drug metabolism - جامعة نزوى · process and their metabolites for drugs before approval....

Those slides adapted from:

• Dr Afaf Mohammed Lecture notes (University of Nizwa)

• The website www. cc.nih.gov/researchers/drug metabolism

• Beale and Block. Wilson and Gisvold’s textbook of Organic and Pharmaceutical chemistry. 2011

• Lemke et al. Foy’s principals of medicinal chemistry. 2008

• Patrick G L. An introduction to medicinal chemistry. 2013

Drug Metabolism

1

Introduction

• Metabolism is the process of biotransformation of endogenous and xenobiotics molecules by enzymes that are present in the liver and extrahepatic sites to be converted into metabolites in order to be excreted from the body.

• The metabolites can be:

pharmacological inactive

pharmacological active

toxic

• Current FDA standards required the identification of metabolism process and their metabolites for drugs before approval.

• Drug metabolism knowledge can be used in design of drugs

2

Sites of Drug Biotransformation

Hepatic microsomal enzymes (oxidation, conjugation)

Extrahepatic microsomal enzymes (oxidation, conjugation)

Hepatic non-microsomal enzymes

(acetylation, sulfation,GSH,

alcohol/aldehyde dehydrogenase,

hydrolysis, ox/red)

3

Cellular localization of metabolic enzymes

• Endoplasmitic reticulum (ER) of intestinal- and liver cells contain P450

• Cytosol contains Phase II metabolic enzymes

4

Phase I

• Phase I metabolism involves chemical

transformations of endogenous or xenobiotics, usually

by enzymes in order to be converted to more polar

molecules.

• Phase I involves oxidation, reduction and hydrolysis

• Most of these reactions occur in the liver, but some (as

hydrolysis) can occur in the other tissues as gut wall

and blood plasma.

5

• Common oxidative metabolisms include:

- Aromatic hydroxylation

- Aliphatic hydroxylation

- Epoxidation of alkenes

- Dealkylation on heteroatoms

- Oxidation of sulfide to sulfoxide to sulfone

- Oxidation of imines to imine oxides

- Oxidation of alcohols to aldehydes

- Oxidation of aldehydes to carboxylic acids

Oxidative Processes

6

-C-

=Oreduction

-C---OH

-C---OH

-C-OH

=

Ooxidation

-NR, OR, and SR NH2, OH, and SHoxidative dealkylation

-N=N-azo reduction

alcohol acid

-NO2

-NH2

-NH2

nitro reduction

-C-OR

=O

O

-C-OH

=

Hydrolysis of ester

-C-NHR

=O

O

-C-OH

=

+ -NHR

Hydrolysis of amide

7

Many Phase I oxidations are mediated by cytochrome P450 enzymes.

Membrane bound monoxygenase proteins - found on the endoplasmic reticulum.

It is consist of:

Heme proteins called CYPP450: responsible for binding of substrate and oxygen

Flavoproteins called NADPH-CYP450 reductase : these are electron carriers, carrying

electrons from NADPH to CYP450.

Phospholipids: facilitate transfer of electrons from NADPH to CYP450 by providing

negative charge environment.

Many iso-forms with different substrate specificities:

Major human CYP’s: 1A2, 2C9, 2C19, 2D6, 3A4

CYP450

8

CYP450 The active site of the protein is hydrophobic and contains iron protoporphyrin (heme).

The iron is the ferric state and coordinated with four nitrogen atoms via tetradentate to the

porphyrin ring. The fifth coordinate is with sulfur of cysteine amino acid that present in

the active site. The sixth coordinate occupied by exchangeable molecules as water which

can be exchanged with strong ligands

Usually ferrous lose water molecule leaving the sixth coordinate open for binding of

strong ligands as oxygen and carbon monoxide. Thus it can form complex with carbon

dioxide that can be absorbed at band of 450 nm.

CYP450 required NADPH (Nicotinamide adenine dinucleotide phosphate) and molecular

oxygen.

9

Structure of P450 Substrate access Heme = catalytic centre

10

Cytochrome P450 catalytic Cycle- An Overview

11

https://www.youtube.com/watch?v=xeS5JnpU2l8

13







Aromatic hydroxylation:-

Hydroxylation usually occurs at the Para-position. Electron rich rings are more easily

epoxidized than rings that have electron withdrawing substituents.

