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?
Biotransformation
Two major sets of pathways (enzymatic) nonsynthetic reactions - Phase I synthetic reactions - Phase II
Liver SER smooth endoplasmic reticulum
Biotransformation
Nonsynthetic Reactions
hydroxylations aromatic, aliphatic, nitrogen dealkylations(N-, S-, P) deaminations N-, S-, P- oxidations oxidoreductases S-replacements oxidases monoamine oxidases epoxidations mixed function oxidases others azo reduction nitro reduction disulfide reduction others
oxidation
reduction
oxidoreductases reductases
hydrolysis
esters amides hemiacetals,acetals, hemiketals, ketals
esterases amidases peptidases lipases hydrolases
BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
hydroxylations
RH + NADPH + H+ + O2
ROH + NADP+ + H2O
The following three reactions involve the central nervous system depressants known as barbiturate O H O N N H CH3 CH2CH2CH CH 2CH 3 CH3 O Amobarbital O H N N H CH 2CH 3 O Phenobarbital O H N N H O O H N N H OH CH2CH2C(CH 3)2 CH2CH3 O O H N N H OH
aliphatic
aromatic
CH2CH3 O
O O H N N H
CH2CH3 CH(CH3)CH2CH2CH3 O
Pentobarbital
CH 2CH3 CH(CH 3)CH 2CHCH3 O OH
aliphatic
dealkylation
CH3 N O
H N O + H O C H
Probably catalyzed via a peroxygenase mechanism.
Cl Diazepam (Valium)
Cl
N Which is the oxidized and which is the reduced product?
deamination
CH2CHNH 2 Amphetamine CH3 CH2C CH3 O + NH3 This type of reaction is catalyzed by deaminases and transaminases. It is normally used for amino acids.
N-, S-, P- oxidation

NH2 Aniline NO2 Nitrobenzene H 3C
O OCNHCH3
O OCNHCH3
CH 3 SCH 3
H 3C
CH 3 SCH 3 O a sulfoxide
Methiocarb (an insecticide)
S-replacement
S C H 3C H 2O Parathion P O C H 3C H 2O Paraoxon O P O
O C H 2C H 3
O C H 2C H 3
epoxidation
Cl Cl CCl 2 Cl Cl Aldrin Both compounds are insecticides. CH 2 Cl Cl Dieldrin Cl CCl2 CH 2 O Cl
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
1-3 proteins depending on organism and site
Fe3+ protoporphryrin ring system
NADPH cyt c cyt b5 FAD FMN cyt P450
cytochrome P450 reductase
OH DRUG CYP450 Fe3+ CYP450 Fe3+ DRUG NADPH + OH CYP450 Fe3+ DRUG CYP450 reductase NADPH + H+ H+ H+ DRUG
Figure 2.4 CYP450 Reaction Sequence
CYP450 Fe3+ DRUG
eCYP450 Fe2+ DRUG
incorporation of oxygen peroxide dioxygen etc.
O
H2O
eCYP450 Fe2+ DRUG CYP450 Fe2+
O2
DRUG
O21-
O2
9

Distribution of CYP450 in Humans
1A2 3A4 2C19

CYP CYP CYP CYP CYP CYP CYP CYP 1A2 2A6 2B6 2C19 2C9 2D6 2E1 3A4
2C9 2D6
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Vivid CYP450 Screening Kits

Product Name Product No. Vivid CYP3A4 Red Screening Kit P2856
Bulk Price Request
Quantity* Price >300 Assays
Price Request
Vivid Vivid Vivid Vivid Vivid Vivid Vivid Vivid Vivid Vivid Vivid Vivid Vivid
CYP3A4 Green Screening Kit CYP3A4 Blue Screening Kit P2858 CYP3A4 Cyan Screening Kit P2968 CYP2C9 Red Screening Kit P2859 CYP2C9 Green Screening Kit CYP2C9 Blue Screening Kit P2861 CYP2D6 Blue Screening Kit P2972 CYP2D6 Cyan Screening Kit P2862 CYP1A2 Blue Screening Kit P2863 CYP2C19 Blue Screening KitP2864 CYP3A5 Green Screening Kit CYP3A5 Blue Screening Kit P2970 CYP3A5 Cyan Screening Kit P2971
P2857 >300 Assays >300 Assays >300 Assays >300 Assays P2860 >300 Assays >300 Assays >300 Assays >300 Assays >300 Assays >300 Assays P2969 >300 Assays >300 Assays >300 Assays
http://www.panvera.com
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4/5
http://www.fda.gov/cder/drug/drugReactions/CERT%20Educational%20Module%201/sld038.htm
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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 UEM - ultra extensive metabolizers
rate of biotransformation Pharmacogenomics

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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 CYP3A4 CYP3A5 etc.
97% identical in aa sequence liver and small intestine stomach
This link goes to a great website on the CYP system.

