EXCRETION

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METABOLISM
EXCRETION
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DRUG EXCRETION RENAL
distal tubule (passive reabsorption from more
acid environment)
glomerulus (filtration)
proximal tubule (active secretion passive
reabsorption)
loop of Henle
collecting duct
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All processes to occur in one circulation-
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GLOMERULAR FILTRATION
Not if bound to protein

• passive process.
• pressure and flow-dependent
• only free drug filtered, protein (albumin
  bound drugs are retained in the bloodstream
  because protein size exceeds the 50Ao
  interstitial pore size. (kidney damage allows
  protein into the urine!).

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TUBULAR SECRETION

• Occurs ONLY in the proximal tubule.
• An active process requires energy and occur
  against a concentration gradient.
• Separate transport systems exist for
• Organic anions (acidic) molecules (mid section
  of the proximal tubule).
• Organic cations (basic) molecules (early and
  mid sections of proximal tubule).
• Can overcome albumin binding
• (higher affinity of transporters is able to
  strip drug).

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BASES
ACIDS
Probenecid
Amiloride
Penicillins (most)
Neostigmine (quaternary ammonium drug)
Quinine
NSAIDS
Cephalosporins (most) Sulfonamides (most)
Histamine H2 antagonists
Morphine
Histamine
Loop diuretics (furosemide/ ethacrynic acid)
Ephedrine/pseudoephedrine
Choline
Thiazide diuretics
Thiamine
Phase II metabolism conjugates
Each transport system exhibits
selectivity, competition and saturation.


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distal tubule (passive reabsorption from more
acid environment)
proximal tubule (passive reabsorption)
loop of Henle
Collecting duct

• REABSORPTION
• passive process passive
• occurs throughout tubule
• lipid soluble drugs and nonionizable drugs are
  most easily reabsorbed
• (i.e. nonionic molecules preferentially diffuse
  across lipid membranes).

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• REABSORPTION contd.
• Secretion of H in distal segment acidifies
  glomerular filtrate from 7.4 to e.g. 5.4. This
  
• (i) reduces the degree of ionization of weak
  acids which favors their reabsorption and
  therefore hinders their excretion (RCOO- H
  RCOOH)
• (ii) increases the degree of ionization of weak
  bases and hinders their reabsorption and
  therefore favors their excretion. (RNH2 H
  RNH3)

(The rate of excretion of D-amphetamine is 20
times higher at pH 5 than at pH 8)

• "Artificial" manipulation of urine pH will alter
  the ion trapping and therefore
• reabsorption characteristics

Weak acids (increased excretion in alkaline
urine) Chlorpropamide, Methotrexate,
Phenobarbital, Salicylates, Sulfonamides Weak
bases (increased excretion in acidic
urine) Dextroamphetamine Ephedrine,
Pseudoephedrine, Mexilitine/Tocainide, Quinine
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• Examples of compounds with high (gt 0.7) renal
  extraction ratios
• penicillins, glucuronides, sulfates, hippurates
• Glucuronides, sulfates, hippurates are
  Phase II drug metabolites - demonstrating that
  metabolism facilitates elimination.

KIDNEY (excretion)
LIVER (metabolism)
GI TRACT (absorption)
passive reabsorption
glomerular filtration
active secretion
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metabolism
absorption
biliary excretion
urinary excretion
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• BILIARY EXCRETION
• Secretion from the hepatocyte into the bile is
  an active process requiring energy,
• and occurs against a concentration gradient.
• Numerous transport systems exist.
• Each transport system exhibits selectivity,
  competition and saturation.
• Serum protein binding does not affect secretion
  (high affinity of transporters
• is able to strip off drug loosely held on
  albumin).

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In clinical studies, avoid looking for drug
in all the wrong places !
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For example, HIV protease inhibitors appear
only minimally in urine
Drug MW Renal excretion drug metabolites
() Saquinavir 767 5 32 Indinavir 7
12 19 118 Ritonavir 721 11
3.57.5 Nelfinavir 664 3 1.51.5
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• excretion from body in stool/feces only
  succeeds
• if drugs are not recirculated through
  enterohepatic
• route

metabolism
2
3
blood
1
4
bile
X
5
excretion
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• NON-URINE, NON-BILE
• Not quantitatively important but may warrant
  consideration.
• Breast milk
• appears to be passive.
• Concentration in milk depends upon
  concentration of free drug in maternal
• plasma
• Important because of chronic drug exposure
  coupled with compromised
• elimination mechanisms in infants.
• Saliva Noninvasive way to monitor drug
  concentrations in plasma ?
• Hair Drug deposition in hair as a way to track
  past drug exposure ?
• Expired air
• Major route for excretion of gaseous
  anesthetics resulting in termination of
• pharmacological activity.
• Drug must have relatively high vapor pressure.
• Ethanol exhalation is a noninvasive way to
  monitor blood ethanol.

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METABOLISM
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METABOLISM OVERVIEW A. Drug metabolism or
"biotransformation" chemical alteration by
enzymes. B Objective of biotransformation is to
promote the excretion of drugs and drug
metabolites by (i) enhancing their
water-solubility and/or (ii) increasing
suitability (recognition) for active secretion in
the kidney. Both are derived primarily from
Phase II (conjugation) where reactions with
natural body constituents add an acidic
functional groups. (e.g., glucuronides,
sulfates, amino acid conjugates) ionized at
physiological pH subject to carrier mediated
active secretion into the bile and urine
(in the proximal tubule of the kidney
nephron). C Some drugs lack suitable chemical
groups for conjugation and must first undergo
Phase I (metabolic transformation) reactions to
provide them. Some drugs are excreted
following metabolic transformations only.