Most phenolic metabolites undergo further phase II metabolism to water soluble

glucuronide or sulfate conjugate which are readily excreted.

R RR

OOH

Arene Arene Oxide Arenol

14

NH

NH

O O

NH

NH

O O

NH

NH

O O

OH

P-HydroxyPhenytoinPhenytoin

O-Glucuronide Conjugate

15

N

HN

Cl

O

4

Cl

Diazepam

p-Chlorobiphenyl

N

S

CH2CH2CH2N(CH3)3

Cl

Chloropromazine

16

Expected

routes for

reactive

epoxide

intermediate

of aromatic

rings

oxidation

17

Dihydrodiol metabolites have been reported in the

metabolism of several aromatic hydrocarbons.

18

Enzymatic conjugation with glutathione plays an

important role not only in detoxification of arene

oxides but also in detoxification of a variety of other

chemically reactive and potentially toxic

intermediates.

19

Oxidation of Olefins: carbon-carbon double bond

Epoxides are minor products owing to further

conversion to 1,2-diol

N

NH2O

N

NH2O

N

NH2O

O HOOH

N

NH2O

HOSG

CarbamazepineCarbamazepine-10,11-epoxide

Trans-10,11 Dihydroxycarbamazepine

CYP450 epoxide hydrolyase

Glutathione transferase

21

Oxidation of benzylic carbon atoms

23

Oxidation of allylic carbon atoms:-

An example of allylic oxidation is the oxidation of

psychoactive component of marijuana (Δ-

Tetrahydrocannabiol)

24

Oxidation at carbon atom ά- to carbonyls and lmines:

25

Oxidation at Aliphatic and alicyclic carbon atoms:- Aliphatic carbon centers are subjected to mixed function oxidation: (ω) Oxidation: - oxidation at the terminal methyl group. (ω-1) Oxidation at carbon atom next to the last carbon.

26

The monosubstituted cyclohexane molecules group is commonly found in many medicinal

agents and also susceptible to mixed function oxidation (alicylic hydroxylation). It occur at

C3 or C4 positions and yield cis or trans stereoisomers

28

Oxidation involving carbon heteroatom systems

Many drugs contain a heteroatom such as nitrogen,

oxygen, or sulfur that is attached to an alkyl group.

When such drugs undergo metabolism these alkyl

groups may be removed in a process called N-(or O-

or S-)-dealkylation.

29


30

Hydroxylation of the carbon attached directly to the heteroatom

(N, O, S), give unstable intermediate which decompose with the

cleavage of the carbon- heteroatom bond.Two products ALWAYS

result from α-carbon atom hydroxylation - an amine and an

aldehyde or ketone.

oxidative N, O and S-dealkylation

Oxidative deamination

hydroxylation and oxidation (N and S only)

R X C

R X C

O

H

R-XH +

O

Where X = N,O,S usually unstable


31

The mechanism for

this transformation

requires that the alkyl

group have at least

one hydrogen on the

carbon directly

attached to the

nitrogen.

α-carbon hydroxylation of aliphatic nitrogen compounds

32

NCH3

CH3

N NCH2

CH3

N N

CH3

NCYP2C19

Spontaneous

OH

H

In general, small alkyl

groups like Me, Et, n-

propyl, i–Propyl, n-butyl

are rapidly removed;

branching on these

substituents slows it down

even more.

CH3

HNCH3

CH3

NH2

CH3

O

CH2

O

NH3

Methampetamine Ampetamine Phenylacetone

Cl

NHCH3

O

Cl

NH2

O

Ketamine Norketamine

N-dealkylation of aliphatic nitrogen compounds

33

N-oxidation of amine compounds

N-oxidation of secondary amines generates the corresponding N-hydroxylamine

metabolites. Often, these hydroxylamine products are susceptible to further oxidation

(either spontaneous or enzymatic) to the corresponding nitrone derivatives.

N-oxidation of tertiary amines and amides yield N-oxide

N-oxidation of tertiary and secondary amines occur by flavin-containing monooxygenase

(FMO)

N-oxidation of primary amines yield hydroxylamine and can be furthered by oxidative

deamination.

