BIOCHEMISTRY

GENERAL MEDICINE

METABOLISM OF FOREIGNCOMPOUNDS

RNDr. Zdeněk DVOŘÁK, PhD.Department of Medical Chemistry and BiochemistryFaculty of Medicine, Palacky University Olomouc

DRUG METABOLISM = XENOBIOCHEMISTRY

Foreign compounds = XENOBIOTICS (lipophilic molecules)= drugs, alkaloids, pesticides, toxic industrial chemicals etc.

BiologicalDefence ofOrganism

ANTIOXIDANT SYSTEM• protection against oxygen radicals,oxidants, ionizing radiation

DETOXICATION SYSTEM• protection against chemicals

DETOXICATION = consecutive increase of molecule polarity to enhance its exctretion= A.D.M.E. (absorption-distribution-metabolism-elimination)= biotransformation

BIOTRANSFORMATION

Phase I.

• oxygenation• reduction• hydrolysis

Phase II.

• conjugation

Phase III.

• transport

Xenobioticdetoxication

activation

Hinderedoxygenation

Reactiveintermediates

GSH; proteinsSH-enzymes; DNA

Covalent bindingand toxicity(neoantigens,mutagens,carcinogenes)

DRUG ACCUMULATION

TOXICITY

CYP1; CYP2CYP3; CYP4

Therapeuticeffects

OXIDIZED METABOLITES

PhysiologicalPathological

GeneticEnvironmental

FACTORS

CONJUGATIONENZYMES

PROTEINS DNA

Cell damage Mutation

TOXICITY

INACTIVATION

ELIMINATION

DETOXICATION

BIOTRANSFORMATION: Phase I. („oxido-reductive“)

1. Oxygenation – hydroxylation (monooxygenases)

HR1

R3R2 OH

R1

R3R2

aliphatic

R1

R2

R1

R2

OH

aromatic

2. Oxidative demethylation (monooxygenases)

OMe OCH2OH OH

HH

O+

O-demethylation

CH2

NH

CH3RCH2

NH

CH2OHRCH2

NH2

RH

HO

+

N-demethylation

BIOTRANSFORMATION: Phase I. („oxido-reductive“)

3. Oxidation – (dehydrogenases)

CH3 CH2

OH CH3

H

OCH3

OH

O

NAD+ NADH + H+ NAD+ NADH + H+

H2O

4. Reduction – (dehydrogenases; reductases)

R1

R2O

R1

R2

H

OH

R1

R2

NO2 R1

R2

NH2

NAD+NADH + H+

5. Hydrolysis – (hydrolases; esterases; amide hydrola ses)

R1O

O

R2

R1NH

O

R2

R1O

O

H

R1O

O

H

R2 OH

R2 NH2

+

+

H2O

H2O

CYTOCHROME P-450: Ubiquitous monooxygenase

HEME PROTEIN - 1 protoporphyrin III. – Fe3+ coordinated to HS-Cys of apoenzyme- integral membrane protein (ER; outer mitochondria )

DETERMINATION – spectrophotometry of a complex with CO: Cyt P450-Fe2+-CO hastypical absorption at 450 nm

450 nmA

λλλλACTIVE SITE - 1. binding site for substrate

- 2. heme – binding and activation of O2

LOCALIZATION - LIVER (+ extrahepatic tissues – placenta; lungs; intestine etc.)- not present in serum, muscle, neurons, erythrocyte etc.

NUMBER - cca 150 enzymes (4 families); 17 representatives in man- classification according primary structure- present in man, animals, plants, fungi, bacteria

CYP1A1

cytochromeP450

family

Sub-family

individualenzyme

CYTOCHROME P-450: Ubiquitous monooxygenase

DIFFERENCES - substrate and reaction specifity- tissue and cell localization- mode of regulation

REGULATION - transcriptional – induction of gene expression (CYP1, 2B, 2C, 3A)- posttranslational (CYP2E1)- polymorphism (CYP2D6) – rapid vs. slow metabolizers- inhibitors, substrates

