drug metabolism

259RESONANCE � March 2014

GENERAL � ARTICLE

Drug MetabolismA Fascinating Link Between Chemistry and Biology

Nikhil Taxak and Prasad V Bharatam

KeywordsDrug metabolism, chemistry,CYP450, toxicity.

Nikhil is a DST InspireFellow and is pursuingPhD in NIPER, Mohali.His research pertains todrug metabolism andtoxicity. His hobbiesinclude playing table

tennis and reading novels.

Prasad V Bharatam is aProfessor in MedicinalChemistry in NIPER,

Mohali. He is interested inareas of theoretical

chemistry, drug metabo-lism, diabetes, malaria andsynthetic chemistry.

Drug metabolism involves the enzymatic conversion of thera-peutically important chemical species to a new moleculeinside the human body. The process may result in pharmaco-logically active, inactive, or toxic metabolite. Drug metabolicprocess involves two phases, the occurrence of which mayvary from compound to compound. In this article, we discussthe basics of drug metabolism, the process, metabolisingorgans and enzymes (especially CYP450) involved, chemistrybehind metabolic reactions, importance, and consequenceswith several interesting and significant examples to epitomizethe same. We also cover the factors influencing the process ofdrug metabolism, structure–toxicity relationship, enzyme in-duction and inhibition.

1. Introduction

Medicines are required for humans to cure diseases but at thesame time, they are foreign objects to the body. Hence, the humanbody tries to excrete them at the earliest. It is highly desirable thatthe medicines get eliminated from the human body immediatelyafter showing their drug action. The longer time the drug spendsin the body, the greater are its side effects. The human body hasa natural mechanism to eliminate these foreign objects (medi-cines). This is mainly facilitated by the process known as drugmetabolism.

Drug metabolism may be defined as the biochemical modifica-tion of one chemical form to another, occurring usually throughspecialised enzymatic systems. It often involves the conversionof lipophilic chemical compounds (drugs) into highly polar de-rivatives that can be easily excreted from the body. (See Figure 1.)

260 RESONANCE ��March 2014

GENERAL � ARTICLE

Since lipid soluble substances undergo passive reabsorption fromthe renal tubules into the blood stream, they accumulate in the bodyand lead to toxic reactions. To avoid the same, the body has beenprovided with an armoury in the form of a metabolic system. Thissystem transforms lipophilic, water-insoluble and nonpolar drugsinto more polar and water-soluble metabolites, easily excretableform the body. Thus, fittingly and aptly, drug metabolism is termedas a detoxification process [1,2]. Some of the metabolites arerequired for drug action. This phenomena is being exploited in thedesign of prodrugs1. However, the samemetabolic process can alsolead to the generation of reactive metabolites (RM), which aretoxic to the human body. This is termed as bioactivation of drugs,which depends specifically on important structural features presentin these compounds [3]. Attempts are being made to bypass theformation of such reactive metabolites. To carry out all thesemetabolic reactions, catalytic machinery present in several organsin the human body is required, as discussed in the next section.

2. Metabolic Organs

The chemistry of drug metabolism needs an elaborate understand-ing – it is a fascinating and a complicated process. The primary siteof drug metabolism is the smooth endoplasmic reticulum of theliver cell. This is because of the presence of large amounts of manyvarieties of enzymes. The drug metabolism happening in the liveris termed as hepatic metabolism. In addition to the liver, everybiological tissue of the body has the ability to metabolize drugs.The drug metabolism process occurring in organs other than the

Figure 1. General pathwayof drug metabolism.

1 See H Surya Prakash Rao,Capping Drugs: Developmentof Prodrugs,Resonance, Vol.8,No.2, 2003.


GENERAL � ARTICLE

liver is called extrahepatic metabolism. The other sites includelungs, kidney, placenta, epithelial cells of gastrointestinal tract,adrenals and skin [1]. However, these sites are involved to a limitedextent in this process. Most drugs (around 70%) undergo metabo-lism, which is catalyzed by enzymes present in the above-men-tioned sites.

3. Chemistry of Drug Metabolism

Drug metabolism is a chemical process, where enzymes play acrucial role in the conversion of one chemical species to another.The major family of enzymes associated with these metabolicreactions is the cytochrome P450 family. The structural featuresand functional activity of these enzymes comprise the bioinorganicaspects of drug metabolism as discussed in this section.

3.1 Bioinorganic Chemistry of Drug Metabolism

The cytochrome P450 (CYP) enzymes are also known as microsomalmixed function oxidases. The CYP enzymes are membrane-boundproteins, present in the smooth endoplasmic reticulum of liver andother tissues. They are the most important enzymes for Phase Ibiotransformation of drugs. These enzymes contain a heme pros-thetic group, where heme group is the iron-porphyrin unit [4,5,6]. Theoxidizing site in these enzymes is the heme centre, and is respon-sible for the oxidation of hydrophobic compounds to hydrophilicor more polar metabolites for subsequent excretion.

These are called CYP450 because the iron in reduced state canbind with high affinity to carbon monoxide and this CO-boundCYP complex shows a large UV absorbance at 450 nm. CYPscatalyze the transfer of one atom of oxygen to a substrate produc-ing an oxidised substrate along with a molecule of water, as shownin the equation below [7].

CYP Sub-H O2 H+ Sub-OH H2O

NADPH NADP+

Substrate/Drug OxidisedSubstrate/Drug

Cytochromescatalyzeoxidation,hydroxylation,sulfoxidation,epoxidation, etc.reactions.

CO-bound CYPcomplex exhibitsstrong absorbanceat 450 nm in UVspectrum; thusenzymes are knownas CYP450.


