3-Biotransformation

8/3/2019 3-Biotransformation

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Chemistry 377 - Drugs and Poisons

2

Liver SER

smooth

endoplasmic

reticulum

Two major sets of pathways (enzymatic)

nonsynthetic reactions - Phase I

synthetic reactions - Phase II

Biotransformation


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oxidation

reduction

hydrolysis

hydroxylations

aromatic, aliphatic, nitrogendealkylations(N-, S-, P)

deaminations

N-, S-, P- oxidations

S-replacements

epoxidations

others

azo reduction

nitro reduction

disulfide reduction

others

Nonsynthetic Reactions

oxidoreductases

oxidases

monoamine oxidases

mixed function oxidases

oxidoreductases

reductases

esters

amides

hemiacetals,acetals,

hemiketals, ketals

esterases

amidases

peptidases

lipases

hydrolases

Biotransformation


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N

N

O

O O

H

H

CH2CH2CH

CH2CH3

N

N

O

O O

H

H

CH2CH3

N

N

O

O O

H

H

CH2CH3CH(CH3)CH2CH2CH3

CH3

CH3N

N

O

O O

H

H

CH2CH2C(CH3)2CH2CH3

OH

N

N

O

O O

H

H

CH2CH3

OH

N

N

O

O O

H

H

CH2CH3CH(CH3)CH2CHCH3

OH

The following three reactions involve the central nervous system depressants known as barbiturat

Pentobarbital

Phenobarbital

Amobarbital

hydroxylationshydroxylations

aliphatic

aromatic

aliphatic

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

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


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N

N

Cl

CH3

O

N

N

Cl

H

OO

CH H

Which is the oxidized and

which is the reduced

product?

+

Diazepam

(Valium)

dealkylation

CH2CHNH2

CH3

CH2C

CH3

O + NH3

Amphetamine

deamination

NH2 NO2

OCNHCH3

H3C CH3SCH3

O

OCNHCH3

H3C CH3SCH3

O

O

Aniline Nitrobenzene

Methiocarb

(an insecticide)a sulfoxide

N-, S-, P- oxidation

This type of reaction is catalyzed bydeaminases and transaminases.It is normally used for amino acids.

Probably catalyzed via aperoxygenase mechanism.



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S

PC H 3C H 2O

O C H 2C H 3

O

O PC H 3C H 2O

O C H 2C H 3

O

Parathion Paraoxon

S-replacement

CCl2 CH2

ClCl

ClCl

CCl2 CH2

ClCl

ClCl

O

Aldrin Dieldrin

Both compounds are

insecticides.

epoxidation



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cytochrome P 450

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 andCyt P 450 reductase

1-3 proteins depending on

organism and site

Fe3+

protoporphryrin

ring system



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cytochrome P450 reductase


NADPHNADPH

FMNFMNFADFADcyt c

cyt b5cyt P450


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OH

CYP450Fe3+

CYP450Fe3+

CYP450Fe2+2+

CYP450

Fe2+CYP450

Fe2+

CYP450Fe3+

CYP450Fe3+

DRUG

DRUG

DRUGDRUG

DRUG

DRUG

DRUG

CYP450

reductase

NADPH + H+

e-

O2

O2

e-

O21-

H2O

Figure2.4

CYP450 Reaction Sequence

O

NADPH + H+

OH

H+DRUG

incorporation of

oxygen

peroxide

dioxygen

etc.



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Distribution of CYP450 in Humans

CYP 1A2

CYP 2A6

CYP 2B6

CYP 2C19

CYP 2C9

CYP 2D6

CYP 2E1

CYP 3A4

3A4

2D6

1A2

2C19

2C9



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Vivid CYP450 Screening KitsProduct Name Product No. Quantity* PriceVivid CYP3A4 Red Screening Kit P2856 >300 Assays Price Request

Bulk Price Request

Vivid CYP3A4 Green Screening Kit P2857 >300 AssaysVivid CYP3A4 Blue Screening Kit P2858 >300 AssaysVivid CYP3A4 Cyan Screening Kit P2968 >300 Assays

Vivid CYP2C9 Red Screening Kit P2859 >300 AssaysVivid CYP2C9 Green Screening Kit P2860 >300 AssaysVivid CYP2C9 Blue Screening Kit P2861 >300 AssaysVivid CYP2D6 Blue Screening Kit P2972 >300 AssaysVivid CYP2D6 Cyan Screening Kit P2862 >300 AssaysVivid CYP1A2 Blue Screening Kit P2863 >300 AssaysVivid CYP2C19 Blue Screening KitP2864 >300 AssaysVivid CYP3A5 Green Screening Kit P2969 >300 AssaysVivid CYP3A5 Blue Screening Kit P2970 >300 Assays

Vivid CYP3A5 Cyan Screening Kit P2971 >300 Assays

http://www.panvera.com



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http://www.fda.gov/cder/drug/drugReactions/CERT%20Educational%20Module%201/sld038.htm


4/5


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rate of biotransformation

PM

EM

UEMpercentofpopulatio

n

phenotype - variants expressed

in >2% of the population

PM - poor metabolizers

EM - extensive metabolizers

UEM - ultra extensive metabolizers

Pha

rmacogeno

mics


phenotype-genetic and environmental

genotype-genetic variations

polymorphism-individual variations


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The CYP3A family is responsible

for 30% of the cytochromes inthe 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

stomach


liver and small intestine

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).


