3 Biotransformation

Chemistry 377 - Drugs and Poisons
BIOTRANSFORMATION
A D Metabolism E
Purpose
To convert lipophilic substances into hydrophilic species for

subsequent excretion.
Where does it occur?
How does it occur?
1
Biotransformation
Two major sets of pathways (enzymatic)
•nonsynthetic reactions - Phase I
•synthetic reactions - Phase II
Liver SER
smooth
endoplasmic
reticulum
2
Biotransformation
Nonsynthetic Reactions
•hydroxylations
aromatic, aliphatic, nitrogen
•dealkylations(N-, S-, P)
oxidation
•deaminations
•N-, S-, P- oxidations oxidoreductases
•S-replacements oxidases
•epoxidations monoamine oxidases
mixed function oxidases
•others
•azo reduction
oxidoreductases
reduction •nitro reduction
reductases
•disulfide reduction
•others
esterases
•esters
amidases
hydrolysis •amides
peptidases
•hemiacetals,acetals,
hemiketals, ketals lipases
hydrolases
3
BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
hydroxylations RH + NADPH + H+ + O2 ROH + NADP+ + H2O
The following three reactions involve the central nervous system depressants known as barbiturates.
O CH3 O OH
H CH2CH2CH H CH2CH2C(CH3)2 aliphatic
N N
CH2CH 3 CH3 CH2CH3
N O N O
O O Amobarbital OH
H H O
O
H H aromatic
N N CH2CH3
CH2CH3
N O N O
O O Phenobarbital H
O H O
H CH2CH3 H CH2CH3
N N aliphatic
CH(CH3)CH2CH2CH3 CH(CH 3)CH 2CHCH3
N N OH
O O O O
H Pentobarbital H
4
dealkylation
CH 3 H
O O Probably catalyzed via a
N N
O peroxygenase mechanism.
+
H C H
N Cl N
Cl
Which is the oxidized and
Diazepam which is the reduced
product?
(Valium®)
deamination
This type of reaction is catalyzed by
CH2CHNH2 CH2C O + NH3 deaminases and transaminases.
It is normally used for amino acids.
Amphetamine CH3 CH3
N-, S-, P- oxidation O O
OCNHCH 3 OCNHCH3
NH2 NO2
Aniline Nitrobenzene H 3C CH 3 H 3C CH3

SCH3 SCH3
Methiocarb a sulfoxide
(an insecticide) O
5
S-replacement
O
C H C H O P O C H C H O P O
3 2 3 2
Paraoxon
Parathion
O C H C H O C H C H
2 3 2 3
epoxidation
Cl Cl
Cl Cl
CCl2 CH2 CCl2 CH2

O
Cl Cl
Cl Both compounds are Cl
Aldrin insecticides. Dieldrin
6
The principal reaction of drug/toxin metabolism is OXIDATION.
The enzymes responsible are oxido-reductases; called mixed-function

oxidases.
cytochrome P 450
Most prominent and

important among these
is the
cytochrome P450
system.
consists of
Cyt P 450 and
Cyt P 450 reductase
Fe3+
protoporphryrin
1-3 proteins depending on
ring system
organism and site
7
NADPH
cyt c FAD cyt P450

cyt b5 FMN
cytochrome P450 reductase
8
Figure 2.4 –
OH
CYP450 Reaction Sequence
DRUG DRUG
CYP450
Fe3+
CYP450 CYP450
incorporation of
oxygen
Fe3+ Fe3+
DRUG DRUG peroxide
NADPH + H+ dioxygen
etc.
OH e-
CYP450
reductase
CYP450
CYP450
Fe3+ NADPH + H+
Fe2+
DRUG
DRUG
.. O : H+
e-
H2O
CYP450 CYP450 O2
Fe2+ Fe2+
DRUG DRUG
O21- O2
9
Distribution of CYP450 in Humans
1A2
3A4
2C19 CYP 1A2

CYP 2A6
CYP 2B6
CYP 2C19
CYP 2C9
CYP 2D6
CYP 2E1
CYP 3A4
2C9
2D6
10
Vivid® CYP450 Screening Kits

Product Name Product No. Quantity* Price
Vivid® CYP3A4 Red Screening Kit P2856 >300 Assays Price Request
Bulk Price Request