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The detailed mechanism of metabolism by CYPs has been de-scribed in the catalytic cycle shown in Scheme 1 [4, 6].

There are more than 300 different CYP enzymes, which havebeen grouped into several families and subfamilies basedon the amino-acid sequence. Out of these, 18 CYPfamilies have been identified in mammals, comprisingmajorly of families CYP1, CYP2 and CYP3. The per-centage contribution of various CYP isoforms in me-tabolism of drugs is given in Figure 2. A selected list ofdrugs which are metabolized by various families of CYPenzymes is shown in Table 1 [5,7].

As discussed before, CYP450 has a heme prosthetic group,whose iron atom can occur in two oxidation states: Fe2+ (reduced)

Scheme1.Thecatalytic cycleof CYP450, leading to theoxidation of substrate.

Figure 2. The percentagecontribution of various CYPisoforms in drug metabolism.

CYP Drugs Metabolised

1A2 Amitriptyline, clozapine2A6 Acetaminophen, amodiaquine, artesunate2C8 Paclitaxel, torsemide2C9 Diclofenac, ibuprofen, phenytoin2C19 Diazepam, omeprazole, phenytoin2D6 Amitriptyline, metoprolol, nortriptyline2E1 Enflurane, halothane3A4 Carbamazepine, erythromycin, zolpidem

Table 1. A selected list ofdrugs metabolized by vari-ous families of CYP enzymes.


GENERAL � ARTICLE

or Fe3+ (oxidized). In these enzymes, the heme prosthetic group isbound to polypeptide chain of several amino acids, through ionicinteractions and one Fe-S (cysteine) covalent bond. The activespecies of CYP450 enzymes is Cpd I, which is responsible for theoxidation reactions by these enzymes. The model of Cpd I (iron-oxo species (Fe=O) coupled to axial cysteine ligand) is shown inBox 1 [8].

A schematic representation of heteroatom oxidation catalyzed byCpd I is shown in Scheme 2. The first step involves the binding of

Box 1. Compound I (Cpd I) of CYP 450: The Active Species

Fe

SH

O

Cpd I is an iron(IV)-oxo heme-porphyrin radical cation species, which participates in the metabolic reactionmechanisms such as hydroxylation, epoxidation, S-oxidation, N-oxidation, etc. In the earlier studies, due to theproblems in detecting and characterizing Cpd I, various suggestions for alternative oxidant species, such asCpd 0 (ferric-hydroperoxo species) and Cpd II (one electron reduced species of Cpd I) were made. The detailedbiomimetic experimental studies showed a higher reactivity of Cpd I than Cpd 0 and therefore ruled out Cpd0 as a possible oxidant. Till date, all studies (computational as well as experimental) agree that only oneoxidant, namely Cpd I is active in CYP 450 enzymes. Since the experimental studies have failed to characterizeCpd I and have yet to resolve the uncertainty associated with the status as the primary oxidant of CYP 450enzymes, theoretical studies have been performed to establish the active species of this enzyme [8].

Figure A. 2D and 3D structures of themodel of Cpd I (iron(IV)-oxo heme-por-phine with SH- as the axial ligand).

Fe

O

S

X

N N

N N

Reactant Cluster Transition State

Fe

O

S

X

N N

N NFe

O

S

X

N N

N N

ProductCluster

Fe

O

S

X

N N

N N

Oxidized Product

X= heteroatom; for example, sulfur.

Cpd I(doublet and quartet)

Scheme 2. The most impor-tant portion of the catalyticcycle of CYP450, leading toheteroatom oxidation of sub-strate.


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substrate in the active site of the enzyme. This leads to theformation of a reactant complex; thereafter, the oxygen transferfrom Cpd I to the heteroatom of the substrate occurs [9]. Thisleads to the product complex where transfer of oxygen to theheteroatom is almost complete; however, the oxygen atom is stillconnected to heme-iron. The product containing the oxidizedheteroatom leaves the active site of the enzyme in the next step.

Other reaction mechanisms of drug metabolism similar to theheteroatom oxidation have been studied. Computational chemis-try methods provide mechanistic details of the bioinorganic chem-istry taking place inside the active site cavity of cytochromes.These methods include quantum mechanical (QM) and quantummechanical/molecular mechanical (QM/MM) methods. The com-putational studies employ the use of active species, Cpd I andutilize theoretical methods in elucidating the mechanism. Forexample, Shaik et al have discussed the process of CH hydroxy-lation, N-oxidation, S-oxidation, epoxidation of olefins, N-dealkylation in various substrates [9,10].

The reactionmechanism of CH hydroxylation is shown in Scheme 3.This involves an initial hydrogen abstraction from the alkane(RH) by Cpd I, followed by radical rebound on the iron-hydroxointermediate to generate the ferric-alcohol complex. Thereafter,this releases the alcohol and restores the resting state (i.e., thewater complex) as shown in Scheme 3.

Thiel and co-workers have further strengthened the mechanisticdetails through the use of QM/MM method, and established the

Cpd I (iron-oxo) isthe active species

of CYP450.

Scheme 3. The H-abstrac-tion and rebound mechanismfor CH hydroxylation.

Fe

O

S

N N

N N

Cpd I

Fe

HO

S

N N

N NFe

O

S

N N

N NFe

OH2

S

N N

N N

R-HHabstraction

RR rebound

R H

HOHROH


GENERAL � ARTICLE

basis behind several metabolic reactions, catalyzed by differentCYP isoforms in the body [10]. Visser and Shaik elucidated anovel mechanism of proton shuttle for CH hydroxylation ofbenzene [11]. A brief description of the mechanism is shown inScheme 4.

On similar lines, our group determined the mechanistic details ofthe process of S-oxidation of thiazolidinediones [12,13], and is asshown in Figure 3.

