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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5933_Библиотеки_им_академика_М_И_Перельмана
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6Drug metabolism 109
C. Hydrolytic reactions
1. hydrolysis of esters and amides
2. miscellaneous hydrolytic reactions
6.2.1 Introduction of a polar functional group
This can be achieved by:
A.
ect introduction of functional group
dir
1. aromatic hydroxylation
2. aliphatic hydroxylation
B. modifying or unmasking existing functionalities
1. reduction of aldehydes to alcohols
2. oxidation of alcohols to carboxylic group
3. reduction of azo and nitro compounds to give NH
4. N-, O-, S- dealkylation to give –NH
, OH and SH groups
2
moieties
2
Phase I metabolites are not sufficiently hydrophilic or inactive. Phase I reactions
provide a functional group or handle to the molecule that can undergo subsequent
phase II reactions.
6.2.2 Phase II reactions
Importance: These reactions form water-soluble conjugated metabolites attaching
small, polar and ioniza
ble endo
genous compounds to phase I metabolite. They are
as follows
– glucuronic acid conjugation
– gulfate conjugation
– conjugation with glycine, glutamine and other amino acids
– glutathione conjugation
– acetylation
– methylation
If the parent compound has polar functional groups such as carboxylic (–COOH),
hydroxyl (–OH), amino (–NH
), they are directly conjugated by phase II enzymes.
2
Methylation and acetylation terminate or alter biological activity of drug molecules.
Glutathione conjugation protects the body against chemically reactive compounds or
metabolites. Phase I and phase II reactions complement one another in detoxifying
and facilitating the elimination of drugs and xenobiotics (Fig.6.1). E.g.: Marijuana
1
-
-tetrahydrocannabinol (1-THC).
110 Komarla Kumarachari Rajasekhar*
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7
CH
3
1
2
6
5
H3C
H
1
-THC
C
3
OH
3
4
O
Hydroxylation
C
5H11
Allylic
Psychoactive constituent of marijuana
(Highly lipophilic)
Fig. 6.1: Detoxification of 1-THC.
CH
OH
2
OH
H
C
3
O
C
H
3
C
5H11
7 – hydroxy - 1-THC
(more polar than parent
molecule)
–
COO
O
R =
CH
OH
OH
OH
3
-Ionised carboxylate group
-3 polar hydroxyl groups
Oxidation
COOH
OH
H
C
3
O
C
H
3
C
5H11
1-THC-7-oic acid
(Ionisable at physiological
pH)
Conjugation
COOR
OR
H
C
3
O
C
H
3
C
5H11
Glucuronide conjugation
at either COOH or
phenolic OH
(more polar, ionazible and
hydrophilict)
6.2.3 Sites of drug biotransformation
The liver is the most important organ for metabolism and majority of drugs undergo
metabolism in the li
ver [4], e.g. propoxyphene, lidocaine, propranolol, meperidine,
nitroglycerine, Etazocine etc. Another important site of metabolism especially for
orally administered dugs is the intestine [5]. Examples include
1. Oral isoproterenol undergoes sulfate conjugation in intestinal wall.
2. Levodopa, chlorpromazine and DES are metabolized in GIT.
3. Ester prodrugs are metabolized by esterases and lipases in the intestine.
4. Sulfadiazine, aromatic azo and nitro drugs are metabolized by bacterial flora in
intestine and colon.
Other tissues include kidney, lungs, adrenal glands, placenta, brain and skin. But
these sites are substrate selective, limited to particular types of reaction and their
metabolic capabilities are not fully understood [6].
6Drug metabolism 111
6.2.4 Role of cytochrome P450 monooxygenases in oxidative
ransformations[7,8]
biot
Mixed-function oxidases or monooxygenases catalyze conversion of molecular
oxy
gen into activated oxygen (Fig.6.2). Important components of this enzyme system
include:
1. Cytochrome P450: responsible for transferring an oxygen atom to the substrate
(R-H).
2. NADPH-dependent cytochrome P450 reductase
3. NADH-dependent cytochrome b
5
Components 2, 3 and cofactors NADPH and NADH supply reducing equivalents (electrons) needed in overall oxidative reaction.
