CH2O

C HO

CH2 O

RO

CH3(CH2)14

O(CH2)14CH3

O

Highly lipophilic conjugates

Acyl-T Acyl-T

Acyl-T

RS CoA

O

¯

Phospholipid conjugates

Mixed triglycerides

Cholesteryl esters

Passive (‘‘down-a-concentration-gradient’’) membrane permeation passive permeation out of cells passive permeation into cells passive accumulation in tissues

Active, carrier-mediated membrane permeation active uptake into cells and tissues (uptake transporters), or active efflux (excretion) from cells (efflux pumps)

Impact on the distribution and excretion of metabolites:

A schematic view

Carrier-mediated transport – a summary of transporters (Giacomini et al., Nature Reviews Drug Discovery 9, 215, 2010)

Passive (‘‘down-a-concentration-gradient’’) membrane permeation passive permeation out of cells passive permeation into cells passive accumulation in tissues

Active, carrier-mediated membrane permeation active uptake into cells and tissues (uptake transporters), or active efflux (excretion) from cells (efflux pumps)

Impact on the distribution and excretion of metabolites:

A schematic view

Metabolites Passive

permeation

Accumulation

in tissues Active uptake Active efflux

Metabolites with modest

decrease in lipophilicity X X X

Metabolites with marked

decrease in lipophilicity X X X

Polar conjugates X X

Highly lipophilic conjugates X

Enterohepatic cycling of exogenous estradiol in post-menopausal women

(Vree & Timmer, Journal of Pharmacy and Pharmacology 50, 857, 1998)

0 5 10 15 20

300

30

100

10

3

1

t1/2 ~ 2h

1st 2nd Time (h)

Serum conc (ng/L) Estrone

Estradiol

Conditions for enterohepatic cycling of glucuronides: • excretable in bile (MW beyond a species-dependent threshold) • hydrolyzable by intestinal glucuronidases • absorbable aglycone

OH

H H

CH3OH

H

OH

H H

CH3

H

O

3. Pharmacological Properties

Drug

Metabolites

Detrimental

effects

Beneficial

effects

Pharmacological scenarios

Drug Metabolite

Active Active

Inactive Inactive X • Drugs having no active metabolite, e.g. Soft drugs Oxazepam

• Drugs having one or more active metabolite(s), e.g. Codeine ⇒ morphine Diazepam ⇒ nordiazepam Tramadol ⇒ O-desmethyltramadol

• Drugs having highly active metabolites, e.g. Cis-platine ⇒ monoaqua and diaqua species Encainide ⇒ O-desmethyl-encainide and 3-methoxy-O-desmethyl encainide Tamoxifen ⇒ 4-hydroxytamoxifen and endoxifen

• Prodrugs

Definitions

• Biologically active compounds (drugs) characterized by a

predictable in vivo metabolism to inactive and non-toxic

moieties, after they have achieved their therapeutic role.

Objectives:

• Short duration of action

• Absence of long-acting toxic residues.

Soft drugs (Bodor & Buchwald, Medicinal Research Reviews 20, 58, 2000)

Esmolol, an ultra-short

acting β-blocking soft drug

t1/2 in human blood

(37oC, pH 7.4) = 23 min

OOH

NH

CH3

CH3

O

O

CH3

OOH

NH

CH3

CH3

O

O

–

The example of Tramadol • μ-receptor affinity: (+) > (–) • other opiate receptors: weak affinities • inhibition of 5HT reuptake: (+) > (–) • inhibition of NA reuptake: (–) > (+)

O-Desmethyltramadol • μ-receptor affinity: (+) > (–) > (+)-tramadol

Analgesia in patients • results from non-opioid and opioid components • (+) ≈ (±) > (–) • weaker in CYP2D6- deficient patients.

OH

H

O

(CH3)2N

CH3

OH

H

OH

(CH3)2N

(–)-(1S,2S)- Tramadol

CYP2D6

(1S,2S)-DMT

OH

H N(CH3)2

O CH3

OH

H N(CH3)2

OH

(+)-(1R,2R)- Tramadol

(1R,2R)-DMT

Drugs having one or more active metabolite(s)

CYP2D6

CYP2D6

CYP3A

CH3

ONH

CH3

OH

CYP2D6

(2C9, 2C19, 3A)

Drugs having highly active metabolites

Tamoxifen

CYP3A4/5 (1A2, 2C9, 2C19, 2D6)

Endoxifen

4-OH-Tam

Activity in patients • appears mainly due to endoxifen.

N-des-Me -Tam

The example of Tamoxifen • 4-OH-Tam and Endoxifen are 30-100 times more active than Tamoxifen. • In chronic patients, plasma levels of Tamoxifen are 30-100 times higher than those of 4-OH-Tam and 5-10 times higher than those of Endoxifen.

CH3

ON

CH3

CH3

CH3

ON

CH3

CH3

OH

CH3

ONH

CH3

Pharmaceutical objectives • Improved solubility • Improved chemical stability • Improved taste, odor • Decreased irritation and pain

Pharmacokinetic objectives • Improved oral absorption • Decreased presystemic metabolism • Improved absorption by non-oral routes • Improved time profile (often increased duration of action) • Organ/tissue-selective delivery of an active agent

Pharmacodynamic objectives • Improved therapeutic index • Activation to a reactive agent

improved acceptability } {

improved bioavailability } {

The case of produgs

Derivatives or analogs with no activity, which undergo biotransformation

to a therapeutically active metabolite (Albert, Nature 182, 421, 1958).