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PHASE I
(metabolic transformations)

drug or other xenobiotic
PHASE II
(conjugations)

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The two step metabolism process proceeds through
a limited number (three major) of functional
groups-
But both happen in a number of ways-

-OH -NH2 -COOH
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Phase I
TOXICITY
product
Chemical reactivity
drug
Phase II

product


Ease of excretion
For many drugs, reaching a state suitable for
conjugation reactions requires passing through a
more chemically reactive intermediate generated
by Phase I metabolism
Highly reactive drug metabolites, usually from
Phase I oxidation, are occasionally produced in
small quantities and these can cause tissue
damage. Mutagenic and carcinogenic epoxides
generated by P450s are good examples of such
intermediates. Reactive intermediates may cause
enzyme inactivation, membrane lipid peroxidations
resulting in membrane alterations, and changes in
DNA. Reactive intermediates have been implicated
in carcinogenesis, tissue necrotic reactions, and
tissue allergic responses.
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Metabolism is by enzymes

drug or other xenobiotic
PHASE II
(conjugations)
Glucuronosyltransferase

Sulfotransferase
Glutathione S-transferase
Amino acid conjugase
Acetyltransferase
Methyl transferase
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Pharmacological and Toxicological Consequences of
Biotransformation 1. the drug can be
inactivated a good body defense mechanism but
bad for therapeutics (In addition to promoting
excretion, metabolism and the increased water
solubility decreases the entry of drugs into
cells). 2. the drug can be changed into a more
active and sometimes more toxic
substance. 3. the drug can be changed into other
chemical entities which can have pharmacological
effects quantitatively or qualitatively different
from the parent drug.


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Sites of Biotransformation. Drug
biotransformation can take place in almost all
organs and tissues (liver, skin, GI tract, lungs,
kidney, blood, etc) however, the liver is
quantitatively the most important tissue for drug
metabolism especially because of its high
expression levels of many drug metabolizing
enzymes. If the liver can rapidly metabolize
the drug following absorption from the GI tract,
a significant portion of the drug may be
inactivated before it can produce a therapeutic
effect.


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Within the liver, the major subcellular site
of drug biotransformation is the endoplasmic
reticulum most drug metabolizing reactions occur
here (cytochrome P450 and FMO, carboxylesterases
and glucuronosyltransferases).
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When liver is homogenized and ruptured cells
are differentially centrifuged, fragments of the
endoplasmic reticulum with drug-metabolizing
capabilities are isolated in the fraction called
microsomes (artifacts of cell disruption).


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(1) MICROSOMAL OXIDASES Oxidation is by enzymes
called monooxygenases. most often a
hemoprotein termed Cytochrome P-450 (CYP), 12
forms less often a flavoprotein termed
Flavin-containing monooxygenase (FMO),
5 forms The two have many characteristics in
common but differ in reaction mechanism and
in substrate selectivity.
NADPH
1
FP
NADPH
1
FMO
O2
rug
2
2
1

4
P450
3
3
O2
rug
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Drugs metabolized to varying extents by
FMOs Psychotropic therapeutics clozapine,
chlorpromazine, fluoxetine, imipramine,
xanomeline. H2 receptor antagonists cimetid
ine, ranitidine. Gastroprokinetic
agent itopride Thioureylene antithyroid agent
methimazole Antihistaminic agents
promethazine, brompheniramine Antiarrhythmic
agent verapamil Antifungal agent
ketoconazole Cancer chemotherapeutic tamoxifen
NSAID benzydamine Others MPTP,
nicotine, aldicarb, trimethylamine,
methionine, tyramine
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Cytochrome P450 exists in many different forms -
Fe
Fe
Fe
2B
2E
2C
Fe
1A
Fe
3A
2D
The nomenclature of CYPs is based on amino acid
sequence homology number gt 40 homology
between members family CYP 1,2,3,
etc letter gt 59 homology between members
subfamily CYP(n) A,B,C,D etc second
number individual identifier based on the order
of discovery independent of animal
species CYP(n,lttr) 1,2,3,4,5,6,etc
third number polymorphic variant CYP(
n,lttr,n) 1,2,3,etc
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alfentanyl, alprazolam, amprenavir, atorvastatin,
budesonide, buspirone, carbamazepine,
clarithromycin, cortisol, cyclophosphamide,
cyclosporine, diltiazem, efavirenz, eletriptan,
erythromycin, 17b-estradiol, fentanyl, gefitinib,
haloperidol, ifosfamide, indinavir, lovastatin,
midazolam, nelfinavir, nevirapine, nifedipine,
nimodipine, nisoldipine, nitrendipine,
progesterone, quinidine, ritonavir, saquinavir
sildenafil, simvastatin, sufentanil, tacrolimus
(FK506), tamoxifen, testosterone, triazolam,
troleandomycin, verapamil, vinblastine,
vincristine, ziprasidone, zolpidem
CYP enzymes show considerable substrate
selectivity-
caffeine, clomipramine, clozapine,
cyclobenzaprine fluvoxamine, haloperidol,
imipramine, mexiletine, olanzapine, sertraline,
tacrine, theophylline, zileuton, zolmitriptan
Fe
Fe
CYP 1A2
CYP 3A4
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amitriptyline, atomexitine, carvedilol,
clomipramine, codeine, desipramine
dextromethorphan, fluoxetine, haloperidol,
hydrocodone, imipramine metoprolol, mexiletine
nortriptyline, ondansetron, oxycodone, paroxetine
propafenone, propranolol, risperidone,
thioridazine timolol,
celecoxib diclofenac flurbiprofen ibuprofen
irbesartan losartan naproxen phenytoin
piroxicam tamoxifen tolbutamide torsemide
warfarin
amitriptyline carisprodol citalopram
clomipramine diazepam imipramine lansoprazole
S-mephenytoin nelfinavir omeprazole
pantoprazole phenytoin voriconazole
buproprion cyclophos- phamide efavirenz ifo
sfamide methadone
Fe
Fe
Fe
CYP2B6 CYP 2C9 CYP 2C19 CYP 2D6
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CYP enzymes also show some promiscuity in
substrate selectivity-
amitriptyline 2C19 2D6 cyclophosphamide
2B6 3A4 efavirenz 2B6 3A4 haloperidol
1A2 2D6 3A4 ifosfamide 2B6 3A4 imipram
ine 1A2 2C19 2D6 mexiletine
1A2 2D6 phenytoin 2C9 2C19 tamoxifen
2C9 3A4
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Cytochrome P450 exists in many different forms -
Fe
Fe
The quantities of different isozymes (and
polymorphic forms) present in humans, are of
increasing importance in understanding drug
interactions, and individual pharmacologic and
toxic responses to standardized doses. Not all
CYPs are present in adequate amounts
Isozyme of P450 in liver drugs
metabolized CYP 1A2 17 2 CYP 2A6
6 CYP 2B6 1 CYP 2C9/19 25 12 CYP
2D6 2 25 CYP 2E1 9 CYP 3A4
40 60
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A variety of reaction names exist for
cytochrome P450 catalyzed oxidations-
drug example
aromatic hydroxylation phenytoin