N-oxidation of primary amines occur by mixed function enzymes while some drugs and

endogenous primary amines compounds metabolized by group of enzymes called

oxidases (MAO).

34

N-oxidation of secondary amines

35

N- oxidation of primary amines

36

Primary Aromatic Amines

Secondary Aromatic amines

37

Tertiary aromatic amines

38

Oxidative N-Dealkylation of amides

39

Acetaminophen

Metabolism Case

40

O-dealkylation

41

Oxidation involving carbon-sulfur systems:-

Carbon-sulfur functional groups are susceptible to

metabolic:

Sulfides can be metabolically oxidized by FMO to sulfoxides

and to sulfones.

• Sulfoxides can be oxidized to sulfones.

• Sulfones, in which sulfur is already in its highest oxidation

state cannot be oxidized any further.

S-dealkylation α-carbon Hydroxylation

42

S-dealkylation

43

Desulfuration

44

S-Oxidation

45

Oxidation of alcohols and aldehydes:-

RCH2OH

NAD+ NADH

Alcohol dehydrogenase RCHO

NAD+ NADH

Aldehyde dehydrogenase

Egs:

Aldeyde oxidase

Xanthine oxidase

RCOOH

2˚ alcohol Ketone

Conjugated

46

Other oxidative biotransformation pathway:

Aromatization or dehydrogenation

O

OH

C

A

CH

CH3CH2

HO

OH

C

A

CH

CH3CH2

Norgesterol 17 -18-homoestradiol

48

Oxidative dehydrohalogenation

49

Reductive reaction:- Reductive processes play an important role in the metabolism

of many compounds containing C=O, NO2 and –N=N groups.

Reduction of aldehydes and ketones

These reductions are carried out by aldo-keto reductases. They are found in liver and

other tissue (kidney). They have broad substrate specificities and require NADPH as

cofactor. Oxido-reductase enzymes that carry out both oxidation and reduction reactions

also are capable of reducing aldehydes and ketones, alcohol dehydrogenase is a NAD +

dependent oxidoreductase which oxidizes ethanol and other aliphatic alcohols to

aldehydes and ketones. This same enzyme system is capable of reducing carbonyl

derivatives to their corresponding alcohols.

50

Azo reduction

Miscellaneouse Reduction:-

Reduction of N-oxide.

52

Reduction of sulfur – containing functional groups:-

53

Hydrolysis of esters and amides;-

Hydrolytic enzymes that catalyze the cleavage of ester and amide

linkages are present in many organs, various tissues and in plasma.

The metabolic products formed (carboxylic acids, alcohol, phenols

and amines) generally are polar and more susceptible to

conjugation and excretion than the parent compounds (esters or

amide drugs).

54

H2NO

N

O

H2N

HN

N

O H2N COOH

esterase

amidase

H2N COOH

slow Hydrolysis

Rapid Hydrolysis

Procainamide

Procaine

55

Drug conjugation pathways (phase II)

Phase II metabolism also called conjugation reactions. A polar molecule introduced to

suitable functional group that present in the parent drug or drug that has undergo phase I

metabolism. The resulting conjugate has more polarity and so can be excreted by urine

or bile.

Hydrophilic moieties such as glucuronic acid, sulfate, or glycine.

Most Phase II reactions occur in the liver.

Many conjugative enzyme accomplish this objective, and these may show stereo

specificity towards enantiomers when a racemic drug is administered.

56

H2N

COOH

OH

N-acetylation or

N-glucuronidation

O-glucuronidation or

O-sulfation

acyl glucuronidation or

amino acid conjugation

57

• Most phase II conjugation are glucuronide conjugates due to the availability of glucuronic acid in the body that derived from D-Glucose.

• Functional groups that are prone to glucuronide conjugation are phenol, alcohol, carboxylic acid and amines

• The primary site for glucuronation is the liver but it can occur in other sites as kidney, intestine, brain and lung.

