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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