Vivid® CYP3A4 Green Screening Kit P2857 >300 Assays
Vivid® CYP3A4 Blue Screening Kit P2858 >300 Assays
Vivid® CYP3A4 Cyan Screening Kit P2968 >300 Assays
Vivid® CYP2C9 Red Screening Kit P2859 >300 Assays
Vivid® CYP2C9 Green Screening Kit P2860 >300 Assays
Vivid® CYP2C9 Blue Screening Kit P2861 >300 Assays
Vivid® CYP2D6 Blue Screening Kit P2972 >300 Assays
Vivid® CYP2D6 Cyan Screening Kit P2862 >300 Assays
Vivid® CYP2C19 Blue Screening Kit P2864 >300 Assays
Vivid® CYP3A5 Green Screening Kit P2969 >300 Assays
Vivid® CYP3A5 Cyan Screening Kit P2971 >300 Assays
http://www.panvera.com
11
4/5
http://www.fda.gov/cder/drug/drugReactions/CERT%20Educational%20Module%201/sld038.htm
12
phenotype - variants expressed

in >2% of the population
genotype-genetic variations
phenotype-genetic and environmental
polymorphism-individual variations
percent of population
EM
PM
UEM
PM - poor metabolizers
EM - extensive metabolizers
rate of biotransformation
UEM - ultra extensive metabolizers
Pharmacogenomics 13
The CYP3A family is responsible

for 30% of the cytochromes in
the liver and 70% in the gut.
Metabolizes more medications and

hormones than any other CYP.
subfamilies
CYP3A3 97% identical in
CYP3A4 aa sequence
CYP3A5
etc. liver and small intestine
stomach
This link goes to a great website on the CYP system.
14
systemic antifungals
macrolide antibiotics
component believed responsible

belongs to the furanocoumarin
family (also known as psoralens).
15
16
Induction - increase in activity

caused by a xenobiotic
also caffeine
http://medicine.iupui.edu/flockhart/clinlist.html
17
BIOTRANSFORMATION
Induction is a complex process.
It can involve nuclear receptors,

response elements, hormone
receptors, second messengers, and
numerous other factors such as
diet and disease states.
St. John’s wort - herbal treatment for depression

component hyperforin binds to pregnane X receptor as well as
to P-glycoprotein - this combination promotes the production
of CYP3A4
18
BIOTRANSFORMATION
Enzyme Induction
O
H H
O N N N
N CYP450 3A4,5,7, 2B6
CYP450
O O
CH3(CH2)3 O C2H5 C6H5 actually affects a transport
phenobarbital protein (P-gp, MDR1)
phenylbutazone
CYP450 1A2 St. John’s wort CYP450 3A4,5,7

actually affects a transport
protein (P-gp, MDR1)
benzo(a)pyrene
CYP450 2E1 CH3CH2OH
19
BIOTRANSFORMATION
Table 3.1 – CYP450 Substrates, Inhibitors, and Inducers*
CYP Substrates Inhibitors Inducers

CYP1A2 caffeine ciprofloxacin cigarette smoke
propanolol fluoxetine phenobarbital
CYP2A6** nicotine tranylcypromine barbiturates
coumarin methoxsalen
CYP2C9 ibuprofen miconazole ethanol
tolbutamide sulfamethoxazole carbamazepine
CYP2C19 diazepam fluoxetine rifampin
omeprazole ritonavir
CYP2D6 codeine paroxetine pregnancy
haloperidol sertraline
CYP2E1 acetaminophen cimetidine ethanol
ethanol watercress isoniazid
CYP3A4/5 bupropion grapefruit juice prednisone
lovastatin erythromycin phenytoin
* This is a very small sample of xenobiotics affecting and being affected by CYP450s.
** CYP2A6 is considered to be an important factor in studies of smoking cessation as well as
processes in the conversion of certain xenobiotics to carcinogens.
20
•hydroxylations
oxidation
•deaminations
•others
•azo reduction
oxidoreductases
reductases
•others
esterases
•esters
amidases
peptidases
hydrolases
21
BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION
azo reduction
H 2N NH2
H2N N N SO 2NH2 +
H 2N
Prontosil® NH 2 H2N SO 2NH2
sulfanilamide
nitro reduction
NO2 NO NHOH NH2

NO2 NH2
chloramphenicol
(antibacterial) CHOH CHOH
HOCH2 CHNHCCHCl2 HOCH2 CHNHCCHCl2
O O
22
BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION
disulfide reduction
S S S
CH3CH2 CH2CH3 CH3CH2
N C SS C N 2 N C SH
CH3CH2 CH2CH3 CH3CH2
Disulfiram (Anatabuse®)
other reductions
C C C C
O
O H
C
C
5+
3+
As
As
23
•hydroxylations
oxidation
•deaminations
•others
•azo reduction
oxidoreductases
reductases
•others
esterases
•esters
amidases
peptidases
hydrolases
24
BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS
esters
COOH COOH
OCCH3 OH HOCCH 3
+ H 2O +
O O
acetylsalicylic acid
amides
H2N S
O O CH3
RCNH RCOH N CH3
S O
CH3
COOH
N CH3 +H2O
O
O
COOH
also
RCNH S
penicillins
CH3 hemiacetals
HOOC N CH3 hemiketals
H
COOH
acetals
ketals
25
Synthetic Reactions
glucuronide formation OH
gluconic acid H CO2H
OH HO
HO
OH COOH HO OPO32-
Remember that H OH
OH HO H H
glucose is a
hemiacetal; OH UTP
O OH
this is the 
form; it is OH COOH PPi
in equilibrium HO O OH
with the open H CO2H
OH
chain aldehyde glucose OH HO UDP
and  form. HO
HO HO O
H OH
glucuronic acid OH H H
UDP
O
OH O
H CO2H UDP H CO2H OH
COH HO HO C
+
HO HO
HO O HO O
H OH H OH
salicylic acid H H H H
a glucuronide
UDP-glucuronide
derivative
26
BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION
acetylation
high energy bond
O O
NH2 SO2NH2 + CH3C SCoenzyme A H 3C C HN SO2NH2
sulfanilamide Acetyl Coenzyme A + CoenzymeA
sulfate ester formation