INDUCTION - increased de novo synthesis of Cyt P-450 in response to exposureto inducers (substrates)- potent inducers are often slowly metabolized substrates- PAH, PCB, rifampicin, phenobarbital, carbamazepine, midazolam…

P450sinduction

ACCELERATED DETOXICATION/METABOLISM• decreased drug effects; loss of therapeutic effects• compensation – increased doses of the drug

INCREASED SYNTHESIS OF REACTIVE METABOLITES• carcinogenesis (PAH – smokers – lungs; PCB - placenta)• immune toxicity; oxygen radicals

R-H + O-O + NADPH + H+ R-OH + H2O + NADP+

• cytochrome P450 mixed function oxidase (MFO)• microsomal drug-metabolizing system (MDMS)

Cytochrome P450 subfamilies

CYP1 CYP2 CYP3 CYP4 CYP11 CYP17 CYP19 CYP21

1A1 2A6 3A3 4A9 11A1 21A21A2 2A7 3A4 4A11 11B1

2B6 3A5 4B1 11B22C8 3A7 4F22C9 4F32C102C182C192D62E1

CYP Content Phenotypic substrates Inducers Inhibitors(pmol/mg) In vivo In vitro

CYP1A1 <0.5 ethoxyresorufin - TCDD, BNF, 3MC αααα-NF omeprazole,lansoprazole

CYP1A2 0.5-33.5 acetanilide caffeine TCDD, BNF, 3MC fura fyllinephenacetin omeprazole propranolol

lansoprazole fluvoxamineCYP2A6 45 coumarin coumarin phenobarbital, dithiocarbama te

dexamethasone pilocarpinerifampicin

CYP2B6 7 cyclophosphamide - phenobarbital orphenadrinerifampicin

CYP2C9 40-277 warfarin warfarin phenobarbital sulfaphena zolerifampicin

CYP2C19 - (S)-mephenytoin (S)-mephenytoin phenobarbital omeprazolerifampicin

CYP2D6 10 debrisoquine dextromethorphan - quinidineCYP2E1 <5-50 chlorzoxazone chlorzoxazone alcohols diethy ldithio-

organic solvents carbamateCYP3A4 81-360 cyclosporin A erythromycin rifampicin trole andomycin

nifedipine cortisol phenobarbital ketokonazoleomeprazole gestodenelansoprazoledexamethasoneothers.

Average content, phenotypic substrates, specific induc ers and inhibitors of human CYP enzymes

BIOTRANSFORMATION: Phase II. („conjugation“)

METABOLITE (phase I.)(chemical)

+ CONJUGATINGREAGENT CONJUGATE

CONJUGATING REAGENT:• endogenous polar compound• product of cellular metabolism• glucuronic acid = glucuronidation• sulphuric acid = sulphatation• glutathione• amino acids = e.g. Hippuric acid• acetyl CoA = acetylation• S-adenosylmethionine = methylation

CONJUGATES:• higly polar, mostly ionized compounds• e.g. Glucuronide; suphate; mercaptouricacid;acylaminoacid; acetylderivative etc.• inactivation by exctretion(urine Mw<400; bile Mw > 400

ENERGY FOR CONJUGATION:• ATP (amino acid)• conjugating reagent – activated donor of conjugating grou p

Conjugation with GLUCURONIC ACID

SUBSTRATES: alcohols; phenols; amines; thiols; carbo xylic acid

DONOR: UDP-GA = Uridine- DiPhospho- Glucuronic- Acid

O

OH

OH

OH

COOH

O P P O CH2

N

NH

O

OHOH

O

O

ENZYME: UDP-glucuronyl transferase

PRODUCTS: O-glucuronidesN-glucuronides

R OH

R NH2

+ UDP-GA UDP+

R NH GA

R O GA

Conjugation with SULPHURIC ACID

SUBSTRATES: alcohols; phenols; arylamines

DONOR: PAPS = 3-Phospo Adenosine-5´- Phospho Sulphate

ENZYME: sulphotransferases; cytosolic

PRODUCTS: sulphates

R OH + PAPS PAP(phosphoadenosinephosphate)+R O SO3

OPOSO3

N

N

N

N

NH2

O

O OH

P

CH2

Conjugation with GLUTATHIONE

SUBSTRATES: aromatic hydrocarbons and heterocycles; hal ogen derivatives;epoxides; etc.