Thus, the mechanism varies for different reactions being cata-lyzed by CYPs; however, the active species (Cpd I) involved is thesame. The mechanisms of these metabolic reactions vary due tothe presence of different functional groups, such as S, N, alkyl,phenyl, alkene, alkyne, etc. Therefore, it becomes essential tounderstand the organic chemistry of metabolic reactions and theinfluence of steric hindrance and briefly structure–toxicity rela-tionship.

Fe

O

S

N N

N N

Cpd I

Fe

O

S

N NH

N N

Proton Shuttle Reprotonation

of phenolateH

H

HH

H

H

H

HH

H

H

Fe

S

N N

N N

HOH

HH

H

HScheme 4. The protonshuttle mechanism for CHhydroxylation in benzene.

Figure 3. The 3D structuresof transition state for S-oxi-dation of thiazolidinedionesby HOONO and Cpd I.


GENERAL � ARTICLE

3.2 Bioorganic Chemistry of Drug Metabolism

To fully understand the concept of drug metabolism, it is essentialto have knowledge of the types of drug metabolism reactions. Asdiscussed earlier, drug metabolism may be a detoxification pro-cess, or a bioactivation process, leading to varied consequences.The detoxification reactions can be divided into two broad cat-egories: Phase I (functionalization) and Phase II (conjugation)reactions [2]. The reactions may occur (i) sequentially, (ii) inde-pendently, or (iii) simultaneously. The bioactivation process ofdrug metabolism will be covered later in Section 3.3. The detailsof both types of detoxification reactions have been described inthe following section [2,7].

3.2.1 Detoxification Reactions: Phase I Reactions: These reac-tions are termed as the nonsynthetic reactions, and include oxida-tion, reduction, hydrolysis, cyclization and decyclization reac-tions. These reactions are carried out mostly by mixed functionoxidases, usually involving CYP450 and occur in the liver. Inthese reactions, a polar group is either introduced or unmasked ifalready present. These reactions are succeeded by Phase II reac-tions. Most of the Phase I products are not eliminated directly;instead they undergo Phase II reactions. Various Phase I reactionsare as follows, with several examples:

(i) Oxidation: This is the most commonly occurring reaction, byvirtue of which hydrophilicty of the substrates is increased via theintroduction of a polar functional group such as –OH. Thisreactionmay occur at several centres in drugs, and thus, oxidationreactions have been classified accordingly.

Oxidation at carbon centre: This includes oxidation at aromaticring, olefinic centre, and aliphatic groups. Important drugs under-going metabolism by this reaction include acetanilide (analge-sic), phenylbutazone (analgesic), valproic acid (antiepileptic),carbamazepine (antiepileptic), and minoxidil (antihypertensive).(See Table 2.)

Drugmetabolismreactions comprise oftwo phases: Phase I(functionalization)reactions such as

oxidation, hydrolysis;and Phase II

(conjugation) reactionssuch as

glucuronidation,sulphate conjugation.

Oxidationreactions are the

most common andvital. These mayoccur at carbon,sulfur or nitrogen

centre.


GENERAL � ARTICLE

ReactionCentre

Chemical Reaction on Drug substrate

Aromatic ring NHCOCH3HO

Acetanilide Paracetamol

NHCOCH3

Olefinic centre

NCONH2

NCONH2

O

NCONH2

HO OH

Carbamazepine Carbamazepine-10,11-epoxide

Trans-10,11-dihydroxycarbamazepine

Aliphaticgroups H3C

HC

CHH2CH3C

COOH

H3CH2C

CHH2C

H2C

COOH

H3CH2C

CHHCH3C

COOH

HO

OH

�-oxidatio

n

�-1-oxidationValproic acid

5-Hydroxy valproic acid(minor product)

4-Hydroxy valproic acid(major product)

Oxidation at carbon-heteroatom systems: This involves reactionon C–N, C–S and C–O systems. The oxidation reactions on C–Nsystems comprise of N-dealkylation, oxidative deamination, for-mation of N-oxide, or N-hydroxylation. The process of N-dealkylation occurs in the substrates having alkyl group attacheddirectly to nitrogen atom. Examples include: methamphetamine(antidepressant) which gets metabolized to amphetamine via N-dealkylation. Amphetamine further gets metabolized tophenylacetone and ammonia via oxidative deamination. (SeeTable 3.)

The reactions in C–S systems may involve S-dealkylation,desulfuration and S-oxidation. The mechanism of S-dealkylationproceeds via �-carbon hydroxylation, e.g., 6-methylmercaptopurine.The barbiturate drug, thiopental undergoes conversion to pentobar-bital via desulfuration, where the cleavage of C=S bond occurs,and the product with C=O is formed. The S-oxidation reaction isobserved in substrates with sulfide unit (-S-). Examples of drugsundergoing S-oxidation are methimazole (antithyroid), cimetidine(antihistaminic), ranitidine (antihistaminic), chlorpromazine

Table 2. Metabolic oxidationon carbon centre of variousdrug molecules.

S-oxidation reactionoccurs in severalclasses of drugs withsulfide unit such asantidiabetics,antihistaminics andantipsychotics leadingto the formation ofsulfoxidemetabolite.


GENERAL � ARTICLE

(antipsychotic), etc. Thiazolidinedione category of antidiabeticdrugs such as rosiglitazone and pioglitazone also undergo S-oxidation reaction. The mechanistic details of S-oxidation havebeen elucidated using theoretical chemistry [12,13].

O-dealkylation, oxidative dehalogenation and oxidative aromati-zation are other important oxidation reactions at carbon centre.Drugs such as phenacetin (analgesic) and codeine (analgesic,antitussive) undergo metabolism by O-dealkylation. Halothane(general anaesthetic) undergoes metabolism by oxidativedehalogenation.

