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Hydrolysis
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Phase: IHYDROLYSIS OF
XENOBIOTICS
ENZYMES
Phase 1 reaction. (Non synthetic phase).
• Oxidation, reduction or hydrolysis.
• Oxidation- MICROSOMAL ENZYMES- located in the Smooth endoplasmic reticulum in Liver and kidney, intestinal mucosa, lung
• Eg:Monooxygenases, Cyp450. Glucoronyl transferases
• Inducible by drugs, diet and other factors
• Lesser polymorphism
Phase II reaction. (Synthetic phase)
• NON MICROSOMAL Enzymes- in cytoplasm and mitochondria of hepatic cells and other tissues ( plasma)
• Eg: flavoprotein oxidases, esterases, amidases, conjugases, all conjugations ( except glucoronidation)(some ,reduction enzymes)
• Not inducible • Genetic polymorphism
(acetyltransferase,pseidocholinesterase)
Hydrolytic Reactions
■ Enzymes: Non-microsomal hydrolases; however, amide hydrolysis appears to be mediated by liver microsomal amidases, esterases, and deacylases
■ Electrophilicity of the carbonyl carbon, Nature of the heteroatom, substituents on the carbonyl carbon, and substituents on the heteroatom influnce the rate of hydrolysis
■ In addition, Nucleophilicity of attacking species, Electronic charge, and Nature of nucleophile and its steric factors also influence the rate of hydrolysis
R1 R2 Name Susceptibility to Hydrolysis
C O Ester Highest
C S Thioester
O O Carbonate
C N Amide
O N Carbamate
N N Ureide Lowest
Table: Naming carbonyl - heteroatom groups
Hydrolyzes (adds water to) esters and amides and their isosteres; the OH from water ends up on the carboxylic acid (or its isostere) and the H in the hydroxy or amine
R1 C R2
O
+
NON MICROSOMAL : ENZYMES IN CYTOSOL (the soluble fraction of the cytoplasm):
• (a) Phase I: alcohol dehydrogenase, aldehyde reductase, aldehyde dehydrogenase, epoxide hydrolase, esterase.
• (b) Phase II: sulfotransferase, glutathione S-transferase, N-acetyl transferase, catechol 0-methyl transferase, amino acid conjugating enzymes.
MITOCHONDRIA. • (a) Phase I: monoamine oxidase, aldehyde
dehydrogenase, cytochrome P450.• (b) Phase II: N-acetyl transferase, amino acid
conjugating enzymes.
LYSOSOMES. Phase I: peptidase.
NUCLEUS. Phase II: uridine diphosphate
HYDROLYSIS The hydrolytic reactions, contrary to oxidative or reductive
reactions, do not involve change in the state of oxidation of the substrate,
But involve the cleavage of drug molecule by taking up a molecule of water.
The hydrolytic enzymes that metabolise drugs are the ones that act on endogenous substances, and their activity is not confined to liver as they are found in many other organs like kidneys, intestine, plasma, etc.
A number of drugs with ester, ether, amide and hydrazide linkages undergo hydrolysis.
Examples are cholinesters, procaine, procainamide, and pethidine.
HYDROLYSIS
Carboxylesterases: the hydrolysis of xenobiotic esters and
amides in humans is largely catalyzed by just two
carboxylesterases called hCE1 and hCE2.
Cholinesterases (AChE and BChE): hydrolyze bambuterol,
chlorpropaine, cocaine, methylprednisolone acetate, heroin,
isosorbide diaspirinate, mivacurium, procaine, succinylcholine,
tetracaine, and other drugs.
Paraoxonases (Lactonases)
Prodrugs and Alkaline Phosphatase
Peptidases
Epoxide Hydrolases
ESTERASES
• Esters, Amides, Hydrazides, and carbamates are hydrolyzed• Hydrolysis in plasma mainly by cholinesterase (nonspecific
acetylcholine esterases, pseudocholine esterases, and other esterases)
• In the liver by specific esterases for particular groups of compounds.
• Rate of Enzymatic hydrolysis of esters and amide: Role in the onset of pharmacological activity and its duration
• SUBCELLULAR LOCALIZATION. Endoplasmic reticulum and cytosol.
• TISSUE DISTRIBUTION. Ubiquitous, liver (centrilobular region), kidney (proximal tubules), testis, intestine, lung, plasma, and red blood cells.