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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
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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. Johns 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
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BIOTRANSFORMATION
Enzyme Induction
O H H
N N
CYP450
CH3(CH2)3
CYP450 3A4,5,7, 2B6

actually affects a transport protein (P-gp, MDR1)
O C 2H 5
O C 6H 5
phenylbutazone
phenobarbital
CYP450 1A2
St. Johns wort CYP450 3A4,5,7

actually affects a transport protein (P-gp, MDR1)
benzo(a)pyrene
CYP450 2E1
CH3CH2OH
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BIOTRANSFORMATION
Table 3.1 CYP CYP1A2 CYP2A6** CYP2C9 CYP2C19 CYP2D6 CYP2E1 CYP3A4/5
CYP450 Substrates, Inhibitors, and Inducers* Inhibitors ciprofloxacin fluoxetine tranylcypromine methoxsalen miconazole sulfamethoxazole fluoxetine ritonavir paroxetine sertraline cimetidine watercress grapefruit juice erythromycin ethanol isoniazid prednisone phenytoin pregnancy ethanol carbamazepine rifampin Inducers cigarette smoke phenobarbital barbiturates
Substrates caffeine propanolol nicotine coumarin ibuprofen tolbutamide diazepam omeprazole codeine haloperidol acetaminophen ethanol bupropion lovastatin
* 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.
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oxidation
reduction
hydrolysis
21
BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION
azo reduction
H 2N H 2N Prontosil N NH2 N S O 2N H 2 H 2N H 2N NH2 + S O 2N H 2 sulfanilamide
nitro reduction
NO2
NO2
NO
NHOH
NH 2
NH 2
chloramphenicol (antibacterial)
CHOH
CHOH HOCH2 CHNHCCHCl2 O
HOCH2 CHNHCCHCl2 O
22
BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION
disulfide reduction
CH3CH 2 CH3CH 2 S N C SS S C N CH2CH 3 2 CH3CH 2 CH3CH 2 S N C SH
CH2CH 3 Disulfiram (Anatabuse)
other reductions
C O C
5+
OH C As 3+
As
23
oxidation
reduction
hydrolysis
24
BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS
esters
COOH OCCH3 O + H2O COOH OH + HOCCH 3 O
acetylsalicylic acid
amides
O RCNH N S O CH3 CH 3 COOH +H 2O O
H 2N O N
CH3 CH3
RCOH O
COOH
penicillins
RCNH HOOC N H
also
S CH3 CH 3 COOH
hemiacetals hemiketals acetals ketals
25
Synthetic Reactions glucuronide formation

gluconic acid OH OH H C O 2H HO HO H OH COOH O OH HO glucuronic acid OH OH H C O 2H HO HO H H O H OH H UDP O H O H OH H UTP PPi O PO 3 2 -
Remember that OH glucose is a hemiacetal; O OH this is the F OH form; it is in equilibrium H O with the open OH chain aldehyde glucose and E form.
OH HO
COOH
OH
O COH +
UDP H C O 2H HO HO H H O H OH H O HO HO H H UDP H C O 2H H O O C OH
salicylic acid
UDP-glucuronide
O OH H a glucuronide derivative
26
BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION
acetylation
high energy bond
O NH2 SO2NH2 + CH3C SCoenzyme A Acetyl Coenzyme A H 3C
O C HN + CoenzymeA SO2NH2
sulfanilamide
sulfate ester formation

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

OH + salicylic acid COH O O H2N CH2COH glycine OH O CNHCH 2COH O
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BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES
O H2N CH C OH
glutathione conjugation GSH

gamma glutamyl cysteinyl glycine
O H2N H2C CH2 C NH HS CH2 CH C HN O H CH C K-glutamyl transpeptidase OH drug aminopeptidase M S O NH2 CH2 CH C OH glycine cystinyl O gamma glutamyldrug S CH C OH glutathione-Stransferase
H2C CH2 O NH CH2 CH C HN O H CH C OH O O NHCCH3 drug O S CH2 CH C O
oxidation
2 GSH
reduction
GS-SG
OH mercapturic acid
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BIOTRANSFORMATION
Complex series of biotransformation steps CYP O GSH S-GS OH S-CH2CHCO 2H NH2 OH
S-CH2CHCO 2H NHCCH3 O H 2O
S-CH2CHCO 2H NHCCH3 OH O
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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 Drug or Poison inactive biotransformed Drug or Poison inactive active less potent TOXIC
In general nonsynthetic precede synthetic reactions nonsynthetic reactions can produce active metabolites synthetic reactions produce inactive metabolites
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BIOTRANSFORMATION
Acetylsalicylic Acid ACTIVE analgesic

C O 2H O CC H 3 O
Salicylic acid ACTIVE analgesic

C O 2H OH
Codeine ACTIVE narcotic analgesic

H H N CH 3
Morphine ACTIVE (more potent) narcotic analgesic

H H N CH 3
H 3 CO
OH
HO
OH
Methanol ACTIVE CNS depressant C H3O H
Formaldehyde TOXIC ( ) H CH
O
Formic Acid TOXIC ( ) H CO H