(aromatic) epoxidation carbamazepine
alicyclic oxidation phenobarbital
O- dealkylation dextromethorphan
N- dealkylation dextromethorphan
(alkyl group removed in dealkylations is usually
methyl or ethyl )
oxidative deamination dextroamphetamine
Sulfoxidation chlorpromazine
Desulfuration thiopental
N- oxidation guanethidine
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BUT all are oxidations (note the appearance of
the oxygen atom)
aromatic hydroxylation C6H5X
HOC6H4XC

(aromatic) epoxidation
C6H5X OC6H5X
alicyclic oxidation RCH3 RCH2OH
O- dealkylation ROCH3 ROCH2OH
ROH HCHO
N- dealkylation RNHCH3 RNHCH2OH
RNH2 HCHO
oxidative deamination R2CHNH2 R2C(OH)NH2
R2CO NH3
sulfoxidation RSR
RS(OH)R RSOR H
desulfuration R2CS R2CO S
N- oxidation R3N R3NOH R3NO
H
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BUT all are oxidations (note the appearance of
the oxygen atom)
aromatic hydroxylation C6H5X
HOC6H4XC

(aromatic) epoxidation
C6H5X OC6H5X
alicyclic oxidation RCH3 RCH2OH
O- dealkylation ROCH3 ROCH2OH
ROH HCHO
N- dealkylation RNHCH3 RNHCH2OH
RNH2 HCHO
oxidative deamination R2CHNH2 R2C(OH)NH2
R2CO NH3
sulfoxidation RSR
RS(OH)R RSOR H
desulfuration R2CS R2CO S
N- oxidation R3N R3NOH R3NO
H
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• PHASE I OXIDATION, -NONMICROSOMAL
• Drug oxidations in non-endoplasmic reticulum
  subcellular compartments can be catalyzed by
  flavoproteins such as pyridine nucleotide linked
  dehydrogenases (e.g. alcohol and aldehyde
  dehydrogenases) and monoamine oxidases
• Dehydrogenase-catalyzed oxidations do not
  involve molecular oxygen. The oxidation of the
  drugs occurs through electron transfer to a
  pyridine nucleotide, usually NAD. Most of the
  dehydrogenases are cytoplasmic in location.
• Monoamine Oxidases oxidize by electron transfer
  to a flavin group and are usually mitochondrial
  in location. Rather than drug oxidation,
  monoamine oxidases are employed in the metabolism
  of neurotransmitters.

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• PHASE I REDUCTION
• Reductive metabolism in the liver endoplasmic
  reticulum can occur
• through the mediation of both cytochrome P450 and
  flavoproteins.
• The most common reductions are
• azo RNNR' ? RNH2 R'NH2 e.g. prontosil
• nitro RNO2 ? RNH2 e.g. chloramphenicol

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PHASE I HYDROLYSIS Hydrolysis reactions are
catalyzed by carboxyl and cholinesterases
esters (RCOOR RCOOH ROH, e.g.
procaine) amides (RCONHR RCOOH RNH2, e.g.
procainamide Hydrolysis of amides is slower than
of esters. Hydrolysis reactions produce two
chemically reactive centers both of which are
suitable for conjugation, if the metabolites are
not first excreted as the Phase I
products. While hydrolysis often results in loss
of pharmacological activity, this is not always
the case. Some pro-drugs are activated by the
reaction- lovastatin to lovastatin
b-hydroxyacid, the active HMG CoA reductase
inhibitor irinotecan (CPT-11) to SN38, the
topoisomerase I inhibitor enalopril to the
active angiotensin-converting enzyme inhibitor.

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PHASE I HYDROLYSIS Carboxylesterases occur in
the plasma and also in the liver where they are
concentrated in the endoplasmic reticulum. They
occur with multiple degrees of glycosylation
leading to confusion on the number of enzymes
that are present. Acetylcholinesterase is
found in dimeric form in RBC membranes and in
tetrameric form in the CNS. Pseudo- or butyryl-
cholinesterase is a tetrameric enzyme found in
the plasma. Carboxyl and cholinesterases are
inhibited by organophosphates because the
phosphorous -oxygen bond formed at the active
site serine residue is resistant to cleavage by
water. It can be de-phosphorylated by
pralidoxime (2-PAM) thereby regenerating enzyme
activity.
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• PHASE I HYDROLYSIS
• Drugs hydrolyzed include
• local anesthetics (cocaine, lidocaine,
  procaine)
• narcotics and analgesics (aspirin, heroin,
  indomethacin, meperidine)
• antitussive (caramiphen)
• parasympatholytics, muscle relaxants and
  vasodilator (pancuronium, succinylcholine)
• neuromuscular blocking agents (atracurium,
  mivacurium)
• antiarrhythmics (lidocaine, procainamide)
• antibiotic (chloramphenicol)
• CNS stimulant (methylphenidate)
• drug used to treat dyslipidemia (clofibrate).

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Phase II
CONJUGATION
COMMON ACCEPTOR GROUPS
REACTION
present either on Drug or Phase I
metabolite
GLUCURONIDATION
UGTs
OH
COOH

NH2
SH
SULFATION
SULTs
OH
NH2
GLUTATHIONE
GSTs
electrophilic centers
AMINO ACID
COOH
ACETYLATION
NAT1, NAT2
NH2
METHYLATION
POMT, COMT, PNMT, HNMT,NNMT TPMT, TMT
OH
NH2
SH
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PHASE II CONJUGATIONS All conjugations involve
the participation of enzymes characterized as
transferases and without exception, they occur in
multiple forms (isozymes), often with differing
substrate selectivities.