R – H + NADPH + O
+ H+ → ROH + NADP+ + H2O
2
Xenobiotic Oxidized metabolite
NADPH-Reduced form of Nicotinamide adenosine dinucleotide phosphate (reducing
agent).
+
NADP
-Oxidized form of Nicotinamide adenosine dinucleotide phosphate (oxidizing
agent).
Cytochrome P450 monooxygenases are chemically heme proteins. The heme portion
is iron containing porphyrin called protoporphyrin–IX and the protein portion is apoprotein. This enzyme system is majorly present in liver and traces in lung, kidney,
skin, placenta, intestine and adrenal cortex. Endoplasmic reticulum (EPR) undergoes homogenization and loses its structure to form small vesicular bodies called
microsomes. Reduced form of this enzyme binds with carbon monoxide (CO) to form
complex. This complex has a spectroscopic absorption maximum at 450nm. Therefore, this enzyme system is named “Cytochrome P450”.
Its ability to metabolize an almost unlimited number of diverse substrates by a
variety of oxidation transformations is a key factor in drug metabolism. This is mainly
due to substrate nonspecificity and multiple forms of enzyme (polymorphism). Some
of these P450 enzymes are selectively induced by various chemicals and drugs like
poly aromatic hydrocarbons (induces cytochrome P448), phenobarbital and tetrachloro dibenzodioxin (induces cytochrome P450).
112 Komarla Kumarachari Rajasekhar*
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Oxidised product
R-OH
Step-6
[P-450(Fe2+)][RH]
CO
proposed activated
oxygen species.
H
2
Step-5
+
2H
[P-450(Fe
Peroxide dianion-P-450
3+
(Fe
) substrate complex.
2+
)][RH]
O
=
2
Step-4
(NADPH /NADH)
P-450
reductase or
reductase
b
5
3+
[P-450(Fe
)]
Oxidised resting
state of
Cytochrome P-450
Substrat
e
R–H
Step-1
[P-450(Fe
3+
)][RH]
P-450 subtrate complex
–
(NADPH)
[P-450(Fe
[P-450(Fe
–
e
dioxygen P-450
2+
)][RH]
CO
chromophore
Step-3b
Step-2
2+
)][RH]
O
2
Step-2
–
e
(NADPH)
[P-450(Fe
Reduced P-450
substrate complex
O
Step-3a
e
Cytochrome
P-450 reductase
2+
)][RH]
Dioxygen or
2
molecular oxygen
substratecomplex
Fig. 6.2: Catalytic reaction cycle of cytochrome P450 monooxygenases.
Step 1: Substrate binding complexation:
Substrate molecule binds to enzyme which is in oxidized resting state (Fe
3+
) and
forms enzyme substrate complex.
3+
Step 2: One electron transfer – reduction of Fe
to Fe
2+
:
One electron from NADPH-dependent cytochrome P450 reductase is transferred to
enzyme–substrate complex. This electron reduces Fe
3+
to Fe2+.
Step 3a: Dioxygen binding – complexation:
Reduced enzyme substrate complex binds to dioxygen (molecular oxygen) and forms
dioxygen enzyme substrate complex.
Step 3b: Chromophore formation – CO binding:
Reduced enzyme substrate complex binds with CO and forms a complex (chromophore) which has maximum spectroscopic absorption at 450nm.
6Drug metabolism 113
Step 4: Reduction of 3a complex – peroxide dianion complex:
Dioxygen enzyme substrate complex undergoes one electron reduction either by
1. cytochrome P450 reductase – NADPH or by
2. cytochrome b
reductase – NADH
5
as a result, peroxide dianion enzyme substrate complex is formed.
Step 5: Formation of activated oxygen complex – activation of complex:
Water is released from the peroxide dianion enzyme substrate complex to form activated enzyme substrate complex. The activated oxygen [FeO
3+
] in this complex is
highly electron deficient and a potent oxidizing agent (Fig.6.3).
Step 6: Oxidation of substrate – regeneration of enzyme:
The activated oxygen [FeO
3+
] is transferred to the substrate and the oxidized substrate
(R-OH) is released from enzyme complex. Oxidized form of cytochrome P450 is regenerated. Many types of oxidative reactions carried out by cytochrome P450 are summarized schematically in Fig. 6.4.