DONOR: GSH = γγγγ-glutamyl-cysteinyl-glycine; tripeptide

ENZYMES: 1. GSH-S-transferases; cytosolic; conjugate remains in cell2. γγγγ-glutamyltranspeptidase; membrane-plasma3. acetyltransferases; cytosolic

PRODUCTS: mercapturic acids OOH

SG

OH

S CH2

CH

NH2

COOH

OH

S CH2

CH

NH

COOH

CH3

O

GSH

epoxide conjugategly-glu

mercapturic acid

AcCoA

CoA-SH

Conjugation with AMINO ACIDS

SUBSTRATES: carboxylic acids

DONOR: glycine or glutamine

ENZYMES: 1. glycine-N-acyltransferase; mitochondria2. glutamine-N-acyltranferase; mitochondria

PRODUCTS: N-acylglycine; N-acylglutamine

COOH

O O

SCoA

O O

NH

CH2

COOH

NH2 CH2

COOH

ATP CoA-SH+

benzoic acid glycine hippuric acid

• first metabolite discovered in human urine (originally in horse urine)• control of toluene abuse

BIOTRANSFORMATION: Phase III. („transport“)

Philosophical question: Are these enzymes really phase III, since they determinewhether drug will get in contact with phase I and phase II?

• important enzymes that control import/export of the drug from the cells• regulation of uptake/efflux• membrane proteins• new approaches to drug design and discovery

• PGP = p-glycoprotein – pumps out drugs from the cells• OATP = organic anions transporting protein – regulates uptake of anionic drugs• OCTP = organic canions transporting protein – regulates uptake of canionic drugs

METABOLISM OF AMPHETAMINE

CH2

CH

CH3

NH2

amphetamine

CH2

CH

CH3

NH2OH

CH2

CH

CH3

NH

OH

CH2

CH3

N

OH

CH2

CH3

NH2

OH

CH CH

CH3

NH2

OH

CH CH

CH3

NH2OH

OH

conjugates

CH2

CH3

NH

CH2

CH3

O glucuronideCH2

CH

CH3

OH

COOH

CONHCH2COOH

CH

CH3

O SO3H

METABOLISM OF ETHANOL

• 90% degraded in the LIVER

CH3 CH2

OH CH3 CH

O CH3 O

O

ethanol acetaldehyde acetate

aldehyde dehydrogenase(cytosol; mitochondria)

NAD+ NADH + H+

alcohol dehydrogenase(cytosol)

NAD+ NADH + H+

NADP+NADPH

CYP2E1; MEOS; Endopl. Ret.

catalase; peroxisomes

H2O2H2O AcetylSCoATCA cycle

Toxic effects of ethanol:• disturbance of liver cell metabolism (NAD+ ----- NADH)• high reactivity of acetaldehyde – adducts with macromolecules• induction of microsomal system (chronic uptake) – radical production, activation of carcinogenes

PERTURBATION OF METABOLISM BY ETHANOL

CH3 CH2

OH CH3 O

O

ethanol acetate

ADH; ALDH

2 x NAD+ 2 x NADH + 2 x H +

dihydroxyacetonephosphate

glycerol3-phosphate

TAG synthesis

Fatty acids

Not availablefor ββββ-oxidation

PYRUVATELACTATE

storage in liverSTEATOSIS

LACTATE ACIDOSIS

HYPERURICEMIA

Not available forgluconeogenesis

HYPOGLYCEMIA

Oxal acetate acetylSCoA

KETO ACIDOSIS

TOXIC EFFECTS OF ACETALDEHYDE

ACETALDEHYDE HS-protein+ ACETALDEHYDE-S-protein

ADDUCT FORMATION• with microtubules – decrease in secretion of plasma proteins = accumulationof proteins in liver = liver enlargement = HEPATOMEGALY

• with some proteins – adducts = antigen properties = immune response = cytokinesALCOHOLIC HEPATITIS