(ii) Reduction: The reduction reactions result in the generation ofpolar functional groups such as amino and hydroxyl, which mayundergo further metabolic reactions. These reactions may occuron several functional groups such as carbonyl, hydroxyl, etc., asshown in Table 4.

Table 3. Metabolic oxidationon carbon-heteroatom sys-tems of various drug mol-ecules.

Reaction Chemical Reaction on Drug Substrate

Deamination CH2CHCH3NH2 H CH

O

Methamphetamine Amphetamine Formaldehyde

Amphetamine

CH2CCH3O NH3

Phenylacetone Ammonia

CH2CHCH3NHCH3

CH2CHCH3NH2

CyclizationCl

N

NH2N

NH2

NH

CH3

H3C

Cl

N

NH2N

N

H2N

H3C CH3

Proguanil (Prodrug) Cycloguanil(Active Antimalarial metabolite)

CYP2C19

[O]HOH

Water

S-dealkylationN

N NH

N

SCH3

N

N NH

N

SCH2OH

N

N NH

N

SH

H CHO

6-Methylmercaptopurine

Hydroxylatedintermediate

6-Mercaptopurine Formaldehyde


GENERAL � ARTICLE

The reduction reaction can take place in aliphatic and aromaticaldehydes and ketones. Drugs such as methadone (analgesic),chloral hydrate (sedative and hypnotic) and naltrexone (manage-ment of alcohol dependence) undergo this metabolic process. TheN-containing compounds having nitro, azo or N-oxide undergothis metabolic reaction. For example: nitrazepam (hypnotic andanxiolytic) and prontosil (antibacterial antibiotic), which getreduced to the corresponding amines. The halogen atom presentin various drugs may undergo reduction via replacement by H-atom, e.g., halothane.

(iii) Hydrolysis: These reactions generally involve a large chemi-cal change in the substrate. The hydrolysis reactions can occur inthe following functional groups, as listed in Table 5. Uponhydrolysis, esters lead to the formation of carboxylic acids andalcohols. Mostly, esters are administered as prodrugs, which onhydrolysis are converted to active forms, e.g., aspirin (analgesic,antipyretic). Drug molecules containing amide functionality un-dergo slow hydrolysis as compared to esters. These reactions arecatalyzed by amidases. The reaction occurs in secondary andtertiary amides, and rarely in primary amides, e.g., procainamide(antiarrhythmic).

Table 4. Metabolic reductionreaction of various drug mol-ecules.

ReactionCentre

Chemical Reaction on Drug Substrate

Carbonyl Cl3C CHO.H2O Cl3C CH2OH

Chloral Hydrate Trichloroethanol

Nitrogen (N=N)N N SO2NH2

NH2

H2N NH2

NH2

H2N SO2NH2H2N

Prontosil 1,2,4- Triaminobenzene Sulfanilamide

Halogens

H

Halothane

C C

Cl

Br F

FF H C C

Cl

H F

FF C C

Br

H

F

H

Cl

F

F

Hydrolysis reactionsare commonlyencountered in themetabolism of esterand amide prodrugs,leading to thegeneration of theactivemetabolites.


GENERAL � ARTICLE

3.2.2 Detoxification Reactions: Phase II Reactions: The PhaseII reactions follow Phase I reactions, and occur mostly in theproducts derived from Phase I reactions. In these reactions, asuitable moiety such as glucuronic acid, glutathione, sulphate,glycine, etc., get conjugated to the metabolites of Phase I reac-tion. The Phase II reactions are the real drug detoxificationpathways. These are also termed as conjugation reactions, be-cause the metabolites are conjugated with the above-mentionedmoieties which are large in size and strongly polar in nature.These reactions are catalyzed by a variety of transferase enzymes,such as uridine diphosphate (UDP)-glucoronsyltransferases,sulfotransferases, glutathione transferases. These transferasesalso exist as a superfamily of enzymes, similar to CYPs, but theydiffer in the number of drugs they metabolize (lesser than CYPs).Various conjugation reactions along with the enzymes involvedand examples of drugs undergoing the same are listed in Table 6.Glutathione conjugation is shown in Scheme 5.

Table 5. Metabolic hydroly-sis reaction of various drugmolecules having differentfunctional groups.

HN

HOO

N

O O

HN

HOO

N-hydroxylation and

rearrangement

Paracetamol NAPQI

GSH conjugation

Conjugated product

HOOCO

NH

S

O

HN COOH

NH2

Scheme 5. A representativescheme showing glutathioneconjugation as the Phase IImetabolic reaction in thebody.

ReactionCentre

Chemical Reaction on Drug Substrate

Esters and ethersOCOCH3

COOH

OH

COOH

CH3COOH

Aspirin Salicylic acid (active)

Amides

H2N COOH

Procainamide p -amino benzoic acid (PABA)

H2NCNH

NO

H2NN


GENERAL � ARTICLE

Conjugated Group Endogenous Enzyme DrugsCofactor

Glucuronidation UDP glucoronic UDP- Chloramphenicol,acid glucuronosyltransferase morphine, paracetamol,

salicylic acid,fenoprofen,desipramine,meprobamate,cyproheptadiene

Sulphation Sulphate Sulfotransferases Paracetamol, salbutamol

Glycine/Glutamine Glycine/glutamine N-acyl transferases Cholic acid,conjugation salicylic acid, nicotinic

acid, phenylacetic acid

Glutathione Glutathione Glutathione S Paracetamol, ethacrynicconjugation -transferases acid

Acetylation Acetyl-CoA N-acetyl transferases Histamine, mescaline,procainamide, p-aminosalicylic acid, isoniazid,phenelzine, hydralazine,dapsone

Methylation S-adenosyl-L- Methyl transferase Morphine, norephedrinemethionine nicotine, histamine, iso-

prenaline, propylthiou-racil

Table 6. The conjugation re-actions along with the en-zymes involved and the ex-amples of drugs metabolizedby the same pathway.