Simplified apoprotein part
L
CH
Fe
N
N
O
3
CH
CH
Substrate
Binding
CH
2
Haeme part with
activated oxygen
3
2
C
H
HOOC
HOOC
Fig. 6.3: Simplified structure of activated enzyme complex.
3
N
N
H–R
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OH
O
Arenols
Arene oxide
Miscellaneous
Oxidations
R
N
R
N
O
N-oxide formation
R
C
H
2
R
N
N-dealkyaltion
HN
R
+
HC
O
R
Oxidative
deamniation
Fig. 6.4: Cytochrome P450 catalyzed reactions.
O
Epoxides
C=C
3+
[FeO]
Activated Oxygen
R
HN
C
S=C
H
O=P
O=C
Desulfuration
S=P
-S- CH
R-O-CH
C
OH
RHO
N
N-hydroxylation
S-dealkylation
O
-SH
3
3
S
R-OH
O-dealkylation
Carbon hydroxylation
Includes benzylic,
Allylic, aliphatic etc.
S-Oxidation
CH
3
6.3 Oxidative mechanisms
6.3.1 Oxidation of aromatic moieties
Aromatic hydroxylation: This is the major route of metabolism for many drugs with
l rings (Fig.6.5) in humans involving the formation of epoxide intermediate [9].
pheny
RR
O
Fig. 6.5: General reaction of aromatic hydroxylation.
R
OH
ArenolArene OxideArene
6Drug metabolism 115
OH
NH
CH
3
O
Propranolol Phenobarbitol Phenytoin
Fig. 6.6: Drugs that undergo aromatic hydroxylation as major metabolic reaction.
CH
3
O
HN
O
N
O
H
CH
O
NH
HN
3
O
Drugs like propranolol (β-blocker), phenobarbital (sedative) and phenytoin (antiepileptic) undergo aromatic hydroxylation as major metabolic reaction (Fig.6.6). Other
drugs that undergo aromatic hydroxylation include phenylbutazone (anti-inflammatory), phenformin (antidiabetic), 17α-ethinyl estradiol (oral contraceptive), S(-)warfarin (anticoagulant), amphetamine (CNS stimulant) etc. In most of the cases aromatic
hydroxylation occurs at para position.
The substituent present on the aromatic ring influences the ease of hydroxylation (Fig.6.7). Aromatic hydroxylation readily occurs in drugs with electron rich or
activated rings. In amphetamine (CNS stimulant) an amino alkyl side chain releases
electrons into the aromatic ring and activates the ring to undergo hydroxylation preferably at para position. Drugs with electron pullers on aromatic ring or deactivated
rings undergo hydroxylation very slowly and in some cases become resistant. Clonidine (antihypertensive) undergoes little aromatic hydroxylation as the ring is deactivated due to presence of two chlorine atoms. Probenecid (uricosuric agent) does not
undergo aromatic hydroxylation, as the aromatic ring is deactivated [10–14].
Probenecid
Clonidine
Amphetamine
CH
NH
2
Fig. 6.7: Effect of substituents on aromatic hydroxylation.
Cl
3
Cl
H
N
+
N
N
H
H
COOH
SO2N(CH2CH2CH3)
2
In drugs with two or more aromatic rings, hydroxylation occurs preferentially in the
more electron rich ring. Diazepam and chlorpromazine are the two drugs that preferably undergo hydroxylation in the electron rich ring (Fig.6.8).
116 Komarla Kumarachari Rajasekhar*
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Diazepam Chlorpromazine
H
O
N
S
Cl
Fig. 6.8: Compounds with two aromatic rings undergoing preferential aromatic hydroxylation.
N
N
CH
2CH2CH2
Cl
N(CH3)
2
6.3.1.1 Environmental pollutants and their toxicity
Environmental pollutants like polychlorinated biphenyls (PCB) and tetrachloro
dibenzo dioxin (T
CDD) carry a higher number of chlorine atoms that are responsible
for resistance to aromatic hydroxylation and high lipophilicity. Therefore, they remain
in active form in a biological system for longer periods resulting in toxicity [15].
R
Spontaneous
rearrangement
O
H
2
R
+
– O H
R
OH
R
NIH
Shift
H
Trans-dihydrodiols
OH
R
H
H
O
R
OH
O
Fig. 6.9: Fate of arene oxide.