• with ornitine decarboxylase – decrease in enzyme activity = diminution of cellproliferation = RETARDED REGENERATION OF DAMAGED LIVER

• with GSH – decrease in GSH level = increased lipid peroxidation of hepatocytemembranes = CELL DEATH

Transcriptional level – increase of collagen synthesis = FIBROSIS - CIRRHOSIS

METABOLISM OF POLYAROMATIC HYDROCARBONSbenzpyrene

O

arene epoxideNAPD+NADPH + H+

P450s

OH

OH

H2O

arene diol

OHphenol

UDPGAPAPS

UDPGAPAPS

CONJUGATES - glucuronides- sulphates

excretion

OH

OHO

NAPD+

NADPH + H+

P450s

arene diol epoxide

GSH

GS

OH

glutathione conjugateGSH

mercapturicacid

excretion

• reactive intermediates – covalent binding to DNA (guanin e) and proteins (-SH) –strong carcinogenes

Regulation of Drug Metabolizing Enzymes

• transcriptional regulation – controlled by „xenoreceptors“

• Aryl Hydrocarbon Receptor (AhR) = dioxin receptor; first discovered xenoreceptor

• orphan receptors – they do not have natural endogenous ligandPXR – pregnane X receptorCAR – constitutive androstane receptor

• other receptors – functional and transcriptional cross-talk („tangle of networks“)VDR – vitamin D receptorRARs – retinoic acid receptors (ligand all-trans RA); forms α, β, γRXR – retinoic X receptor (ligand cis-RA); forms a, b, gGR – glucocorticoid receptorothers (e.g. FXR, LXR, SHP etc.)

• compounds which activate xenoreceptors (agonists; ligands) induce expressionof drug metabolizing enzymes phase I, II and III. - INDUCERS

• wide spectrum of structurally unrelated compounds – alkaloids, drugs, antibiotics,environmental polutants, cigarette smoke, food constituents etc.

DRUG INTERACTIONS• pharmacodynamic are predictable whereas pharmacokinetic are not

• P450 INDUCTION – increase in enzyme number; accelerated metabolism• P450 INHIBITION – decrease in enzyme activity; retardation of metabolism

• EXAMPLE 1 : cyclosporin A + ketoconazole = immune supressive cyclosporine Ais metabolized by CYP3A4. Antifungicide ketoconazole is strong inhibitor ofCYP3A4. When the two drugs are administered together, i.e. in transplant patients,cyclosporine metabolism is reduced, and patient suffer/die due to cyclosporinenephrotoxicity. Solution: decrease the dose of cyclosporine A.

• EXAMPLE 2 : cyclosporin A + rifampicin = immune supressive cyclosporine Ais metabolized by CYP3A4. Antibiotic rifampicin is strong inducer of CYP3A4.When the two drugs are administered together, cyclosporine metabolism isaccelerated, and patient suffer/die due to organ rejection (not enough CsA).Solution: increase the dose of cyclosporine A.

• EXAMPLE 3 : Drinking too much of alcohol = induction of CYP2E1. The day after,taking a pill of paracetamol. Paracetamol is converted by CYP2E1 to hepatotoxicquinone. Heeling hang-over with paracetamol increased the risk of liver damage!!!

• EXAMPLE 4 : Cigarette smoke contains PAHs - PAHs induce CYP1A1/2 in lung-CYP1A1/2 converts PAHs to carcinogenes – lung cancer!!! (similarly grilled meatcontains PAHs that induce intestinal CYP1A1/2 – conversion to carcinogenes –colorectal cancer!!!

Regulation of Drug Metabolizing Enzymes

GR

CARPXR

CYP2A6 CYP2C8

CYP2B6 CYP2C9

AhR

CYP1A1 CYP1A2

CYP1B1

CYP3A4 CYP2C19

N-AcT

Phase II Phase III

Signaling by AhR

DREDREDREDRE

AhRhsp90

hsp90p23

XAP2L

L

L

L

L

L LL

LL

AhRhsp90

hsp90p23

XAP2

L

hsp90

hsp90p23

XAP2

AhRL

ARNT

CYP1CYP1CYP1CYP1A1AhR

LARNT