Thus, we have listed both Phase I and II reactions, and thefunctional groups on which different reactions take place in thebody. We have covered the detoxification pathways of drugmetabolism; however, we still have to understand the toxiceffects of drug metabolism via bioactivation process.

3.3 BioactivationReactions:Chemistry of ReactiveMetabolitesand Adverse Drug Effects

The drug metabolism process can lead to the generation ofReactive Metabolites (RM) in some instances, which further leadto toxicity. The toxicity may vary from genotoxicity to immune-


GENERAL � ARTICLE

mediated Adverse Drug Reactions (ADRs). These adverse drugreactions are major obstacles of drug therapy, and to clinical drugdevelopment. The organ most affected by adverse drug reactionsis the liver, which is responsible for most of the metabolicreactions [3]. These RMs are specifically termed as ChemicallyReactive Metabolites (CRMs) because they possess a chemicallyreactive group, which leads to ADRs.

3.3.1 Structural Alerts: Several functional groups associatedwith the generation of CRMs constitute the structural alerts.Somewell-known structural alerts are epoxides, furan, thiophene,anilines, anilides; arylacetic acids; hydrazines; thiophenes; ter-minal alkenes; nitroaromatics; quinones, quinone-methide, etc.,as shown in Figure 4.

However, just the presence of these substructures that may formCRMs may not indicate the type and severity of adverse reac-tions. Many drugs present in the market contain one or the otherstructural alert, because of their higher benefit-to-risk ratio, e.g.,aniline sulphonamides as anti-infective agents. In some cases,toxic effects are not seen to a large extent owing to extensiveclearance. For example, raloxifene (for treatment of osteoporo-sis) gets cleared by glucoronidation and sulphation of phenolicOH groups, thereby minimising the amount of quinone andquinone-methide reactive metabolites formed [14].

The knowledge of structural alerts has been utilised successfullyin the early phase of drug discovery, as in the case of bromofenac(ocular pain relief) that contained aniline, arylacetic acid and abromophenyl ring as structural alerts.

A structural alert which has caught everyone’s attention is thecarboxylic acid. Most of the Non-Steroidal Anti-InflammatoryDrugs (NSAIDs) such as aspirin, diclofenac, etc., contain a

O

ORNHO S C CR C CHRR1

O

R R1RHN

Figure 4. Structural alertsassociated with the genera-tion of reactive metabolites inthe body.

Majority of adversedrug reactions occurowing to the formation

of reactivemetabolites. Theseare formed in the

metabolism of drugswith functional groups

such as furan,thiophene, epoxide,

etc.


GENERAL � ARTICLE

carboxylate function, and cause a wide range of ADRs. There-fore, it is important to understand the importance of severalfunctional groups which may lead to toxic reactions in the body.This can be represented by structure–toxicity relationships asdiscussed in the next section.

3.3.2 Structure–toxicity relationship: Several methods can beutilized to qualify certain functional groups as the structuralalerts. One methodology uses DEREK software. The computerprogram DEREK (an acronym for Deductive Estimation of Riskfrom Existing Knowledge) is designed to assist chemists andtoxicologists in predicting likely areas of possible toxicologicalrisk for new compounds, based on an analysis of their chemicalstructures. It is a knowledge-based expert system to identifyvarious structural alerts, and the predictions are based in part onvarious alerts that describe structural features or toxicophoresassociated with toxicity. However, no in silico tools are availablethat can be utilized in predicting the occurrence of bioactivationand further toxicity in a drug candidate. Computational tools suchas MetaSite [3] which can predict metabolic transformations ofdrug candidates, are available. It is a computational procedurewhich provides the structure of the metabolites formed with aranking derived from the site of metabolism predictions. It pre-dicts ‘hot spots’ in the molecule to help chemists focus theirdesign of compounds to reduce CYP-mediated metabolism. Italso highlights the molecular moieties that help to direct themolecule in the cytochrome cavity such that the site of metabo-lism is in proximity to the catalytic centre. However, it cannotpredict the bioactivation pathway of a drug candidate.

Since the prediction of bioactivation pathways of possible drugcandidates is a daunting task, a knowledge of structural alertswould aid in an efficient drug design, such that the structural alertexisting in a toxic predecessor, would be absent in a successfulsuccessor drug candidate. This type of relationship is referred toas ‘structure–toxicity’ relationship. Table 7 lists a set of examplesof drug molecules, where the presence or absence of a functionalgroup or an atom, results in toxic or therapeutic activity [3,14,15].

Molecularmodelingmethods have seenresurgence in theprediction ofmetabolic reactionsand metabolites indrugs, which helpsthe chemists in theproper design ofmolecules.

Computationaltools help inidentifyingstructural alerts,and thus, becomeuseful during theearly stages ofdrugdiscovery.


GENERAL � ARTICLE

3.3.3 Consequences of Reactive Metabolite Formation: In mostcases, the reactive metabolites (RMs) formed by CYP-mediatedmetabolism lead to the inactivation of enzyme, which may bereversible or irreversible in nature. The irreversible inactivation orinhibition of enzyme is termed as mechanism-based inactivation or

Table 7. Differential metabo-lism of drugs, where one istoxic and the other non-toxic,representing structure–toxic-ity relationship.