All conjugation reactions, except with
glutathione involve the participation of
energy-rich or activated co-substrates-

CONJUGATION REACTION COSUBSTRATE ---------------
--------------------------------------------------
---------------- GLUCURONIDATION UDP-glucuronic
acid SULFATION PAP-sulfate GLUTATHIONE Glutath
ione AMINO ACID Coenzyme A-Aminoacid ACETYLATI
ON Acetyl-Coenzyme A METHYLATION S-adenosylmet
hionine
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Fraction of clinically used drugs metabolized by
the major phase 2 enzymes-
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(1) GLUCURONIDATION. UDP-glucuronosyltransfera
ses (abbreviated UGTs) are microsomal enzymes
that catalyze the transfer of glucuronic acid
from a uridine diphosphoglucuronic acid (UDPGA)
cofactor to (most often) a hydroxyl
phenol group (e.g. acetaminophen) (forms
an ether glucuronide). carboxyl group
(e.g. valproate, naproxen) (forms
an ester glucuronide). amine group (e.g.
sulthiazole) (forms
an N-glucuronide). The UDPGA is generated from
the abundant carbohydrate supply in the liver via
glucose-1-phosphate. UDP-glucuronosyltransferases
occur in multiple forms in two families-
UGT1A1, UGT1A3 through UGT1A10. UGT2B4,
UGT2B7, UGT2B10, UGT2B15, UGT2B17) The two
families have different gene structures-


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è
The unique gene structure for UGT1As
GENE

mRNAs

independent genes for UGT2Bs
è
6
5
4
3
2
1
UGT2B15 GENE (4q13)
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Actual size of the UGT1A1 locus-
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(1) GLUCURONIDATION. The formation of the
glucuronide does not involve the acid group of
glucuronic acid so the conjugate retains acid and
ionized character at physiological pH, providing
dramatic enhancement of water solubility, and
passive excretability.
Glucuronides are also actively secreted into bile
and in the proximal tubule of the kidney.


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UGT1A1 bilirubin, estradiol, estriol,
ethinylestradiol, thyroxine, triiodothyronine.
UGT1A3 amitriptyline, cyproheptadine,
clofibrate, fenoprofen, ibuprofen, ketoprofen,
ketotifen, naproxen, valproic acid.
UGT1A4 amitriptyline, chlorpheniramine,
cyproheptadine, dapsone, desmethylclozapine),
diphenhydramine, doxepin, imipramine,
ketotifen, lamotrigine, loxapine, meperidine,
promethazine, pyrilamine, trifluorperazine,
tripelennamine. UGT1A5 UGT1A6 acetaminophen,
serotonin, valproic acid. UGT1A7 UGT1A8 mycophen
olic acid, propofol UGT1A9 bumetanide, dapsone,
diethylstilbestrol, diflunisal, estrone,
furosemide, (hydroxytamoxifen), ibuprofen,
ketoprofen, labetalol, naproxen, propranolol,
thyroxine, triiodothyronine. UGT1A10 estradiol,
estrone. UGT2B4 estriol, bile
acids. UGT2B7 buprenorphine, codeine, estriol,
hydromorphone, morphine (both phenolic
3-hydroxyl alcoholic 6-hydroxyl groups),
nalbuphine, nalmefene, nalorphine, naloxone,
naltrexone, NSAIDs (naproxen, ketoprofen,
ibuprofen, fenoprofen) oxymorphone, oxazepam,
all-trans-retinoic acid UGT2B10/11 none
known UGT2B15 17b-hydroxyandrogens UGT2B17/28 Bo
ld putative probe substrate, Italic
endogenous substrate.
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(2) SULFATION. The enzymes catalyzing the
sulfate conjugations are a family of cytosolic
enzymes termed sulfotransferases (abbreviated
SULTs)- Designation Common identifier 1A1
1A2 phenol form low expression phenol form,
respectively 1A3 catecholamine/dopamine
form, thermolabile form 1B1 Thyroid
hormone form 1C2, 1C4 1E1 estrogen
form 2A1 DHEA (dehydroepiandrosterone)
form 2B1a/2B1b 3b of hydroxysteroids
form 4A1 brain form


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SULTs have substrate (drug/hormone) selectivity,
but with considerable inter-enzyme overlap-
hydroxyarylamines 1A1, 1A2 acetaminophen 1A1,
1A3 apomorphine 1A1 1A3 minoxidil 1A1 geni
stein, naringenin 1A1 1E1 4-hydroxytamoxifen
1A1 1E1 iodothyronines 1A1,
1B1 thyroxine (T4) 1E1 catechols 1A1, 1
A2, 1A3, 1B1 dopamine 1A1 1A3 1B1 epinephrine
1A1 1A3 norepinephrine, 5-hydroxytryptamine 1A3
tyramine, albuterol, isoproterenol, dobutamine
1A3 N-hydroxyacetylaminofluorene 1A2
1C2, 1C4 17b estradiol (E2),
estrone 1A1, 1E1 2A1 17a ethinylestradiol 1A1
1E1 2A1 diethylstilbestrol
(DES) 1A1 1E1 cholesterol 2A1, 2B1 pre
gnenolone, dehydroepiandrosterone (DHEA)
1E1 2A1, 2B1 17a hydroxypregnenolone 2B1
Testosterone, androsterone cortisol,bile acids
2A1 dihydrotestosterone 2B1 etiochola
nolone 2A1 2B1 italics minor contributor
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(2) SULFATION. The sulfate conjugates are
ionized at physiological pH and easily
eliminated. The 3'-phosphoadenosine-5'-phosphosul
fate (PAPS) cofactor from which the sulfate group
is transferred is generated from ATP and
inorganic sulfate. The sulfate can be derived
from the sulfur containing amino acids, cysteine
and methionine. It is often limited in
availability, making this a low capacity pathway
Sulfate conjugation is an important
alternative to glucuronidation for phenolic
compounds and occasionally arylamines. Because
PAPS availability within the cell may be
limited, this conjugation pathway decreases in
importance with higher xenobiotic or phenolic
metabolite concentrations.


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Remember, alternative but similar strategies
(metabolic pathways) often exist -
-
-
UDP
COO
COO
O
O
OH
Phase I metabolite or drug
OH
UGT
O
HO
HO
OH
OH
UDP
-
-
ADP
SO3
SO3
Phase I metabolite or drug
O
O
SULT
ADP
example
acetaminophen (Tylenol)
acetaminophen-glucuronide acetaminophen-sulfate
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(Electrophile)
(nucleophile)
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(3) GLUTATHIONE CONJUGATION Glutathione
transferases (abbreviated GSTs) are predominately
in cytosol, as dimeric proteins, and have
overlapping substrate selectivity. The soluble
GSTs fall into seven classes (alpha, mu, pi,
theta, zeta, omega, sigma) and in man there are 4
alpha forms, 5 mu forms and 2 theta forms.
Isozymes are expressed differently in different
organs. Liver concentrations of glutathione
(g-glutamylcysteinyl glycine) are high (gt5 mM).
Conjugation with nucleophilic glutathione is an
important reaction for sequestering reactive
(toxic) electrophilic metabolites (e.g. epoxides)
which may be generated (e.g. by cytochrome P450
oxidations). Secondary metabolic products of
glutathione conjugates include mercapturic acids.
They are formed by the sequential removal of
glutamate and then glycine from the glutathione
portion, followed by acetylation of the amino
group of the residual cysteine. It is
mercapturic acids which are found in the urine.