NH2
HO + N N O
O N N O SO
O
Sulfates are carried as O S O PO O
phosphoadenosine- O
phosphosulfate derivatives O O The enzymes catalyzing
(PAPS) - a high energy form. this type of reaction are
HO O called sulfotransferases.
O P O
27
BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES
methylation
OH OH
HO CHCH2NH 2 HO CHCH2NHCH3 CH3O CHCH2NH2
HO HO HO
norepinephrine epinephrine metanephrine
Hg 2+ CH 3 Hg + (CH 3 ) 2 Hg
dimethylmercury
amino acid conjugation

OH O OH
+ H2N CH2COH O
COH CNHCH2COH
salicylic acid glycine
O O
28
BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES

O
H2N CH C OH
glutathione conjugation
O H2C
GSH H2N CH C OH
glutathione-S-
CH2
transferase
H2C O
gamma glutamyl gamma
CH2 glutamyl- NH
cysteinyl glycine drug S CH2 CH
C O
NH C O
cystinyl H
HS CH2 CH HN
CH
C O
C O
H
HN
CH OH
glycine O
C O
NHCCH3
γ-glutam yl OH NH2
transpeptidase drug S CH2 CH
drug S CH2 CH
am inopeptidase M C O
C O
oxidation
OH
2 GSH GS-SG OH
m ercapturic
acid
reduction
29
BIOTRANSFORMATION
Complex series of biotransformation steps -

GSH S - C H C H C O H
2 2
O
S - G S
C Y P
N H
2
O H
O H
S - C H C H C O H
2 2
S - C H C H C O H
2 2
N H C C H
3
N H C C H
3
O H
O
O
H O
2
30
BIOTRANSFORMATION
Resistance to biotransformation
P-glycoprotein - responsible for transport of xenobiotics out of the cell
(implicated in multidrug resistance)
part of a group of efflux proteins

170 kDa transmembrane, ATP-dependent protein 31
BIOTRANSFORMATION
Results of biotransformation
more potent
active less potent
active
Drug biotransformed TOXIC
or Poison Drug or Poison
inactive
inactive
In general -
•nonsynthetic precede synthetic reactions
•nonsynthetic reactions can produce active metabolites
•synthetic reactions produce inactive metabolites
32
BIOTRANSFORMATION
Acetylsalicylic Acid Salicylic acid
ACTIVE analgesic ACTIVE analgesic
CO2H CO2H
OCCH3 OH
Codeine Morphine
ACTIVE narcotic analgesic ACTIVE (more potent) narcotic analgesic
H N CH3 N CH3
H
H H
H3CO O OH O
HO OH
Methanol Formaldehyde Formic Acid

ACTIVE CNS depressant TOXIC (N ) TOXIC (N )
CH3OH HCH HCOH
O O
33
BIOTRANSFORMATION
Factors affecting rates of biotransformation
Pathways
oxidation hydrolysis acetylation

Lidocaine Isoniazid
analgesic antitubercular
Lorazepam glucuronide formation Other drugs in this structural/pharmacological class

tranquilizer go through oxidation first. This is important when
prescribing combinations of drugs which might
compete for the enzymes of biotransformation.
malathion
Oxidation Hydrolysis
rapid in insects slow in insects
slow in humans rapid in humans
ACTIVE INACTIVE
TOXIC (N ) Hydrolysis
slow in insects
rapid in humans
34
BIOTRANSFORMATION
Factors affecting rates of biotransformation

enzyme kinetics
an enzyme is a catalyst
k1 k2
Km = (k2+k-1)/k1
E + S ES E + P
k-1 Vmax = k2[ET]
velocity ([S]/t) or [P]/t)
velocity ([S]/t) or [P]/t)
1/v
Vmax
1/Vmax
[S] [S] -1/Km 1/[S]
Km = [S] at 1/2 Vmax 1/v = Km/Vmax(1/[S]) + 1/Vmax
35
BIOTRANSFORMATION
Km is the [substrate] at 1/2 Vmax.
kcat/Km is the catalytic efficiency Vmax 1