ESTERASES
SUBSTRATES. Esters and amides.REACTIONTYPE. Hydrolysis:• (a) Hydrolysis of esters: R1–CO–OR2 R1–COOH + R2–OH• (b) Hydrolysis of amides: R1–CO–NH-R2 R1–COOH + R2–NH2• Hydrolysis of amides can occur by amidases in the
liver and in general, enzymatic hydrolysis of amides is slower than that of esters.
• Amides , also hydrolyzed by esterases with a much slower rate than the corresponding esters.
Carboxylesterases and cholinesterases
• Serine esterases • The catalytic site contains a nucleophilic serine residue- participates
in hydrolysis of various xenobiotic, endobiotic substrates. • Carboxylesterases : serum, liver, intestine, and other tissues• Cholinesterases in blood (and muscles depending on the route of
xenobiotic exposure) • Collectively determine the duration and site of action of certain
drugs. • Eg: Procaine( ester—is rapidly hydrolyzed, a local anesthetic.)• Procainamide, the amide analog of procaine, ( hydrolyzed slowly;
cardiac arrhythmia.) • The hydrolysis of xenobiotics by carboxylesterases , other hydrolytic
enzymes is not always a detoxication process
1. Dimethyl ester of succinic acid carboxyl esterase methanol and succinic acid-epitehlium degeneration
2. Vinyl acaetate carboxyl esterase acetate and acetaldehyde- nasal tumors
CARBOXYLESTERASES• 60 kDa glycoproteins • Wide variety of tissues, including serum. ( in liver is associated with
the endoplasmic reticulum, also in lysosomes and cytosol. ) • The hydrolysis of xenobiotic esters and amides : largely catalyzed by
just two carboxylesterases called hCE1 and hCE2.• Human plasma does not contain carboxylesterases• Butyrylcholinesterases and Paraoxonases : responsible for the
hydrolysis of amide and ester-containing compounds in the plasma • Also hydrolyze Endogenous compounds : palmitoyl-CoA,
monoacylglycerol, diacylglycerol, retinyl ester, platelet- activating factor, and the synthesis of fatty acid ethyl ester ther esterified lipids.
Mechanism : analogous serine-proteases. • A charge relay among : a.a. residue : glutamate ,histidine,
nucleophilic residue :serine
Aldridge: Basis of their interaction with OP compounds, • A-esterases : hydrolyze OP compounds• B-esterases: inhibited by OP compounds • C-esterases : do not interact with OP compounds as. AConfusing : • because it divides the paraoxonases into the A- and C esterase class • The human paraoxonase hPNO1 hydrolyzes OP compounds and so
can be classified as an A-esterase• hPON2 and hPON3 can be classified as C-esterases because they do
not• hydrolyze OP compounds, nor are they inhibited by them)• Furthermore, carboxylesterases and cholinesterases, two distinct
classes• of hydrolytic enzymes, are both B-esterases according to Aldridge
because both are inhibited by OP compounds
ESTERASES
CHOLINESTERASES (AChE and BChE) • Acetylcholinesterase (AChE) : High activity toward acetylcholine• Butyrylcholinesterase (BChE, /Pseudocholinesterase): High activity
toward acetylcholine and butyrylcholine (and propionylcholine)• BChE can also hydrolyze: bambuterol, chlorpropaine, cocaine,
methylprednisolone acetate, heroin, isosorbide diaspirinate, mivacurium, procaine, succinylcholine, tetracaine
• Eserine is an inhibitor of both enzymes• BW84C51 is a selective inhibitor of AChE • Iso-OMPA is a selective inhibitor of BChE • Exist in six different forms with differing solubility in in three
states: soluble (hydrophilic), immobilized (asymmetric), and amphiphilic globular (membrane-bound through attachment to the phospholipid bilayer)
• Six forms : monomer (G1), dimer (G2), tetramer (G4), tailed tetramers (A4), double tetramers (A8), and triple tetramers (A12).
• All forms are expressed in muscle.• AChE, major form in brain: tetramer G4 (anchored with a
20-kDa side chain containing fatty acids)
• The major form in erythrocytes : the dimer G2 (anchored with a glycolipid-phosphatidylinositol side chain).
• BChE: major form in serum is the tetramer G4 (a
glycoprotein with Mr 342 kDa).• In both AChE and BChE, the esteratic site (containing the
active site serine residue) is adjacent to an anionic (negatively charged) site that interacts with the positively charged nitrogen on acetylcholine and butyrylcholine.
