metabolism of foreign comounds - univerzita … of foreign...drug metabolism = xenobiochemistry...
TRANSCRIPT
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