Vmax 1 enzyme 1
enzyme 1 Vmax 2
[velocity]
Vmax 2 enzyme 2
[velocity]
enzyme 2
K m1
K m2 [substrate]
[substrate]
Lineweaver-Burke plot
1/velocity enzyme 2
double-reciprocal plot
1/Vmax enzyme 1
1/Vmax
-1/Km -1/Km 1/[substrate]
36
BIOTRANSFORMATION
substrate x
1/velocity
1/Vmax substrate y
1/Vmax 1/velocity substrate z
-1/Km -1/Km 1/[substrate] 1/Vmax
substrate x
1/Vmax
competitive inhibition
-1/Km -1/Km 1/[substrate]
uncompetitive inhibition
37
BIOTRANSFORMATION
E + S ES E + P
E + I EI irreversible inhibition
E + S ES E + P E + S1 ES E + P1
+ + “dueling substrates”
competitive
I EI reversible inhibition S2 ES2 E + P2
E + S ES E + P E + S ES E + P
+ + +
uncompetitive mixed inhibition
EIS EIS
reversible inhibition I I I
38
BIOTRANSFORMATION
k k
1 2
E + S E S E + P
1 1 1
k
-1
k ' k '
1 2
+ S E S E + P
2 2 2
k '
-1
HOCH2CH2OH CH3OH CH3CH2OH

ethylene glycol methanol ethanol
39
BIOTRANSFORMATION
40
BIOTRANSFORMATION
Microfluidic and Fluorescence-Based Oxygen Biosensor System
Test disk contains 48 wells.

485 nm 615 nm
Disk is spun to mix the components.
Look at difference in fluorescence
between a blank and treated system.
to air vent from loading channel

O2 O2
O2
O2 medium
O2
silicone
total volume of well is 15L membranes for CYP rxn
dye quenched by O2
NADPH + H+ + RH + O2 NADP+ + ROH + H2O
excited dye
41
BIOTRANSFORMATION
sample biotransformation problem

H
N O
C OCH3
CH3 O
N A. C
O
O
C OCH3 CH3
H3C
N O
O
C B.
C OCH3
cocaine
O
C.
O
C
CH3
O
N O CH3
C N O
OCH3
D.
C
OH OCH3
O
C CH3
42
Drug Administered
Absorption
Drug Concentration Drug Metabolized

Drug in Tissues Pharmacokinetics
in Systemic or Excreted
of Distribution
Circulation
Distribution Elimination
Drug Concentration
at Site of Action
Pharmacologic Effect Pharmacodynamics
Clinical Response
Toxicity Efficacy
adapted from http://www.pharmGKB.org/do/serve?id=loe#pd

43

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3 Biotransformation

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Chemistry 377 - Drugs and Poisons

To convert lipophilic substances into hydrophilic species for

Where does it occur?

How does it occur?

Two major sets of pathways (enzymatic)

•nonsynthetic reactions - Phase I

•synthetic reactions - Phase II

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

hydroxylations RH + NADPH + H+ + O2 ROH + NADP+ + H2O

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

N-, S-, P- oxidation O O

Aniline Nitrobenzene H 3C CH 3 H 3C CH3

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

CCl2 CH2 CCl2 CH2

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

The principal reaction of drug/toxin metabolism is OXIDATION.

The enzymes responsible are oxido-reductases; called mixed-function

Most prominent and

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

cyt c FAD cyt P450

cytochrome P450 reductase

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Distribution of CYP450 in Humans

2C19 CYP 1A2

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Vivid® CYP450 Screening Kits

Bulk Price Request

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

phenotype - variants expressed

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

The CYP3A family is responsible

Metabolizes more medications and

This link goes to a great website on the CYP system.

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

component believed responsible

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Induction - increase in activity

Induction is a complex process.

It can involve nuclear receptors,

St. John’s wort - herbal treatment for depression

CYP450 1A2 St. John’s wort CYP450 3A4,5,7

CYP450 2E1 CH3CH2OH

Table 3.1 – CYP450 Substrates, Inhibitors, and Inducers*

CYP Substrates Inhibitors Inducers

BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION

NO2 NO NHOH NH2

HOCH2 CHNHCCHCl2 HOCH2 CHNHCCHCl2

BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION

BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS

BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION

NH2 SO2NH2 + CH3C SCoenzyme A H 3C C HN SO2NH2

sulfanilamide Acetyl Coenzyme A + CoenzymeA

sulfate ester formation

BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES

HO CHCH2NH 2 HO CHCH2NHCH3 CH3O CHCH2NH2

amino acid conjugation

BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES

Complex series of biotransformation steps -

part of a group of efflux proteins