• The enzyme responsible for the transfer of gulucorinate moiety is UDP-glucuronyltransferase (UGT)

Glucuronide Conjugates

58

O

HOOC

HOHO

HO

-D-glucose-1-phosphate

UTP PP

Uridine-5-diphospho- -D-glucose(UDPG)phosphorylase

UDPG dehydrogenase

2NAD+

2NADH + 2H

OUDP

UDP-glucuronate

UDP-glucuronyl-transferase

R XH

O

HOOC

HOHO

OH

XR

glucuronide conjugate

Glucuronide conjugation mechanism

59

UDP-glucuronate

NHCOCH3

OH

UGTO

HOOC

HOHO

OH

O NHCOCH3

Acetaminophen

UDP-glucuronate

COOHCO

O

COOH

OHOH

HO

O

Ibuprofen

UGT

O-glucuronide

Acyl-glucuronide

60

H2N

COOH

OH

O

HOOC

HOHO

OH

O-HN

COOH

OH

UGT

Amino salicylic acid

UDP-glucuronate

N-glucuronide

61

Sulfate Conjugation

This type of conjugation involves the addition of sulfate group

The source of sulfate group come a cofactor 3-phosphoadenosine 5-phosphosulfate (PAPS).

The reaction of sulfate conjugation catalyzed by enzyme called sulfotransferases.

Sulfotransferases that present in the cytosol associated with conjugation of phenolic steroids, neurotransmitters and xenobiotics

Membrane-bound sulfotransferases located at Golgi apparatus are associated with glycosaminoglycans, glycoproteins and tyrosine of peptides.

The metabolites of this conjugation is highly water soluble as the pH of sulfonate group is about 1-2 and also totally ionized at physiological pH.

62

NHCCH3

OCH2CH3

=O

NCCH3

OCH2CH3

=O

OSO3-

Phenacetin N-HydroxyphenacetinO-Sulfate conjugate of

N-Hydroxyphenacetin

NCH2CH3

OCH2CH3

=OHO

64

CONJUGATION WITH AMINO ACIDS:

Conjugation with amino acids is an important metabolic route in the metabolism of the

carboxylic acids prior to elimination.

Glycine, the most common amino acid, forms water soluble ionic conjugates with

aromatic, arylaliphatic and hetero cyclic carboxylic acids.

These amino acid conjugates are usually less toxic than their precursor acids and are

excreted readily into the urine and bile.

The xenobiotic must first activated to its CoA thioester before reacting with the amino

acid.

65

Amino acid conjugation

66

GLUTATHIONE OR MERCAPTURIC ACID CONJUGATESUTATHIO:-

Glutathione is a tripeptide (γ – glutamyl cysteineyl glycine) found in most

tissues.

Gultathione conjugation is important in detoxifying various environmental

toxins or electrophilic alkylating agents that result from phase I.

Glutathione through thiol group can react with electrophilic groups such

as epoxides, alkylhalides, sulfonates, disulfoxides and radical species to

form glutathione conjugate. This can undergo further metabolism to

mercapturic acid which can be excreted in the bile.

The enzyme that catalyzed glutathione conjugation is glutathione

transferase.

68

Examples of GSH conjugation

70


71

OP

S

OCH3

OH3C

NO2

GSH

OP

S

OCH3

-O

NO2GSCH3 +

OHP

S

OCH3

OH3C

NO2SG+

Methyl parathion

Pathway a

Pathway b

S-Methylglutathione

S-para nitrophenylglutathion


72

Acetylation

Acetylation is principally a reaction of amino groups involving the transfer of acetyl group to primary aliphatic and aromatic amines, amino acids, hydrazines and sulfonamide groups.

The acetyl group utilized in N-acetylation of xenobiotics is supplied by acetyl CoA. The transfer of this group to the amino substrate is carried out by soluble N-acetyl transferase present in the hepatic reticulendothehial system.

73

Acetylation

74

METYLATION

Methylation is important in the biosynthesis of endogenous compounds as

epinephrine and melatonin and inactivation of other compounds as

norepinephrine, dopamine, serotonin and histamine

Methylation does not produce water-soluble metabolites except in quaternary

ammonium metabolites.

The coenzyme involved in methylation is S-adonesylmethionine (SAM). The

activated methyl group is transferred by enzymes called methyltransferases.