Zanamivir (A highly hydrophilic drug

administered in aerosol form)

OOH

O

OH

OH

OHH

NH

NH

NH2

NH

O

Oseltamivir (An orally available prodrug

whose rapid in vivo hydrolysis yields the active drug)

OCH2CH3

O

NH2

NH

O

O

OH

O

NH2O

O

NH

Oseltaminir as example of

a carrier-linked prodrug (carrier = promoiety)

Olsetamivir acid (active metabolite)

(promoiety)

N

R'S

R

R = -COOCH3

R' = -Cl

N

R'SO

OCH3

R

R = -CO

R' = -F

N

R'SO

R

N

R'SO

R

O

N

R'

R

S

HOOC

OH

N

R'SO

R

N

R'

R

SH

HOOC

N

R'

R

SH

HOOC

+ –

Clopidogrel Prasugrel

Clopidogrel and prasugrel as examples of bioprecursors (Dansette et al., Chem Res Toxicol 25, 348, 2012)

CYP (minor route)

CYP

H2O

CES (major route)

PON

GSH reactive sulfenic

acid

Mechanism of action: Irreversible antagonism of platelet ADP receptors by formation of a covalent S-S bridge

PON

Types Mechanism(s) of Adverse Drug Reactions (ADRs)

1) On-target ADRs

Predictable in principle and generally dose-dependent. Based on the pharmacology of the drug and its metabolite(s), often an exaggerated response or a response in a non-target tissue.

2) Off-target ADRs

Predictable in principle and generally dose-dependent. Resulting from the interaction of the drug or a metabolite with a non-intended target.

3) ADRs involving reactive

metabolites

Predictable in principle and generally dose-dependent. A major mechanism is covalent binding to macromolecules (adduct formation) resulting in cytotoxic responses, DNA damage, or hypersensitivity and immunological reactions. A distinct (and synergetic) mechanism is the formation of reactive oxygen species (ROS) and oxidative stress.

4) Idiosyncratic drug reactions

(IDRs)

Unpredictable, apparently dose-independent, and rare (< 1 case in 5000). They might result from a combination of genetic and external factors, but their nature is poorly understood. IDRs include anaphylaxis, blood dyscrasias, hepatotoxicity, and skin reactions.

4. Toxicological Relevance

UGT

SULT

GST

Drugs or chemicals

Adducts Proteins, DNA

NQO, etc

CYP, PER,

PGHS

Free radicals

O2 O2• - Oxidative

stress

Electrophiles

Nucleophiles

GSH conjugates

Glucuronides Sulfoconjugates

Quenching GSH

O2

Reductases

Detoxification GSH, GST, Catalase, etc

SOD

Proximal mechanisms of metabolic toxification and detoxification

Acyl-Coa ligases; Glycine N-acyltransferases CYP 2E1 Deshydrogenases

CH3

CH2OH

CHO

COOH

NH

O COOH

Fast metabolism (ca. 80% of dose) to inert metabolites

The compared toxicities of toluene and benzene

• The acute toxicity of benzene and toluene is comparable and due to their solvent properties. • The chronic toxicity of the two solvents is vastly different due to their different metabolism.

EH

CYP

2E1

Slow metabolism to highly toxic metabolites (leukemia!)

O

OHHOH

H

OH

OHOH

OH

OH

OO

O

O

OH

OH

GS

OHOH

GS

CYP

2E1

DD

PER

PER

CYP

CYP

The case of benzene

GST

GST

Adducts

Adducts

Why leukemia ? Bone marrow is rich in myeloperoxidases !

OH NH

NH

O

NH2

O SH

OOH

O

O

O

OH NH

NH

O

NH2

O S

OOH

O

Detoxification versus toxification : The double role of Glutathione (GSH)

SGOH

OH

SNH

OH

O

CH3

O OH

OH

S

O

OR

etc.

a mercapturic acid

Glu Cys Gly

NH-COCH3

OH

The case of paracetamol

NH-COCH3

O-Glucuronide

NH-COCH3

O-Sulfate

NH-COCH3

OHNu

NH-COCH3

OHS-Glutathione

NH-COCH3

OHS

NH-COCH3

COOHN-acetyl-para- quinoneimide

O

N-COCH3

CYP ca. 5% major (ca. 60%) minor (ca. 30%)

+

Endogenous nucleophiles

Necrosis Hepatic failure

Death Adducts

OO

O

OHOH

OHGS

OHOH

OOH

OOH

GS

OHSG

O

OHSG

OHOH

O

OHOH

OHOH

The case of 1,3-butadiene

EH

GST

CYP

GST

EH

EH ADH

CYP EH

GST

GST

CYP

CYP

OHSG

OHGS

GST

01. Csp3 oxidation (6.3%)

02. Csp2–Csp oxidation (15.0%)

03. -CHOH ↔ >C=O or >C=O → -COOH

(2.5%)

05a. >NH, >NOH oxid (8.2%)

14. Other hydrol (3.8%)

07a. S oxidat (9.5%)

06a. Oxidation to quinones (41%)

23. Sulfon (3.8%)

24. GSH conj (2.7%)

Which reactions formed toxic or reactive metabolites?

Xenobiotic

toxification

Inert metabolite detoxification

Inert metabolite detoxification detoxification

Inert oxygen

Reactions with Biosystems

O2

Activated oxygen Toxicant

Cellular damage

Toxic effects

Healing

Death

repair Removal of lesion Chemical lesion

(critical)

(protective)

The interplay of toxification and detoxification

Vol. 1, 2008, 320 pages Vol. 2, 2010, 590 pages

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