BIOTRANSFORMATION

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BIOTRANSFORMATION
Metabolism
A D
E
Purpose
To convert lipophilic substances into hydrophilic
species for subsequent excretion.
Where does it occur?
How does it occur?
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Biotransformation
Two major sets of pathways (enzymatic)

• nonsynthetic reactions - Phase I

• synthetic reactions - Phase II

Liver SER
smooth endoplasmic reticulum
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Biotransformation
Nonsynthetic Reactions

• hydroxylations
• aromatic, aliphatic, nitrogen
• dealkylations(N-, S-, P)
• deaminations
• N-, S-, P- oxidations
• S-replacements
• epoxidations
• others

oxidation reduction hydrolysis
oxidoreductases oxidases monoamine oxidases
mixed function oxidases

• azo reduction
• nitro reduction
• disulfide reduction
• others

oxidoreductases reductases
esterases amidases peptidases lipases hydrolases

• esters
• amides
• hemiacetals,acetals, hemiketals, ketals

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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
hydroxylations
aliphatic
aromatic
aliphatic
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
dealkylation
Probably catalyzed via a peroxygenase mechanism.
deamination
This type of reaction is catalyzed by deaminases
and transaminases. It is normally used for amino
acids.
N-, S-, P- oxidation
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
S-replacement
epoxidation
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
The principal reaction of drug/toxin metabolism
is OXIDATION.
The enzymes responsible are oxido-reductases
called mixed-function oxidases.
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
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
Figure 2.4 CYP450 Reaction Sequence
OH
incorporation of oxygen peroxide dioxygen etc.
NADPH H
e-
OH
CYP450 reductase
NADPH H

O
..
H
e-
O2
H2O
O21-
O2
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
Vivid CYP450 Screening Kits Product Name Product
No. Quantity Price Vivid CYP3A4 Red Screening
Kit P2856 gt300 Assays Price Request Bulk Price
Request Vivid CYP3A4 Green Screening
Kit P2857 gt300 Assays Vivid CYP3A4 Blue
Screening Kit P2858 gt300 Assays Vivid CYP3A4
Cyan Screening Kit P2968 gt300 Assays Vivid
CYP2C9 Red Screening Kit P2859 gt300 Assays Vivid
CYP2C9 Green Screening Kit P2860 gt300
Assays Vivid CYP2C9 Blue Screening
Kit P2861 gt300 Assays Vivid CYP2D6 Blue
Screening Kit P2972 gt300 Assays Vivid CYP2D6
Cyan Screening Kit P2862 gt300 Assays Vivid
CYP1A2 Blue Screening Kit P2863 gt300
Assays Vivid CYP2C19 Blue Screening
Kit P2864 gt300 Assays Vivid CYP3A5 Green
Screening Kit P2969 gt300 Assays Vivid CYP3A5
Blue Screening Kit P2970 gt300 Assays Vivid
CYP3A5 Cyan Screening Kit P2971 gt300 Assays
http//www.panvera.com
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
4/5
http//www.fda.gov/cder/drug/drugReactions/CERT20
Educational20Module201/sld038.htm
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
phenotype - variants expressed in gt2 of the
population
genotype-genetic variations
phenotype-genetic and environmental
polymorphism-individual variations
PM - poor metabolizers
EM - extensive metabolizers
UEM - ultra extensive metabolizers
Pharmacogenomics
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
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.
This link goes to a great website on the CYP
system.
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
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BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION
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
CYP450 3A4,5,7, 2B6
CYP450
actually affects a transport protein (P-gp, MDR1)
St. Johns wort CYP450 3A4,5,7
CYP450 1A2
actually affects a transport protein (P-gp, MDR1)
CYP450 2E1 CH3CH2OH
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BIOTRANSFORMATION
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Nonsynthetic Reactions

• hydroxylations
• aromatic, aliphatic, nitrogen
• dealkylations(N-, S-, P)
• deaminations
• N-, S-, P- oxidations
• S-replacements
• epoxidations
• others

oxidation reduction hydrolysis
oxidoreductases oxidases monoamine oxidases
mixed function oxidases

• azo reduction
• nitro reduction
• disulfide reduction
• others

oxidoreductases reductases
esterases amidases peptidases lipases hydrolases

• esters
• amides
• hemiacetals,acetals, hemiketals, ketals

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azo reduction
nitro reduction
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disulfide reduction
other reductions
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Nonsynthetic Reactions

• hydroxylations
• aromatic, aliphatic, nitrogen
• dealkylations(N-, S-, P)
• deaminations
• N-, S-, P- oxidations
• S-replacements
• epoxidations
• others

oxidation reduction hydrolysis
oxidoreductases oxidases monoamine oxidases
mixed function oxidases

• azo reduction
• nitro reduction
• disulfide reduction
• others

oxidoreductases reductases
esterases amidases peptidases lipases hydrolases

• esters
• amides
• hemiacetals,acetals, hemiketals, ketals

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BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS
esters
amides
also hemiacetals hemiketals acetals ketals
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Synthetic Reactions
glucuronide formation
Remember that glucose is a hemiacetal this is
the ? form it is in equilibrium with the open
chain aldehyde and ? form.
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BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION
acetylation
sulfate ester formation
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BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES
methylation
amino acid conjugation
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BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES
glutathione conjugation
GSH
gamma glutamyl cysteinyl glycine
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BIOTRANSFORMATION
Complex series of biotransformation steps -
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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
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BIOTRANSFORMATION
Results of biotransformation
Drug or Poison
biotransformed Drug or Poison

• In general -
• nonsynthetic precede synthetic reactions
• nonsynthetic reactions can produce active
  metabolites
• synthetic reactions produce inactive metabolites

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BIOTRANSFORMATION
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BIOTRANSFORMATION
Factors affecting rates of biotransformation
Pathways
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BIOTRANSFORMATION
Factors affecting rates of biotransformation
enzyme kinetics
an enzyme is a catalyst
1/v Km/Vmax(1/S) 1/Vmax
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BIOTRANSFORMATION
Km is the substrate at 1/2 Vmax.
kcat/Km is the catalytic efficiency
Lineweaver-Burke plot double-reciprocal plot
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BIOTRANSFORMATION
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BIOTRANSFORMATION
E S ES E P
irreversible inhibition
I
S2
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BIOTRANSFORMATION
HOCH2CH2OH ethylene glycol
CH3OH methanol
CH3CH2OH ethanol
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BIOTRANSFORMATION
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BIOTRANSFORMATION
Microfluidic and Fluorescence-Based Oxygen
Biosensor System
Test disk contains 48 wells. Disk is spun to mix
the components. Look at difference in
fluorescence between a blank and treated system.
485 nm
615 nm
from loading channel
to air vent
O2
O2
O2
O2
medium
O2
silicone
membranes for CYP rxn
total volume of well is 15?L
dye quenched by O2
NADPH H RH O2 NADP ROH H2O
excited dye
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BIOTRANSFORMATION
sample biotransformation problem
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Drug Administered
Absorption
Drug Concentration in Systemic Circulation
Drug Metabolized or Excreted
Drug in Tissues of Distribution
Pharmacokinetics
Distribution
Elimination
Drug Concentration at Site of Action
Pharmacodynamics
Pharmacologic Effect
Clinical Response
Toxicity
Efficacy
adapted from http//www.pharmGKB.org/do/serve?idl
oepd