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http://medicine.iupui.edu/flockhart/clinlist.html

also caffeine


Induction - increase in activity

caused by a xenobiotic


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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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Enzyme Induction

NNO

OCH3(CH2)3

phenylbutazone

N N

O

O O

H H

C2H5 C6H5

phenobarbital

benzo(a)pyrene

CYP450CYP450 3A4,5,7, 2B6

CYP450 1A2

CYP450 2E1 CH3CH2OH

St. Johns wort CYP450 3A4,5,7

BIOTRANSFORMATION

actually affects a transport

protein (P-gp, MDR1)

actually affects a transport

protein (P-gp, MDR1)


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Table3.1 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 asprocesses in the conversion of certain xenobiotics to carcinogens.

BIOTRANSFORMATION


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oxidation

reduction

hydrolysis

hydroxylations

aromatic, aliphatic, nitrogen

dealkylations(N-, S-, P)deaminations


S-replacements

epoxidations

others

azo reduction

nitro reduction

disulfide reduction

others


oxidoreductases

oxidases

monoamine oxidases


oxidoreductases

reductases

esters

amides


hemiketals, ketals

esterases

amidases

peptidases

lipases

hydrolases


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BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION

H 2N

N H 2

N N S O 2N H 2

H 2N S O 2N H 2

H 2N

H 2N

N H 2

Prontosil

sulfanilamide

+

NO2 NO NHOH NH2NO2

CHOH

CHNHCCHCl2HOCH2

O

NH2

CHOH

CHNHCCHCl2HOCH2

O

chloramphenicol

(antibacterial)

azo reduction

nitro reduction


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N C

S

SS

S

C NCH3CH2

CH3CH2

CH2CH3

CH2CH3N C

SCH3CH2

CH3CH2

SH

Disulfiram (Anatabuse)

2

C C C C

O

C C

O H

As 3+As5+

disulfide reduction

other reductions

BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION


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oxidation

reduction

hydrolysis

hydroxylations

aromatic, aliphatic, nitrogen

dealkylations(N-, S-, P)deaminations


S-replacements

epoxidations

others

azo reduction

nitro reduction

disulfide reduction

others

esters

amides


hemiketals, ketals


oxidoreductases

oxidases

monoamine oxidases


oxidoreductases

reductases

esterases

amidases

peptidases

lipases

hydrolases


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BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS

COOHOCCH3

O

COOHOH

O

acetylsalicylic acid

+HOCCH 3

+ H2O

N

S

O

O

CH3

CH3

COOH

N

SH2NO

O

CH3

CH3

COOH

HOOC N

S

O

H

CH3

CH3

COOH

RCOH

+H2O

penicillinsRCNH

RCNH

esters

amides

also

hemiacetalshemiketals

acetals

ketals


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BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION

NH2 SO2NH2

O

CH3C SCoenzyme A H N SO2NH2

high energy bond

sulfanilamide Acetyl Coenzyme A

+ CH 3C

O

+ CoenzymeA

acetylation

HO N

N N

N

NH2

O

SO

OHO

POOS

OO

OO

O

P OO

O

O

O

O

The enzymes catalyzingthis type of reaction are

called sulfotransferases.

Sulfates are carried as

phosphoadenosine-

phosphosulfate derivatives(PAPS) - a high energy form.

+

sulfate ester formation


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HO

HO

CHCH2NH2

OH

HO

HO

CHCH2NHCH3 CH3O

HO

CHCH2NH2

OH

dimethylmercury

(CH3)2HgCH3Hg+Hg2+

metanephrineepinephrinenorepinephrine

OH

COH

H2N CH2COH

O

O OH

CNHCH2COH

O

O

salicylic acid

+

glycine

BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES

methylation

amino acid conjugation


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glutathione conjugation

BIOTRANSFORMATION - SYNTHETIC - OTHER TYPES

GSH

2 GSH GS-SG

oxidation

reduction

H2N CH C

H2C

OH

O

CH2

C

NH

O

CH

C

CH2

HN

O

HS

CH

C

H

OH

O

H2N CH C

H2C

OH

O

CH2

NH

O

CH

C

CH2

HN

O

S

CH

C

H

OH

O

NH2

CH

C

CH2

OH

O

S

NHCCH3

CH

C

CH2

OH

O

S

O

gammaglutamyl-

cystinyl

glycine

drug

glutathione-S-transferase

drugdrug

K-glutamyl

transpeptidase

aminopeptidase M

mercapturicacid

gamma glutamyl

cysteinyl glycine


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O S-GS S-CH2CHCO2H

OH OH

NH2

S-CH2CHCO2H

OH

NHCCH3

O

S-CH2CHCO2H

NHCCH3

O

GSHCYP

H2O

Complex series of biotransformation steps -

BIOTRANSFORMATION


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Resistancetobiotransformation