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1
2
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(4) OTHER CONJUGATIONS. These are reactions
which do not contribute much to enhanced
excretability through an increase in water
solubility, but mask reactive centers.
(Acetylation demonstrated this well with some
early sulfonamides where the acetylated
metabolites were sufficiently less water soluble
that they precipitated as crystals in the urine
resulting in renal damage). The three
conjugations are Acetylation, Methylation
Amino acid conjugation


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• ACETYLATION
• Acetylation reduces water solubility and
  excretability, but masks reactive amine (e.g.
  isoniazid) or sulfonamide (e.g. sulfanilamide)
  groups.
• Catalyzed by two N-acetyltransferases, NAT1 and
  (liver)NAT 2. NAT2 shows genetic polymorphisms,
  giving fast and slow acetylator phenotypes ( of
  slow is higher in middle-eastern, lower in asian
  populations).
• Acetyltransferases are located in the cytosol.
• Acetyl coenzyme A (a thioester) co-substrate
  is readily available from intermediary
  metabolism.
• Acetyltransferases show a limited degree of
  substrate selectivity.
• Drugs acetylated include p-aminobenzoic acid,
  aminoglutethimide, p-aminosalicylic acid,
  dapsone, hydralazine,isoniazid, procainamide, and
  sulfamethoxazole.

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NAT Acetyl
CoA
Acetylated
NAT
CoA
O
O
O
S
NH
O
2
NH
S
CH
C
O
3
sulfanilamide
NH
2
NH
CH
O
3
2
C
NH
NH
O
2
NH
NH
O
C
isoniazid
NAT
C
CH
O
N
3
N
C
NH
NH
2
NH
NH
N
N
N
N
hydralazine
67

• METHYLATION
• Methyl conjugation reduces water solubility and
  excretability, but serves to mask reactive
  functional groups, most often amines and
  catechols.
• Methylations are catalyzed by O-, N- and
  S-MethylTransferases and each has multiple forms-

68
M-Site Enzyme Location Substrates O ca
techol (COMT) cytosolic epinephrine,
norepinephrine, dopamine, L-dopa,
a-methyldopa, catechol (A ring) estrogens,
catechol metabolites from Phase I phenol
(POMT) microsomal phenols (not
catechols) N histamine (HNMT) cytosolic
imidazole ring of histamine and related
compounds nicotinamide (NNMT) pyridines/indole
s, nicotine, tryptophan, serotonin
phenylethanolamine (PNMT) norepinephrine (to
epinephrine) S thiopurine (TPMT) cytosolic
6-mercaptopurine, 6-thioguanine,
azathioprine thiol (TMT) microsomal
aliphatic-SH disulfiram, D-penicillamine,
captopril
69

• AMINO ACID CONJUGATION
• Amino acid conjugation gives little net change
  in water solubility
• Coenzyme A and glycine/glutamine co-substrates
  are readily available. (The reaction is
  catalyzed in two steps, an ATP dependent
  formation of an acylcoenyzme A thioester on the
  carboxyl group of the drug e.g. salicylic acid
  followed by coenzyme A displacement by an amino
  acid.

