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Transcript of 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
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
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 allylic carbon atoms:-
An example of allylic oxidation is the oxidation of
psychoactive component of marijuana (Δ-
Tetrahydrocannabiol)
24
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
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
Oxidation involving carbon heteroatom systems
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
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
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
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
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
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
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
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
Examples of GSH conjugation
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
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
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
Species and strain differences
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)
Species and strain differences
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)
Species and strain differences
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
It is preferential formation of one stereoisomeric metabolite over the other by creation of new asymmetric center.
Product stereoselectivity
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 %.
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
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