PredecessorDrug

Toxic Reaction Pathway Successor Drug(devoid oftoxicity)

Clozapine

(hepatotoxicity,agranulocytosis)

Clozapine Reactive iminium

GSH

NH

NN

N

Cl P450

N

NN

N

ClNH

NN

N

Cl

NH

NN

N

Cl

Covalent adducts

SG

SG

S

NN

N(CH2)2

Cl

OCH2CH2OH

Quetiapine

X

Quetiapine(rareagranulocytosis)

Halothane(hepatotoxin)

Halothane Reactive acyl chloride

Cl

F3C Br

P450 Cl

F3C Br

OH

HBr

O

F3C Cl

~20%

Isof luorane

Cl

F3C O

P450

HOCHF2

O

F3C Cl

~ 0.2%

F

FCl

F3CO

F

F

OH

Desf luorane

F

F3C O

P450

HOCHF2

O

F3C F

~ 0.02%

F

FF

F3CO

F

F

OH

Isofluorane,desfluorane(nohepatotoxicity)

Tolcapone(idiosyncratichepatotoxicity)

HO

Tolcapone

HO

HO

HOO

NCN

O

NO2

NO2

Entacopone

HO

HO

NH2

HO

HO

HN

O

O

HO

N

OX

P450 GSH

HO

HO

HN

O

SG

Reactive Quinone-imine

NAT

Entacopone(nohepatotoxicity)


GENERAL � ARTICLE

inhibition (MBI) of CYPs. The name is apt because a mechanismprecludes enzyme inactivation [3,6]. This inactivation may be classi-fied into two categories based on the mechanism:

(i) Formation of metabolic–intermediate complex (MIC) be-tween reactive species and heme-iron, via coordination: Thealiphatic and aromatic amines (nortriptyline, antidepressant;tamoxifen, anticancer) generate reactive nitroso (R-N=O) inter-mediate after various metabolic transformations, such as N-dealkylation, N-hydroxylation, etc. On the other hand, 1,3-benzdioxole moiety containing drugs lead to the generation ofreactive carbene intermediate. Both the intermediates are nucleo-philic in nature, and form a coordination bond with heme-iron asshown in Figure 5. This coordination is termed as the metabolic–intermediate complex (MIC), which leads to inhibition of CYPactivity.

Scheme 6 shows the formation of reactive carbene intermediatefrom 1,3-benzdioxole moiety. Drugs such as stiripentol(antiepileptic), paroxetine (antidepressant), etc., possess thismoiety, which acts as a perpetrator, and leads to mechanism-based inhibition of CYPs [3,6].

O O

MI Complex

R

Carbene

Fe

S

N N

N N

RNO

MI Complex

Fe

S

N N

N N

Nitroso

Figure 5. A representation ofcoordination between reac-tive carbene/nitroso speciesand heme-iron, leading to for-mation of metabolic–interme-diate complex (MIC).

O

O

O

O OHH

R R

O

OMI Complex

RP450 P450

H2O Carbene

Scheme 6. Schematic path-way for the formation of reac-tive carbene species, start-ing from 1,3-benzdioxolemoiety.


GENERAL � ARTICLE

(ii) Covalent modification of reactive species with heme oramino-acid residues: Drugs containing the thiophene, furan, ole-fin, phenyl group may lead to the mechanism-based inhibition ofCYPs by generating reactive epoxide intermediate. The epoxidering opening by nucleophilic active site residues lead to forma-tion of a covalent bond, thus inhibiting CYP activity. Otherreactive species leading to MBI via covalent bond formationinclude quinone-imine and quinone-methide. Scheme 7 shows theformation of reactive epoxide intermediate from furan moietypresent in a few drugs such as L-754,394 (HIV protease inhibi-tor).

Paracetamol is the other example of a drug, whose metabolitequinone-imine (N-acetyl p-benzoquinone-imine; NAPQI) is re-active, and depletes GSH levels by binding covalently to livermacromolecules (Scheme 8). This results in hepatic necrosis inthe body.

This CYP inactivation further translates into drug–drug interac-tions (DDIs), which could be deleterious to the body. For

O

P450

O

O

OHO

HO O

OS NH2

COOH

cis-2-butene-1,4-dial

Covalent Modification

P450 INACTIVATION

Nucleophilic amino acids (cysteine)

Scheme 7. Schematic path-way for the formation of reac-tive epoxide intermediate andnucleophilic adducts leadingto MBI of CYPs, starting fromfuran moiety.

NH CO

OH O

N CO

NH CO

OHSG

NAPQI

N-hydroxylation

and rearrangement

GSH conjugation

Toxic reactions with proteins and nucleic acids

Scheme 8. Schematic path-way for the metabolism ofparacetamol to the toxicNAPQI metabolite.


GENERAL � ARTICLE

example, increased cases of myopathy (muscular weakness) andrhabdomyolysis (breakdownproducts of damagedskeletalmusclesreleased in blood stream) have been observed in hypertensivepatients who are being administered a calcium-channel blockerand a potent CYP3A4 inactivator mibefradil together withsimvastatin. Here the mechanism for DDI involves the inactiva-tion of CYP-catalyzed metabolism process of simvastatin bymibefradil, which further results in the elevation of plasma levelsof statin, leading to death. Owing to such fatal consequences,mibefradil was withdrawn from the market later [3].

Further examples include tadalafil, a drug used for treating erec-tile dysfunction, which is reported to be metabolized by CYP3A4and also has inhibitory effects on CYP3A4. The inhibitory effectsare proposed to be due to the presence of 1,3-benzodioxole group,which after metabolism gives rise to the corresponding carbeneintermediate followed by its coordination with the heme iron asdiscussed before. Therefore, it has potential adverse effects onpatients in its long term use. Similarly, the antidepressant drug,paroxetine acts as the potent inhibitor of CYP2D6 by generatingreactive carbene intermediate, leading to a coordination bondformation with heme-iron. Since enzyme inactivation leads topotential DDIs, mechanism-based inactivation of major humanCYP enzymes by new compounds is being routinely assessed ina drug-discovery paradigm.