GSH
Toxic eect
Glutathione adducts
OH
GS
R
Macromolecular adduct
OH
M
6Drug metabolism 117
6.3.1.2 Formation and fate of arene oxide
Arene oxide is formed when a double bond in aromatic moiety is epoxidized. It binds
cov
alently with nucleophilic groups in proteins, DNA and RNA leading to toxicity
(Fig.6.9). However, it can be detoxified in three ways i.e. spontaneous rearrangement
to arenols, enzymatic hydration to trans-dihydrodiols and enzymatic conjugation
with glutathione (GSH).
Arene oxide undergoes spontaneous rearrangement into arenol metabolite
(Fig.6.10). This is accompanied by a novel intramolecular hydride (deuteride) migration called “NIH shift” (NIH-National Institute of Health shift or 1, 2-deuteride shift).
Arene oxide ring (epoxide ring) opens in the direction that generates most resonance
stabilized carbocation (the charge on C-3 is resonance stabilized by methoxy group).
In this mechanism, deuterium is retained in the molecule [16].
OCH
3
+
D
OH
D
OCH
OCH
3
H
O
species
3
NIH
Shift
+
H
–
DO
OCH
3
–H
D
H
O
Dienone 3-deuterio-4-hydroxy
anisole
OCH
3
D
4- Deuterio anisole Arene oxide Zwitter ionic
Fig. 6.10: Spontaneous rearrangement of arene oxide into arenol metabolites (NIH shift).
Arene oxide is detoxified by nucleophilic attack of water on the epoxide (arene
oxide) and yields inactive trans–dihydrodiol metabolites (Fig.6.11). Hepatic microsomal epoxide hydrase is the catalyst in this reaction and is inhibited by chemicals
like cyclohexene oxide and trichloro propeneoxide (Fig. 6.12). Trans-dihydrodiols
obtained from arene oxide undergo enzymatic dehydrogenation and conjugate with
glucuronic acid. Naphthalene and benzo[a]pyrene are the examples for enzymatic
dehydrogenation. Glutathione-S-transferases (GSH-S-transferases) catalyze the conjugation of arene oxide with glutathione [17].
RR
Arene oxide
[toxic]
Detoxification
Enzymatic hydration
Epoxide hydrases
O
OH
OH
Trans-dihydrodiols
[non toxic]
Fig. 6.11: Detoxification of arene oxide via enzymatic hydration.
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Cl
Cl
Cl
O
1,1,1-trichloro propene-2,3-oxideOCyclohexene oxide
Fig. 6.12: Inhibitors of hepatic microsomal epoxide hydrase.
Arene oxides are highly electrophilic and reactive metabolites that react with nucleophilic groups present in biomacromolecules like DNA, RNA and other cellular components. This reaction carries undesired modifications in biomacromolecules leading to
irreversible damage and cellular toxicity [18]. Bromobenzene is a classic example of
cellular toxicity caused by arene oxide metabolite (Fig.6.13).
Br
Br
4 - Bromobenzene Epoxide metabolite
Fig. 6.13:
Cellular toxicity of bromobenzene.
Br
GSH
M
O
Covalent binding with
macromolecules
OH
SG
6.3.2 Oxidation of olefins
The metabolic oxidation of olefinic double bonds leads to corresponding epoxide
ane). These epoxide metabolites are more stable than arene oxide metabolites
(oxir
formed from aromatic compounds. Epoxide metabolites also undergo enzymatic
hydration by epoxide hydrases into trans-1,2-dihydrodiols or 1,2-diols. In addition
to enzymatic hydration, epoxides undergo conjugation reaction with glutathione
[19]. Carbamazepine (antiepileptic) undergoes olefinic oxidation and yields epoxide
metabolite which is pharmacologically active. Other drugs (Fig.6.14) which undergo
olefinic oxidation are protryptyline (antipsychotic), cyproheptadine (H
-antihista-
1
minic), alcofenac (anti-inflammatory) and secobarbital (sedative).
Isolation of GSH or mercapturic acid metabolites in a biological system provides
indirect evidence for the formation of epoxides (Fig.6.15). For example, styrene in
rats produces two isomeric mercapturic acid metabolites resulting from nucleophilic
attack of GSH on styrene epoxide [20].
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