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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
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
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
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
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