Till now, the basic aspects of drug metabolism, bioinorganic andbioorganic chemistry of drug metabolism, detoxification andbioactivation pathways of drug metabolism have been covered.Since the intensity, therapeutic efficacy, toxicity, biological half-life and duration of pharmacological action of any drug is depen-dent on the rate of its metabolism, the determination of factorsinfluencing this process becomes essential.

4. Factors Influencing Drug Metabolism

Physicochemical parameters of the drug, biochemical factors likeCYP induction or inhibition influence drug metabolism, and lead

The inactivation ofCYPs leads todisruption of theenzymatic machinery,thus, hampering themetabolism of drugs.This leads to potentialdrug-drug interactionsand adverse drugreactions.


GENERAL � ARTICLE

to variations in the drug action in humans. Some important factorsare discussed here [2,16].

4.1 Physicochemical Properties of the Drug

These include the molecular size, shape, lipophilicity, acidity/basicity, electronic characteristics, pKa, etc. These propertiesinfluence the interaction of drug with the metabolizing enzymesand control the drug action.

4.2 Biochemical Factors

4.2.1 Metabolic Enzyme Induction: Any process that increasesthe rate of metabolism of a drug is termed as the metabolicenzyme induction, which results in a decrease in the duration andintensity of the drug action. Agents which carry out such effectsare termed as enzyme inducers. This increase in drug metabolismarises usually due to the increased synthesis of enzyme protein.For example: barbiturates (anxiolytics) are the inducers whichenhance the metabolism of coumarins, phenytoin, cortisol, test-osterone, etc., because of induction. Similarly, alcohol (CNSstimulant) increases the metabolism of pentobarbital, coumarinsand phenytoin because of induction. Environmental chemicalssuch as pesticides (DDT), and polycyclic aromatic hydrocarbonspresent in cigarette smoke are well known to be enzyme inducers.

4.2.2 Metabolic Enzyme Inhibition: Some chemical speciesblock the catalytic site of cytochromes and decrease the catalyticconversion of drugs to metabolites. This process is known asenzyme inhibition, which results in an increase in duration of thedrug in the body, thereby, leading to the accumulation of drug inthe body and also an increase in toxicity. For example: metacholine(anti-asthmatic) inhibits the metabolism of acetyl choline bycompeting with it for cholinesterase. Similarly, isoniazid (antitu-bercular) inhibits the metabolism of phenytoin. Such influence ofone drug on the metabolism of another drug leads to drug–druginteractions. Environmental chemicals such as heavy metals in-cluding nickel, mercury, arsenic are known to be potent enzymeinhibitors.

Enzyme induction andinhibition are oppositeprocesses with variedeffects on metabolicprocess and severeconsequences on the

drug action on thebody.

The rate of drugmetabolism is

influencedby severalfactors, such asphysicochemical

properties of drugs,biochemical andbiological factors

which thereby affectthe therapeutic

efficacy, toxicityandduration of

pharmacologicalaction of the drug.


GENERAL � ARTICLE

4.3 Biological Factors

The biological factors include species differences, strain differ-ences, pharmacogenetics, ethnic variations, gender differences,age, etc. Other biological factors include diet, pregnancy, hor-monal imbalance and presence of disease states in the individu-als. For example, the activity of drug-metabolising enzymesdecrease in people with cardiac failure. Similarly, hormonalfactors during pregnancy affect the metabolic process, usually inthird trimester, such as in case of anticonvulsant drugscarbamazepine and phenytoin. The circadian rhythm of an indi-vidual is also a major influencing factor on drug metabolism.Genetic polymorphism (two or more variants of an enzymeencoded by a single locus within the population) has appeared tobe the common phenomenon, leading to variations in metabolicprocess in humans. This results in a higher or lower activity forone form of polymorphic enzyme as compared to the other form(enzyme isoforms specificity). The enzyme polymorphism is aninherited process, and thereby a major cause of inter-individualdifferences with respect to the rate of drug metabolism. Theindividuals are classified accordingly as poor metabolizers andextensive metabolizers. For example, Caucasians are poormetabolizers as compared to Asians and blacks for CYP2D6 (anisoform of CYP450) [4]. All these factors have their variedinfluence on the rate of metabolic process, which has a criticalinfluence on its outcome, which may vary from a therapeutic totoxic activity as described in the next section.

5. Importance and Consequences of Drug Metabolism

The drug metabolism or biotransformation process results in avariety of consequences as listed below [1,2,16]:

• Pharmacological inactivation of drugs: Here, the metaboliteformed has little or absolutely no pharmacological activity. Themetabolite of phenytoin (antiepileptic), p-hydroxy phenytoin hasno pharmacological activity.

• No effect on pharmacological activity: Here, the metabolites

Geneticpolymorphism (two ormore variants of anenzyme encoded by asingle locus within thepopulation) hasappeared to be thecommonphenomenon, leadingto variations inmetabolic process inhumans.


GENERAL � ARTICLE

formed after biotransformation processes have equal and similaractivity to that of the original drug. Nortriptyline possesses equalactivity as that of the parent drug, amitriptyline (antidepressant).

• Toxicological activation of drugs: Here, the metabolites formedafter biotransformation process have a high tissue reactivity, thuscausing toxicity, which may include necrosis, hepatitis, etc. N-acetyl p-benzoquinoneimine which is a metabolite of paracetamolcauses hepatotoxicity. Other examples such as tadalafil havebeen discussed earlier.