Carboxylesterases and cholinesterases• In blood and tissues play an important role in
limiting the amount of OP compounds that reaches AChE in the brain
• ChE inhibition : is the mechanism of toxicity of OP and carbamate insecticide
• 70-90% loss of AChE activity is lethal to mammals, insects, and nematodes
• An inverse relationship between serine esterase activity and susceptibility to the toxic effect of OP compounds
• Factors that decrease serine esterase activity potentiate the toxic effects of OP compounds
• Eg-susceptibility of animals to the toxicity of parathion, malathion, and diisopropylfluorophosphate (DFP) is inversely related to the level of serum esterase activity (which reflects both carboxylesterase and BChE activity).
• Esterases are not the only enzymes involved in the detoxication of OP pesticides.
• Certain OP compounds are detoxified by cytochrome
P450, flavin monooxygenases,and glutathione transferases.
• Paraoxonases, enzymes that catalyze the hydrolysis of certain OP compounds, appear to play only a minor role in determining susceptibility to OP
ESTERASES
POLYMORPHISM.• Approximately 2% of Caucasians have defective serum
cholinesterase activity
SPECIES DIFFERENCES• Activity is higher in small laboratory animals such as the rat
and mouse than in humans.
PARAOXONASES (LACTONASES) • .Calcium-dependent enzymes containing a
critical sulfhydryl (-SH) group; as such they are inhibited by EDTA, metal ions (Cu andBa), and various mercurials such as phenylmercuric acetate (PMA)
• Catalyze the hydrolysis of a broad range of organophosphates, organophosphinites, aromatic carboxylic acid esters, cyclic carbonates, and lactones
Three paraoxonases : hPON1, hPON2, hPON3. hPON1 : • Liver microsomes and plasma, where it is associated exclusively
with high-density lipoprotein (HDL) • protects against atherosclerosis by hydrolyzing specific derivatives
of oxidized cholesterol and/or phospholipids in atherosclerotic lesions
• Appreciable arylesterase activity and the ability to hydrolyze the toxic oxon metabolites of OPC insecticides
• hPON2 : several tissues, not in plasma
• hPON3: serum and liver and kidney microsomes
ALKALINE PHOSPHATASE• Luminal surface of the enterocytes lining the wall of the small
intestine.• Hydrolysis of the prodrugs releasing the active drug at the
surface of the enterocytes, where it can be readily absorbed.• Clinical applications in the treatment of certain cancers. • Eg: to activate prodrugs in vivo and thereby generate potent
anticancer agents in highly selected target sites (e.g., at the surface of tumor cells, or inside the tumor cells themselves)
• Eg: prodrugs, such as fosphenytoin and fosamprenavir
• The hydrolysis of valacyclovir to the antiviral drug acyclovir is catalyzed by a human enzyme named valacyclovirase (genesymbol: BPHL)
PEPTIDASE• Recombinant peptide hormones, growth factors, cytokines,
soluble receptors, and humanized monoclonal antibodies : administered parenterally are hydrolyzed in the blood, Lysosomes and tissues by a variety of peptidases: Aminopeptidases and Carboxypeptidases
• Hydrolyze amino acids at the N- and C-terminus, respectively• Endopeptidases, which cleave peptides at specific internal
sites (trypsin, for example, cleaves peptides on the C-terminal side of arginine or lysine residues)
• Peptidases cleave the amide linkage between adjacent amino acids, function as amidases.
• ,The active site of peptidases : serine or cysteine residue, which initiates a nucleophilic attack on the carbonyl moiety of the amide bond.( like carboxylesterases)
EPOXIDE HYDROLASES• Found in the microsomal fraction of virtually all tissues, including the liver, testis,
ovary, lung, kidney, skin, intestine, colon, spleen, thymus, brain, and hear• Catalyze the transaddition of water to alkene epoxides and arene oxides
(oxiranes),which can form during the cytochrome P450-dependent oxidation of aliphatic alkenes and aromatic hydrocarbons, respectively
• FIVE distinct forms of epoxide hydrolase in mammals:• Microsomal epoxide hydrolase (mEH, which is the product of the gene EPHX1),
soluble epoxide hydrolase (she, the gene product of EPHX2), cholesterol epoxide hydrolase, leukotriene A4 (LTA4, the gene product of LTA4H) hydrolase, and hepoxilin hydrolase ( last three- hydrolyze endogenous epoxides exclusively, and have virtually no capacity to detoxify xenobiotic oxides)
• mEH hydrolyzes a wide variety of xenobiotics with an alkene epoxide / arene oxide.