Example of methyltransferase enzymes are:

Catechol-O-methyl transferase (COMT)

Phenol-O-methyltransferase

N-methyltransferase

S-methyltransferase 76

METYLATION:-

77

Factors affecting drug metabolism

Age

Species and strain differences

Gender differences

Enzyme induction

Enzyme inhibition

Stereochemical aspects of drug metabolism

79

The enzymes system at birth especially in preterm and neonates are

functionally not well developed, especially for oxidation and for

conjugation with glucuronic acid.

The drug like chloramphenicol is unable to get conjugated due to

deficiency of glucuronyltransferase. This lead to accumulation of toxic

metabolites and formation of fatal grey baby syndrome in neonates .

Hyperbilirubinemia in infant is due to inability of the infant to metabolize

bilirubin by glucuronide conjugation.

80

Age

In elderly metabolism is reduced because liver mass and liver blood

flow are decreased. This may account for the physiological changes.

Other factors like genetics and environmental lead to slow metabolic

inactivation of drugs. However, slow metabolism must be differentiate

if it is due to diseases related to liver and kidney.

Drugs persist for longer time and in higher concentration that the dose

must be lowered e.g. tricyclic antidepressants, antiarrhythmic drugs.

Age

81


Different animal species may bio-transform a particular xenobiotic by similar or different

metabolic pathways.

NH

HN

O O

NH

HN

O O

OH

NH

HN

O O

OH

S(-)-p-Hydroxyphenytoin

R(+)-m-Hydroxyphenytoin

( Man)

( Dog)

Phenytoin

82

NH2

NH2

O

HO

p-Hydroxyamphetamine

(Rat)

Oxidative Deamination

Aromatic Hydroxylation

Phenylacetone

(Man, Rabbit and

Guinea pig)


83

N

C-NH-NH2

O

Eskimos and Asians Rapid acetylators so there will be

inadquate theraputic response

N

C-NH-NH2

O

Egyptions and

mediterranianSlow acetylators might show toxic effect

Even with the same species there could be differences in metabolism (strain difference)


84

The rate of metabolism of xenobiotic show some variation between male and

female which may be due to endogenous sex hormones or hydrocortisone or

their synthetic equivalence may affect the activity of the metabolic enzymes

e.g.

N-demethylation of erythromycin was significantly higher in female than

males.

N-demethylation of mepridine was depressed during pregnancy and for

woman taking oral contraceptives.

Gender differences

85

It is either increase in the amount of newly synthesized enzyme or increase in the activity of the

enzyme both result in increase in the rate of drug metabolism and decrease duration of action.

This will affect the pharmacokinetics and pharmacodynamic of a drug with clinical implication for the

therapeutic actions of a drug and increased potential for drug interactions.

As a result of induction, a drug may be either metabolized more rapidly to metabolites that are more

potent, more toxic or metabolites that are less active than the parent drug.

For example phenobarbital induce microsomal enzyme so when given with warfarin it increases its

rate of metabolism so decrease its anticoagulant affect.

Phenobarbital and rifampin enhance the metabolism of oral contraceptive estrogen.

Polycyclic aromatic hydrocarbons, pesticides, cigarette smoke all are inducers of microsomal cytp-450

enzyme.

Enzyme Induction

86

Inducing Agent Drug Affected Potential Outcome

Cigarette Smoke Theophylline Asthma Attacks

Rifampin Coumarin Thrombosis

Tegretol OCs Pregnancy

St. John`s Wort Protease inhibitor

Increased Viral Load

Clinical Examples: Induction

87

• It is the decrease in the rate of metabolism of xenobiotics by using

cytochrome P450 inhibitors. This will lead to accumulation of drug and

several side effects.

• Cyp450s inhibitors are divided into:

Reversible inhibition.

Metabolite intermediate complexation of cyp450.

Mechanism-based inhibition of cyp450.

Enzyme inhibition

88

This is the result of reversible interaction at the heme-iron active center

of cyp450 or the lipophilic site on the apoprotein or both.

Chloramphenicol inhibit CYP450 and thus affect the metabolism of

phenytoin and tolbutamide.

Cimetidine inhibit CYP450 and thus affect the metabolism of

phenytoin.

Reversible Inhibition

89

Alkyl amine drugs have the ability to undergo cyp450 mediated oxidation to nitroso

alkane metabolites. This process is called metabolite intermediate complexation.