70
Redundancy in drug metabolism
N
C
O
N
C
O
sulfation (SULT) ether glucuronidation (UGT)
OH
71
DRUG-CAUSED VARIATIONS IN DRUG METABOLISM (
includes chemicals other than prescription and
OTC drugs!) Can arise from either, or a
combination of concomitant
"drug"administration, past "drug"
exposure. Concomitant increased metabolism
(activation) rare. decreased metabolism
(inhibition) common. Past increased
metabolism (induction). decreased
metabolism (inhibition, sometimes from
toxicity). Most variations have been examined
through cytochrome P450's
72
ALTERED DRUG METABOLISM INHIBITION Reports of
clinically significant drug interactions related
to inhibition at cytochrome P450s
amiodarone cimetidine clarithromycin fluoxetine fl
uvoxamine itra/keto-conazole nefazadone indin/nelf
in-avir riton/saquin-avir troleandomycin
amiodarone, cimetidine, clomipramine,
fluoxetine, haloperidol, methadone, paroxetine,
quinidine, ritonavir
cimetidine fluoroquinolones fluvoxamine ticlopidin
e
fluoxetine, fluvoxamine ketoconazole lansoprazole
omeprazole ticlopidine
amiodarone fluconazole isoniazid ticlopidine
Fe
Fe
Fe
Fe
CYP 1A2 CYP 2C9 CYP 2C19 CYP 2D6
CYP 3A4
73
Listings such as the above often do not indicate
the mechanism of inhibition. As a consequence of
the body only having a limited number of enzymes
with which to metabolize a 100s-1000s drugs and
"foreign' chemicals (xenobiotics) the mechanism
is often COMPETITIVE-
CYP2C19
CYP3A4
74
For drugs that can interact strongly with part of
the enzyme in addition to the substrate binding
site (e.g., the heme moiety), the inhibition can
be NON-COMPETITIVE (cannot be displaced by
another substrate).
75
Significant inhibition by such drugs tends to
occurs at relatively low drug (inhibitor)
concentrations
76
(No Transcript)
77
There is great interest and concern about drugs
that demonstrate unusual inhibition kinetics-
they initially show competitive kinetics but
with time become non-competitive. With
metabolism, the drug gains the ability to
interact at other than the substrate binding
site, in some cases binding in a covalent manner
to the protein or heme. These are
mechanism-based or "suicide" inhibitors.
78
(No Transcript)
79
Mechanism-based inhibition of CYP3A4 Antibiotics
Clarithromycin Erythromycin Isoniazid
Troleandomycin Anticancer drugs Irinotecan
SN-38 Tamoxifen N-desmethyltamoxifen Anti-
HIV agents Amprenavir Delavirdine DPC
681 L-754,394 Nelfinavir Ritonavir
Antihypertensive agents Dihydralazine
Diltiazem N-desmethyl diltiazem Mibefradil
Nicardipine Verapamil Steroids and their
modulators 17a-Ethynylestradiol Gestodene
Mifepristone Raloxifene
Herbal constituents Bergamottin 6',
7'-Dihydroxybergamottin Glabridin
Oleuropein Resveratrol Silybin Miscellaneou
s Diclofenac Fluoxetine (-)-Hydrastine
K11002 K11777 Midazolam Tabimorelin
80
In many cases, drugs inhibit more than one
cytochrome P450, although the relative
inhibitory activity may differ. (Underlines in
the chart below demonstrate multiple CYPs
affected) CYP Inhibitory "drug"
1A2 cimetidine, ciprofloxacin, fluvoxamine,
levofloxacin, ticlopidine 2B6 thiotepa 2C9 amiod
arone, fluconazole, fluoxetine, fluvastatin,
imatinib, metronidazole, paroxetine,
zafirlukast 2C19 cimetidine, felbamate,
fluoxetine, fluvoxamine, isoniazid, ketoconazole,
lansoprazole, omeprazole, ticlopidine 2D6
amiodarone, chlorpheniramine, cimetidine,
fluoxetine, haloperidol, imatinib, indinavir,
paroxetine, quinidine, ritonavir, terbinafine,
ticlopidine 3A4 amiodarone, clarithromycin,
diltiazam, erythromycin, fluconazole,
(grapefruit juice), imitanib, indinavir,
isoniazid, itraconazole, ketoconazole,
nefazadone, ritonavir, troleandomycin, verapamil
81
In many cases, drugs inhibit more than one
cytochrome P450, although the relative
inhibitory activity may differ. (Underlines in
the chart below demonstrate multiple CYPs
affected) CYP Inhibitory "drug"
1A2 cimetidine, ciprofloxacin, fluvoxamine,
levofloxacin, ticlopidine 2B6 thiotepa 2C9 amiod
arone, fluconazole, fluoxetine, fluvastatin,
imatinib, metronidazole, paroxetine,
zafirlukast 2C19 cimetidine, felbamate,
fluoxetine, fluvoxamine, isoniazid, ketoconazole,
lansoprazole, omeprazole, ticlopidine 2D6
amiodarone, chlorpheniramine, cimetidine,
fluoxetine, haloperidol, imatinib, indinavir,
paroxetine, quinidine, ritonavir, terbinafine,
ticlopidine 3A4 amiodarone, clarithromycin,
diltiazam, erythromycin, fluconazole,
(grapefruit juice), imitanib, indinavir,
isoniazid, itraconazole, ketoconazole,
nefazadone, ritonavir, troleandomycin, verapamil
82
In many cases, drugs inhibit more than one
cytochrome P450, although the relative
inhibitory activity may differ. (Underlines in
the chart below demonstrate multiple CYPs
affected) CYP Inhibitory "drug"
1A2 cimetidine, ciprofloxacin, fluvoxamine,
levofloxacin, ticlopidine 2B6 thiotepa 2C9 amiod
arone, fluconazole, fluoxetine, fluvastatin,
imatinib, metronidazole, paroxetine,
zafirlukast 2C19 cimetidine, felbamate,
fluoxetine, fluvoxamine, isoniazid, ketoconazole,
lansoprazole, omeprazole, ticlopidine 2D6
amiodarone, chlorpheniramine, cimetidine,
fluoxetine, haloperidol, imatinib, indinavir,
paroxetine, quinidine, ritonavir, terbinafine,
ticlopidine 3A4 amiodarone, clarithromycin,
diltiazam, erythromycin, fluconazole,
(grapefruit juice), imitanib, indinavir,
isoniazid, itraconazole, ketoconazole,
nefazadone, ritonavir, troleandomycin, verapamil
83
Consequences of inhibition If metabolism of a
drug results in inactivation, inhibitors will
likely increase the pharmacological effect. In
those instances where metabolism results in
bioactivation of a drug, inhibitors will
decrease the pharmacological effect.
84
ALTERED DRUG METABOLISM STIMULATION Like
inhibition, has been most extensively studied
with cytochrome P450 (to a more limited extent
with UGTs, GSTs, transporters). Enhaced activity
can result from increased catalytic efficiency of