• sEH hydrolyzes some xenobiotic epoxides and oxides, such a trans-stilbene oxide, Important role in the hydrolysis of endogenous fatty acid epoxides, such as the epoxyeicosatrienoic acids (EETs) that are formed by epoxidation of arachidonic acid by cytochrome P450
•
EPOXIDE HYDROLASES• play an important role in detoxifying electrophilic epoxides
that might otherwise bind to proteins and nucleic acids and cause cellular toxicity and genetic mutations
• Many epoxides and oxides are intermediary metabolites formed during the cytochrome P450-dependent oxidation of unsaturated aliphatic and aromatic xenobiotics.
• These electrophilic metabolites might otherwise bind to proteins and nucleic acids and cause cellular toxicity and genetic mutations.
• In general, sEH and mEH are found in the same tissues and cell types that contain cytochrome P450.
• For example, the distribution of epoxide hydrolase parallels that of cytochrome P450 in liver, lung, and testis.
• Both enzymes are located in the centrilobular region of the liver (zone 3), in Clara and type II cells in the lung, and in Leydig cells in the testis.
EPOXIDE HYDROLASES• Epoxide hydrolase is one of several proteins (so-called
preneoplastic antigens) that are overexpressed in chemically induced foci and nodules that eventually develop into liver tumors.
• Several alcohols, ketones, and imidazoles stimulate microsomal epoxide hydrolase activity in vitro.
• Epoxide hydrolase cannot be inhibited by antibodies raised against the purified enzyme, but it can be inhibited by certain epoxides, such as 1,1,1-trichloropropene oxide and cyclohexene oxide, and certain drugs, such as valpromide (the amide analog of valproic a cid) and progabide, a γ –aminobutyric acid (GABA) agonist.
• These ltwo drugs potentiate the neurotoxicity of carbamazepine by inhibiting epoxide hydrolase, leading to increased plasma levels of carbamazepine 10,11-epoxide and presumably the more toxic 2,3-epoxide
Carboxylesterases and epoxide hydrolases
• Exhibit no primary sequence identity
• But they share similarities in the topology of the structure and sequential arrangement of the catalytic triad.
• Both are members of the α/β-hydrolase fold enzymes, a superfamily of proteins that include lipases, esterases, and haloalkane dehydrogenases
R1 C
O
O R2 R1 C
O
OH HO R2
R1 C
OHN R2 R1 C
O
OH H2N R2
O C O R2R1
O
HO C O R2R1
O
OH HO C OHR2
O
HO O C O O HH
+++
Carbonate Carbonic acid derivative Carbonic acid
O C NR1
O
HO C NR1
O
OH HO C OH
O
HN O C O O HH
+++
Carbamate Carbamic acid derivativeCarbonic acid
R2
R3
R2
R3
R2
R3
N C N
O
HO C N
O
NH HO C OH
O
HN O C O O HH
+++
Urea derivative Carbamic acid derivativeCarbonic acid
R3
R4
R3
R4
R2
R3
R1
R2
R1
R2
R1 CHN N
OR2
R3
R1 C OH
O
H2N NR2
R3
+
Hydrazide Hydrazine
Ester hydrolysis
Amide hydrolysis (slower)
Carbonate hydrolysis
Carbamate hydrolysis
Urea hydrolysis
Hydrazide hydrolysis
The Reactions
Drug Examples
H3COO
O
N
CH3
O
Cocaine
OHO
O
N
CH3
O
H3COO
N
CH3
HO+
Benzoylecgonine Methylecgonine
H3C O
O
O
OH
H3C OOH
O
OH
OH+
Aspirin Salicylic Acid
CH3
CH3N
H2N
O
O
CH3
CH3N
H2N
O
HN
Procainamide
Procaine
H2N
O
OH
Slow Hydrolysis
Rapid Hydrolysis
OH
OH3C O
CH3
Cl
O
N
Indomethacin
CH3
CH3
CH3
CH3
O
N
HN
Lidocaine
O
O
N
O
ON
NH2
N
NH3C
H3C
Prazosin
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