P-glycoprotein - responsible for transport of xenobiotics out of the cell

(implicated in multidrug resistance)

part of a group of efflux proteins170 kDa transmembrane, ATP-dependent protein

BIOTRANSFORMATION


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Resultsofbiotransformation

Drug

or Poison

biotransformed

Drug or Poison

active

inactive

active

inactive

more potent

TOXIC

less potent

In general -

nonsynthetic precede synthetic reactions

nonsynthetic reactions can produce active metabolitessynthetic reactions produce inactive metabolites

BIOTRANSFORMATION


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ACTIVE analgesicanalgesic

Salicylic acidAcetylsalicylic Acid

ACTIVE

OCCH3

CO 2H

O

OH

CO 2H

ACTIVE (more potent)ACTIVE narcotic analgesicnarcotic analgesicMorphineCodeine

OH3CO O H

H

H N CH 3

OH O O H

H

H N CH 3

ACTIVE TOXICTOXICCNS depressantFormic Acid

FormaldehydeMethanol

( ) ( )

C H3O H H CH

O

H C O H

O

BIOTRANSFORMATION


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Factors affecting rates of biotransformation

Pathways

Other drugs in this structural/pharmacological classgo through oxidation first. This is important when

prescribing combinations of drugs which might

compete for the enzymes of biotransformation.

tranquilizer

glucuronide formationLorazepam

acetylation

antitubercular

IsoniazidanalgesicLidocaine

hydrolysisoxidation

malathion

Oxidation Hydrolysis

rapid in insects

slow in humans

slow in insects

rapid in humans

ACTIVE INACTIVE

TOXIC Hydrolysis

slow in insects

rapid in humans

( )

BIOTRANSFORMATION


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Factors affecting rates of biotransformation

Vmax = k2[ET]

Km = (k2+k-1)/k1

an enzyme is a catalyst

velocity(([S

]/(t)or([P]/(t)

[S]

Vmax

Km = [S] at 1/2 Vmaxvelocity(([S

]/(t)or([P]/(t)

[S]

1/v

1/[S]

1/v = Km/Vmax(1/[S]) + 1/Vmax

1/Vmax

-1/Km

E + S ES E + P

k1

k-1

k2

BIOTRANSFORMATION

enzyme kinetics


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[substrate]

[velocity]

Vmax 1

Vmax 2

enzyme 1

enzyme 2

kcat/Km is the catalytic efficiency

[substrate]

[velocity]

Vmax 1

Vmax 2enzyme 1

enzyme 2

Km2

Km1

Km is the [substrate] at 1/2 Vmax.

1/velocity

1/[substrate]-1/Km -1/Km

1/Vmax

1/Vmax

enzyme 2

enzyme 1

Lineweaver-Burke plot

double-reciprocal plot

BIOTRANSFORMATION


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1/velocity


1/Vmax

1/Vmax

substrate x

substrate y

competitive inhibition

1/velocity


1/Vmax

1/Vmaxsubstrate x

substrate z

uncompetitive inhibition

BIOTRANSFORMATION


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dueling substrates

competitivereversible inhibitionEI

E + S ES E + P

E + I EI

E + S ES E + P

+

I

E + S1 ES E + P1

+

S2 ES2 E + P2

irreversible inhibition

E + S ES E + P

+

I EISuncompetitivereversible inhibition

E + S ES E + P

+

IEIS

+

I mixed inhibition

BIOTRANSFORMATION


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E+S1

ES1

E+P1

ES2

E+P2

+S2

k1

k1'

k -1

k -1'

k2

k2'

HOCH2CH2OH

ethylene glycol

CH3OH

methanolCH3CH2OH

ethanol

BIOTRANSFORMATION


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BIOTRANSFORMATION


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Microfluidic and Fluorescence-Based Oxygen Biosensor System

from loading channelto air vent

dye quenched by O2

O2

O2

O2O2

O2

excited dye

silicone

membranes for CYP rxn

medium

485nm 615nm

total volume of well is 15QL

Test disk contains 48 wells.

Disk is spun to mix the components.

Look at difference in fluorescence

between a blank and treated system.

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

BIOTRANSFORMATION


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N

C

OC

O

OCH3

O

CH3

HN

C

OC

O

OCH3

O

N

C

OC

O

OCH3

O

CH3

N

C

OH

OCH3

O

CH3

N

C

O C CH3

O

OCH3

O

CH3

H3C

cocaine

A.

B.

C.

D.

sample biotransformation problem

BIOTRANSFORMATION


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adapted from http://www.pharmGKB.org/do/serve?id=loe#pd

Drug Administered

Drug in Tissues

of Distribution

Drug Concentration

in Systemic

Circulation

Drug Metabolized

or Excreted

Drug Concentrationat Site of Action

Pharmacologic Effect

Clinical Response

Distribution Elimination

Toxicity Efficacy

Absorption

Pharmacokinetics

Pharmacodynamics

Documents

3-Biotransformation