• Pharmacological activation of drugs: By this process, prodrugs(inactive) are metabolised to highly active drugs. Prodrug is theinactive derivative that is converted to the active component invivo. These are generally devised when the drugs possess unat-tractive physicochemical and undesired pharmacokinetic proper-ties. These are used to improve patient acceptability, improveabsorption, alter metabolism, biodistribution and elimination.Ampicillin (antibiotic) is the active form of pivampicillin.

• Changed pharmacological activity: Here, the therapeutic activ-ity displayed by the metabolite formed is different from that of theoriginal drug. Isoniazid (antitubercular) is the metabolite of ipro-niazid (antidepressant).

6. Conclusions

Drug metabolism is a fascinating and useful link between chem-istry and biology, yet a complicated one. It may result in detoxi-fication or bioactivation of drugs. The detoxification processinvolves two phases of drug metabolism: Phase I converts highlylipophilic drug molecules to polar metabolites, whereas Phase IIinvolves conjugation reactions. The metabolism includes a vari-ety of reactions such as oxidation, reduction, hydroxylation,dealkylation, etc. Most of the metabolic reactions are carried outby a large family of heme-containing CYP enzymes. The activespecies of these enzymes is Cpd I (iron-oxo heme porphine withcysteine as the axial ligand), which is responsible for majoroxidation and hydroxylation reactions. Besides detoxification

Drugmetabolism trulylinks chemistry andbiology in a unique

and splendid manner,however,unraveling

the link is acomplicated task. Aspecial emphasis ondrug metabolism is anecessity in the fast

moving era of adversedrug reactions and

DDIs.


GENERAL � ARTICLE

reactions, these enzymes can result in the formation of reactivemetabolites which lead to toxicity and drug–drug interactions.Hence, extensive studies on drug metabolism to elucidate thescientific principles are in progress.

Acknowledgement

We would like to thank the reviewer for very valuable andthorough comments, which immensely helped in shaping thisarticle to suit the Resonance readers.

Suggested Reading

[1] M Coleman, Human Drug Metabolism: An Introduction, 1stEd., John Wiley & Sons, UK, pp.13–18, 2010.

[2] G G Gibson and P Skett, Introduction to Drug Metabolism, 3rd Ed.,Nelson Thornes Publishers, UK, pp.1–194, 2001.

[3] A S Kalgutkar and M T Didiuk, Structural Alerts, Reactive Metabo-lites, and Protein Covalent Binding: How Reliable Are These At-tributes as Predictors of Drug Toxicity?, Chemistry and Biodiversity,Vol.6, pp.2115–2137, 2009.

[4] H Liston, J Markowitz and C Devane, Drug Glucuronidation inClinical Psychopharmacology, Journal of Clinical Psychopharmacol-ogy, Vol.21, pp.500–515, 2001.

[5] E G Hrycay and S M Bandiera, Preclinical Development Handbook,Ed. S Cox, John Wiley & Sons, Inc., UK, pp.627–696, 2008.

[6] N Taxak and P V Bharatam, An Insight into the Concept and Detailsof Mechanism-Based Inhibition of CYP450, Current Research andInformation in Pharmaceutical Sciences, Vol.11, pp.62–67, 2010.

[7] B Bryant and K Knights, Pharmacology for Health Professionals, 3rdEd., Elsevier Pty. Ltd., Australia, pp.128–145, 2010.

[8] P R O de Montellano, Cytochrome P450: structure, mechanism, andbiochemistry, 3rd Ed., Kluwer Academic/Plenum Publishers, NewYork, 2005.

[9] B Meunier, S P de Visser and S Shaik, Mechanism of OxidationReactions Catalyzed by Cytochrome P450 Enzymes, Chemical Re-views, Vol.104, pp.3947–3980, 2004.

[10] S Shaik, S Cohen, Y Wang, H Chen, D Kumar and W Thiel, P450enzymes: their structure, reactivity, and selectivity-modeled by QM/MM calculations., Chemical Reviews, Vol.110, pp.949–1017, 2010.

[11] S P de Visser and S Shaik, A proton-shuttle mechanism mediated bythe porphyrin in benzene hydroxylation by cytochrome p450 en-zymes, Journal of American Chemical Society, Vol.125, pp.7413–7424, 2003.


GENERAL � ARTICLE

[12] N Taxak, V Parmar, D S Patel, A Kotasthane and P V Bharatam, S-Oxidation of Thiazolidinedione with Hydrogen Peroxide,PeroxynitrousAcid,andC4a-Hydroperoxyflavin:ATheoreticalStudy,Journal of Physical Chemistry A, Vol.115, pp.891–898, 2011.

[13] P V Bharatam and S Khanna, Rapid Racemization inThiazolidinediones: A Quantum Chemical Study, Journal of PhysicalChemistry A, Vol.108, pp.3784–3788, 2004.

[14] K B Park, A Boobis, S Clarke, C E P Goldring et al., Managing thechallenge of chemically reactive metabolites in drug development,Nature Reviews Drug Discovery, Vol.10, pp.292–306, 2011.

[15] A S Kalgutkar, Handling reactive metabolite positives in drug discov-ery: What has retrospective structure–toxicity analyses taught us?,Chemico-Biological Interactions, Vol.192, pp.46–55, 2011.

[16] M Coleman, Human Drug Metabolism: An Introduction, 1st Ed., JohnWiley & Sons, UK, 2010.

Address for CorrespondencePrasad V Bharatam

Department of MedicinalChemistry

National Instituteof Pharmaceutical Educationand Research (NIPER)

Sector-67S A S Nagar 160 062

Punjab, India.Email:

[email protected]

drug metabolism

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