existing enzyme (activation) but this is
relatively rare. The stimulation of enzyme
activity is most often the result of an
increased production of enzyme, a process called
INDUCTION. Induction in humans may necessitate a
change in the therapeutic dosage regimen of
drugs with time. Enzymes and isozymes vary in
their degree of inducibility most inducible
CYPs (most), UGTs, GSTs,. most refractory
CYP2D6, FMOs, SULTs. A specific enzymes or
isozyme may be induced by a range of drugs . One
drug may induce more than one enzyme.
85
Because induction arises from the synthesis of
new enzyme, the increase in activity is not an
immediate event, and occurs over a period of
days. (Returning to normal following inducing
drug exposure also takes a similar time
course). Induction is not open-ended, there
appear to be limits to changes in the total
enzymes of a given function (e.g. UGTs or
CYPs). Inducing agents tend to have good lipid
solubility and a relatively long biological half
life, (i.e. they gain access to the liver and
remain there for a considerable period of time).
If the drug (D) or xenobiotic enters the liver
cell and is adequately metabolized, it is
discharged as metabolites and it does not
induce-
86
liver cell
nucleus
D R E-enz- yme
gene
cytosolic receptor
DNA
D
D
ribosome
drug
enzymes
metabolites
M
87
With inefficient clearance from the cell, often
from high doses or slow metabolism, drug
accumulates and some is able to bind to a
cytosolic receptor protein which has a high
affinity for the accumulating drug. The binding
initiates a conformational change in the
receptor, (sometimes displacing a chaperone
protein, sometimes facilitating phosphorylation),
which is then able to translocate into the
nucleus. (For some drugs, induction may be
initiated by direct interaction with proteins in
the nucleus or bound to DNA). In the nucleus,
the receptor-agonist complex is able to link with
a partner nuclear factor(s), (often a retinoic
acid binding protein) and the heterodimeric
complex interacts with a regulatory region of DNA
(DRE drug response element). This brings all
the necessary factors together to initiate the
transcription of mRNA to a select number of drug
metabolizing enzyme genes. The mRNA molecules
move out of the nucleus and are translated into
new proteins (induced enzymes) on the ribosomes
attached to the endoplasmic reticulum.
88
Enzyme and drug selectivity for induction
therefore resides at the cytosolic receptor and
the DRE (i.e., does the drug bind a suitable
receptor and does the enzyme gene have the
necessary recognition sequence for the occupied
receptor complex).
Cytosolic Nuclear DNA response
Human CYP Genes Inducer receptor coregulator
element regulated
Rifampicin PXR RXR PXRE
1A2, 2B6, 2Cs, 3A4
89
Cytosolic Nuclear DNA response
Human CYP Genes Inducer receptor coregulator
element regulated
Rifampicin PXR RXR PXRE
1A2, 2B6, 2Cs, 3A4
90
Multiple enzyme induction by one drug is
demonstrated by rifampicin. Rifampicin treatment
increases the metabolism of theophylline
(CYP1A2), warfarin and phenytoin (CYP2C9), and
mephenytoin (CYP2C19) and numerous CYP3A4
substrates. The gene of each CYP induced has the
requisite response element for the receptor that
rifampicin binds to.
91
Other receptors, nuclear factors and response
elements (1) Human
Cytosolic Nuclear DNA response CYPs
Inducer receptor coregulator
element regulated Polycyclic aromatic AhR
Arnt XRE 1A2
hydrocarbons
Abbreviations AhR aromatic hydrocarbon
receptor. (Agonist binding releases the
receptor from a sequestering complex
comprising the AhR receptor itself, a 23kDa
co-chaperone protein (p23), an X-associated
protein (XAP2) and two 90kDa heat shock proteins
(hsp90). Arnt AhR nuclear translocator XRE
Xenobiotic response element, DNA sequence
T(T/A)GCGTG
92
Other receptors, nuclear factors and response
elements (2) Human
Cytosolic Nuclear DNA response CYPs
Inducer receptor coregulator
element regulated Phenobarbital CARb
RXR PBRE 2B6, 2C9, 2C19, 3A4
Abbreviations CARb constitutively active
androstane receptor, (androstane displacement by
drug allows dimerization) RXR 9-cis retinoic
acid receptor. PBRE phenobarbital
responsive enhancer
93
Other receptors, nuclear factors and response
elements (3) Human
Cytosolic Nuclear DNA response CYPs
Inducer receptor coregulator
element regulated Peroxisome PPAR
RXR PPRE 4As proliferators
Abbreviations PPAR peroxisome proliferator
activated receptor. RXR 9-cis retinoic acid
receptor. PPRE peroxisome proliferator
response element, DNA sequence
PuGGTCAnPuGGTCA
94
Enzyme and drug selectivity for induction
therefore resides at the cytosolic receptor and
the DRE (i.e., does the drug bind a suitable
receptor and does the enzyme gene have the
necessary recognition sequence for the occupied
receptor complex). These two conditions are met
for the following
barbiturates carbamazepine efavirenz nevirapine ph
enytoin rifabutin rifampicin ritonavir St. Johns
Wort
carbamazepine char-grilled meats omeprazole rifamp
icin tobacco,
carbamazepine rifampicin
rifampicin phenobarbital
rifampicin phenobarbital phenytoin
95
Many drug metabolizing enzyme genes have several
of the response elements. Not shown above are
the receptors and response elements for
endogenous compounds (these are less well
understood). There is evidence for CYP
regulation by (i) glucocorticoid receptors (GR)
- CYP2C9, CYP2C19, CYP3A4 (ii) vitamin D
receptors VXR (activated by 1,25
dihydroxyvitamin D3 and certain lipophilic bile
acids) - CYP2B6, CYP2C9 CYP3A4. (iii)
hormones receptors. CYP3A4 can be up-regulated by
growth hormone and down regulated by thyroid
hormone.
96
Consequences of induction If metabolism of a
drug results in inactivation, induction will
likely decrease the pharmacological effect. In
those instances where metabolism results in
bioactivation of a drug, induction will likely
increase the pharmacological effect.
97