O
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BIOTRANSFORMATION
Factors affecting rates of biotransformation Pathways

oxidation hydrolysis acetylation
Lidocaine
analgesic
Isoniazid
antitubercular Other drugs in this structural/pharmacological class go through oxidation first. This is important when prescribing combinations of drugs which might compete for the enzymes of biotransformation.
Lorazepam glucuronide formation

tranquilizer
malathion Oxidation rapid in insects slow in humans ACTIVE TOXIC ( ) Hydrolysis slow in insects rapid in humans INACTIVE Hydrolysis slow in insects rapid in humans
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BIOTRANSFORMATION
Factors affecting rates of biotransformation

enzyme kinetics
an enzyme is a catalyst k2 E + P Km = (k2+k-1)/k1 Vmax = k2[ET]
k1 E + S k-1 ES
velocity (([S]/(t) or ([P]/(t)
velocity (([S]/(t) or ([P]/(t)
1/v
Vmax
1/Vmax [S] -1/Km 1/[S]
[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
[velocity]
Vmax 1
[velocity]
enzyme 1 Vmax 2 enzyme 2 Km 1

[substrate]
enzyme 1 Vmax 2 enzyme 2
Km 2
[substrate]
Lineweaver-Burke plot double-reciprocal plot
1/velocity 1/Vmax
enzyme 2 enzyme 1
1/Vmax
-1/Km
-1/Km
1/[substrate] 36
BIOTRANSFORMATION
1/velocity 1/Vmax 1/Vmax
substrate x
substrate y 1/velocity 1/Vmax 1/Vmax substrate x
substrate z
-1/Km
-1/Km
1/[substrate]
competitive inhibition
-1/Km -1/Km
1/[substrate]
uncompetitive inhibition
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BIOTRANSFORMATION
E + S E + I EI
ES
E + P
irreversible inhibition
E + S + I EI
ES
E + P
E + S1 + S2 ES2
ES
E + P1 E + P2
competitive reversible inhibition
dueling substrates
E + S
ES +
E + P EIS
E + S + I EIS
ES + I
E + P mixed inhibition
uncompetitive reversible inhibition I
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BIOTRANSFORMATION
k1
k2
E + S1 + S2
k -1 k1' k -1'
ES 1 ES 2
k2 '
E + P1 E + P2
HOCH2CH2OH ethylene glycol
CH3OH methanol
CH3CH2OH ethanol
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BIOTRANSFORMATION
40
BIOTRANSFORMATION
Microfluidic and Fluorescence-Based Oxygen Biosensor System

485 nm 615 nm Test disk contains 48 wells. Disk is spun to mix the components. Look at difference in fluorescence between a blank and treated system. from loading channel
O2 O2
to air vent
O2
O2 O2
medium silicone
total volume of well is 15QL
membranes for CYP rxn dye quenched by O2 excited dye
NADPH + H+ + RH + O2
NADP+ + ROH + H2O
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BIOTRANSFORMATION
sample biotransformation problem

H N O C CH3 N O C OCH 3 O cocaine C. CH3 N O C OCH 3 D. OH N CH3 O C OCH3 O C CH3 O O C A. H3C N B. CH3 O C OCH 3 O C O OCH 3 O C O
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Drug Administered Absorption Drug in Tissues of Distribution Drug Concentration in Systemic Circulation Drug Metabolized or Excreted Pharmacokinetics
Distribution Drug Concentration at Site of Action
Elimination
Pharmacologic Effect
Pharmacodynamics
Clinical Response Toxicity Efficacy
adapted from http://www.pharmGKB.org/do/serve?id=loe#pd
43

3 Biotransformation

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Chemistry 377 - Drugs and Poisons

Where does it occur?

How does it occur?

Chemistry 377 - Drugs and Poisons

Liver SER smooth endoplasmic reticulum

Chemistry 377 - Drugs and Poisons

esters amides hemiacetals,acetals, hemiketals, ketals

esterases amidases peptidases lipases hydrolases

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

ROH + NADP+ + H2O

CH 2CH3 CH(CH 3)CH 2CHCH3 O OH

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Probably catalyzed via a peroxygenase mechanism.

N Which is the oxidized and which is the reduced product?

N-, S-, P- oxidation

Methiocarb (an insecticide)

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Fe3+ protoporphryrin ring system

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

NADPH cyt c cyt b5 FAD FMN cyt P450

cytochrome P450 reductase

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Figure 2.4 CYP450 Reaction Sequence

CYP450 Fe3+ DRUG

eCYP450 Fe2+ DRUG

incorporation of oxygen peroxide dioxygen etc.

eCYP450 Fe2+ DRUG CYP450 Fe2+

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

1A2 3A4 2C19

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Vivid CYP450 Screening Kits

Quantity* Price >300 Assays

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

phenotype - variants expressed in >2% of the population

PM - poor metabolizers EM - extensive metabolizers UEM - ultra extensive metabolizers

rate of biotransformation Pharmacogenomics

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

subfamilies CYP3A3 CYP3A4 CYP3A5 etc.

97% identical in aa sequence liver and small intestine stomach

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

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Chemistry 377 - Drugs and Poisons

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

Induction - increase in activity

Chemistry 377 - Drugs and Poisons

Chemistry 377 - Drugs and Poisons

CYP450 3A4,5,7, 2B6