CYP450 complexation inhibition

90

Certain drugs are oxidized by cyp450 generating metabolites that can bind irreversibly to

the enzyme e.g. cyclophosphoamide and its conversion to acroline and phosphoramide

mustared. Spironolacton and its thio-metabolite that alkylates heme, choramphenicol and

its oxidative dechlorination to an acyl moiety that alkylates cytp450 apoprotein.

Mechanism-based inhibition

91

• Food have a great effect on intestinal CYP450s enzymes.

• Cauliflower, cabbage, spinach may induce some of CYP450 or inhibit others.

• Two days eating cooked Brussels sprouts decrease the hydroxylation of testosterone

• Grape fruit juice inhibits some of the CYP450 enzyme increasing the bioavailability of

some drugs like felodipine a calcium channel blocker

• (8oz) of grape fruit juice decrease sulfoxidation of omeprazole.

• St. John’s Wort induces CYP450 and decrease the effectiveness of contraceptives and

warfarin

Food Interactions

92

Stereochemical Aspects of Drug metabolism

In addition to the physicochemical factors that affect xenobiotic metabolism, stereochemical factors play an important role in the biotransformation of drugs. Most of the metabolizing enzymes show stereoselectivity when one stereoisomer enters into biotransformation pathway preferentially, but not exclusively. Metabolic stereochemical reactions can be categorized as follows:-

Substrate stereoselectivity

Product stereoselectivity

Regiostereoselctivity

93

Substrate Stereo selectivity

It is the preference of one stereoisomer for metabolizing enzyme or

metabolic process than the other e.g. the preferred decarboxylation of S-α-

methyldopa to S-α-methyldopamine, with almost no reaction for R-α-

methyldopa

HO

HO

C

CH3

COOH

S(-)- -MethyldopaS(-)- -Methyldopamine

HO

HO

C

CH3

HL-aromatic a.a.

decarboxylase

94

Substrate Stereo selectivity

95

It is preferential formation of one stereoisomeric metabolite over the other by creation of new asymmetric center.


96

In phenytoin, both rings are prone to p-hydroxylation. In humans, para

hydroxylation of the S ring occur at 90 % while at ring R only 10 %.

97


Selective metabolism of one of the similar functional groups that are positioned in different regions of the molecule.

NH

N

O2N

NO2

5

7 NH

N

H2N

NO2

5

7

98

Regiostereoselctivity

HO

HO

HN

OH

H3CO

HO

HN

OH

Dobutamine

99

Extra hepatic metabolism

Intestinal metabolism:-

The intestinal mucosa is enriched with cypt450 isoforms, glucuronosyl transferases,

sulfo transferases, and glutathione S-transferases. The highest concentration of cyp450s

occurs in the duodenum with a gradual tapering into the ileum.

100

Lung metabolism:-

Lung may play an important role in the metabolic elimination or activation of injected

or inhaled xenobiotics.

Nasal metabolism:-

Many metabolic enzymes are present in the nasal mucosa like cyp450, dehydrogenases

and other conjugation enzymes. The nasal decongestant essences, anesthetics, alcohols,

nicotine and cocaine have been shown to be metabolized by in vitro nasal cyp450.

Drug metabolism studies • This done by synthesizing the drug with an isotope label as Deuterium (2H or D),

tritium (3H or Tr), carbon-13 C13 and carbon-14 C14

• Metabolites containing isotopes as tritium and carbon-14 can be detected easily by measuring the metabolites β-radiation.

• Metabolites containing isotope as deuterium can be detected by mass spectrometry.

• Metabolites containing isotopes as carbon 13 can be detected by NMR spectroscopy.

• Labelled drug can be subjected to in vivo and in vitro tests. It is important that the animal for in vivo test should be chosen correctly as there is a variation between different species.

• For in vivo tests, the labelled drug is administered to a test animal and then taking samples for analysis. Analysis can be done with HPLC with radioactivity detector.

• For in vitro tests, this can be done by commercial individual cyp450 or perfused liver system, microsomal liver fraction or pure enzyme.

101

drug metabolism - جامعة نزوى · process and their metabolites for drugs before approval....

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