PHARMACOGENETICS
drug or other xenobiotic
PHARMACOGENOMICS
PHASE II
(conjugations)
RELATED TO DRUG
Glucuronosyltransferase

Sulfotransferase
METABOLISM
Glutathione S-transferase
Amino acid conjugase
Acetyltransferase
Methyl transferase
98
Drug half life varied more in fraternal (right)
than identical (left) twins
99
Many drug metabolizing enzyme polymorphisms
(allelic frequency gt 1) are monogenic and
easily seen
100
PHARMACOGENETICS AND DRUG METABOLISM
Phase I HYDROLYSIS One of the earliest drug
metabolism variations. Psuedocholinesterase, an
enzyme present in plasma, is responsible for the
hydrolysis of drugs that include procaine,
tetracaine, cocaine, heroin, and the
neuromuscular blocking agent, succinylcholine.
Some patients showed exaggerated muscle
relaxation response to succinylcholine and the
incidence varied with populations. It was found
that the enzyme in the abnormal responders had
reduced activity towards the drug because of a
lowered affinity (higher Km). Upon
investigation, the variant enzyme was also more
resistant to the inhibitory effects of
dibucaine on benzoylcholine hydrolysis ( a
local anesthetic that is not a substrate) and
this gave rise to a test for the variants
(dibucaine number inhibition under specified
benzoylcholine and dibucaine concentrations
normal 78, variant 4)
101
Changes traced to an aspartate70 to glycine 70
substitution. (a single base change will
convert Asp to Gly, GAC to GGC or
GAT to GGT) Nonsynonymous SNPs, (single
nucleotide polymorphisms) are those that result
in amino acid changes. In synonymous SNPs there
is no AA change due to code redundancy (e.g.,
GCT and GCC both code for alanine). Frequency
of abnormal psuedocholinesterase gene
(chromosomal location 3q26) is- 2 in
British, Greek, Portuguese, North African, Jewish
and some Asiatic groups. rare
in Africans, Australian aborigines, Filipinos,
and Orientals other than Japanese
absent in Eskimos, South American Indians and
Japanese.
102
Changes traced to an aspartate70 to glycine 70
substitution. (a single base change will
convert Asp to Gly, GAC to GGC or
GAT to GGT) Nonsynonymous SNPs, (single
nucleotide polymorphisms) are those that result
in amino acid changes. In synonymous SNPs there
is no AA change due to code redundancy (e.g.,
GCT and GCC both code for alanine). Frequency
of abnormal psuedocholinesterase gene
(chromosomal location 3q26) varies with the
population examined - 2 in British,
Greek, Portuguese, North African, Jewish and
some Asiatic groups. rare in
Africans, Australian aborigines, Filipinos, and
Orientals other than Japanese
absent in Eskimos, South American Indians and
Japanese.
103
Phase II ACETYLATION (Another early
identified drug metabolism variation). Isoniazid
was (and still is) used in the chemotherapy of
tuberculosis. It is metabolized by acetylation
104
Phase II ACETYLATION (Another early
identified drug metabolism variation). Isoniazid
was (and still is) used in the chemotherapy of
tuberculosis. It is metabolized by acetylation,
and it was found that at prolonged high doses,
patients showed different toxicities some showed
peripheral neuritis and others liver toxicity.
Upon examination it was found that the two
groups differed in their rate of
metabolism Patients were termed'fast slow
acetylators
105
Phase II ACETYLATION (Another early
identified drug metabolism variation). Isoniazid
was (and still is) used in the chemotherapy of
tuberculosis. It is metabolized by acetylation,
and it was found that at prolonged high doses,
patients showed different toxicities some showed
peripheral neuritis and others liver toxicity.
Upon examination it was found that the two
groups differed in their rate of
metabolism Patients were termed'fast slow
acetylators The phenotype arises from genetic
polymorphisms in the hepatic enzyme
N-acetyltransferase (NAT2). (chromosomal location
8p22) The polymorphism incidence varies with
ethnic population. Slow metabolizer status is
most common in Middle Eastern populations
(70) intermediate in Caucasian populations
(50) lowest in Asian populations
(lt25). Numerous NAT2 allelic variants have been
identified. Alleles bearing altered amino acids
include-
106
Amino Acid Change(s) Allele(s) in
common other NAT25A-J I114T, K268R
(B,C,F,G,H,I), R197Q (E,J), I287T (H),
NAT26A-E R197Q K268R (C) L137F
(I) NAT27A,B G286E NAT210 E167K NAT212A-D
K268R D122N (D) NAT214A-G R64Q K268R
(C,E,F,G), I114T (C,F), R197Q (D) NAT217
Q145P NAT218 K282T NAT219  R64W The most
common mutation in Caucasians (the basis of the
NAT25 allele) is T341C (thymine to cytosine)
giving Ile114Thr (isoleucine to threonine).
This change decreases the Vmax, with no change
in Km (affinity). The most common mutation in
Chinese (the NAT27 allele) is G857A (guanine to
adenine) giving Gly286Glu (glycine to
glutamate). This changes (decreases) enzyme
stability.
107
Amino Acid Change(s) Allele(s) in
common other NAT25A-J I114T, K268R
(B,C,F,G,H,I), R197Q (E,J), I287T (H),
NAT26A-E R197Q K268R (C) L137F
(I) NAT27A,B G286E NAT210 E167K NAT212A-D
K268R D122N (D) NAT214A-G R64Q K268R
(C,E,F,G), I114T (C,F), R197Q (D) NAT217
Q145P NAT218 K282T NAT219  R64W The most
common mutation in Caucasians (the basis of the
NAT25 allele) is T341C (thymine to cytosine)
giving Ile114Thr (isoleucine to threonine).
This change decreases the Vmax, with no change
in Km (affinity). The most common mutation in
Chinese (the NAT27 allele) is G857A (guanine to
adenine) giving Gly286Glu (glycine to
glutamate). This changes (decreases) enzyme
stability.
108
The variation in isoniazid N-acetyltransferase
activity was also a key element in the
development of the field of toxicogenetics.
Slow acetylator phenotype with isoniazid was
associated with peripheral neuritis. The
neuropathy arises from a chemical reaction
(hydrazone formation) between the higher levels
of isoniazid present in slow acetylators and
pyridoxine (vitamin B6). Slow acetylator status
can also be linked to other toxicities- 1-3
of slow acetylators administered hydralazine or
procainamide exhibit drug induced lupus
erythematosus Fast acetylator phenotype with
isoniazid was associated with hepatic damage-
109
CH
3
CH
CH
3
CH
CH
3
3
3
C
O
C
O
O
C
NAT2
C
O
O
C

-H20
NH
NH
NH
NH
N
2
CYP
_
N
OH
NH
NH
NH
N
2
H20
H
critical liver nucleophile
H
C
O
C
O
C
O
HO
N
N
N
more hydrolyzed acetylated metabolite further
metabolized
more reactive (toxic) carbonium ion generated
more acetylated metabolite
110
DEHYDROGENATIONS observable pharmacogenetics !
ALDH
ADH
CH3COOH
CH3CH2OH
CH3CHO
NAD
NAD
acetic acid
acetaldehyde which is the primary cause of the
peripheral vasodilation, nausea etc
The body metabolizes absorbed ethanol
to
to
111
ALCOHOL DEHYDROGENASES (ADH) A cytosolic dimeric
protein of two 40-kDa subunits,
(????????????????????).???????????, are each 1
amino acid different Class 1 alcohol
dehydrogenases (main liver forms) are composed of
??? or ????????, or ????or ???? ? ?2 subunit
is able to metabolize ethanol more rapidly
producing acetaldehyde more quickly. Frequency
of ?2 subunit presence is highest in
Japanese/Chinese populations (85), less in Swiss
(20), German (12), Black American (10),
English (8), and Caucasion American (5), and
absent in Asian Indians and Native Americans
112
ALDEHYDE DEHYDROGENASES (ALDH) ALDHs are
divided into three classes of which class II has
the highest affinity (Low Km) for acetaldehyde
50 of Japanese, Chinese, and Vietnamese
populations have Glutamate (GAA or GAG)
changed to a Lysine (AAA or AAG) at
residue 487 giving low activity. If both
ADH and ALDH genetic variations are present. . .
. . Pacific Rim Populations high incidence of
rapid formation and slow removal of acetaldehyde
following ethanol ingestion, giving rise to a
high EtOH intolerance
113
METHYLATION pharmacogenetics

• 6-mercaptopurine, (Purinethol) is it is an
  antineoplastic agent used principally in the
  maintenance therapy of acute lymphocytic leukemia
  and is metabolized (inactivated) by thiopurine
  methyltransferase (TPMT).

TPMT
SH
S-adenosylmethionine (SAM)
ATP methionine
N
N
N
N
H

• TPMT is encoded by a single gene (6p22.3), with
  alleles for low and high activity of 6 and 94
  frequency (respectively) -
• there is a trimodal distribution of TMPT
  activity of low (0.3 of the population),
  intermediate (11.1) and high (88.6).

114
(No Transcript)
115

• 12 polymorphisms are associated with low TPMT
  activity -
• The most common allele
• in Caucasians