PHASE I: REDUCTION OF XENOBIOTICS

Certain metals (e.g., pentavalent arsenic) and xenobiotics containing an aldehyde, ketone, alkene, disulfide, sulfoxide, quinone, Noxide, hydroxamic acid, amidoxime, isoxazole, isothiazole, azo, or nitro group are often reduced in vivo Although it is sometimes difficult to ascertain whether the reaction proceeds enzymatically or nonenzymatically by interaction with reducing agents (such as the reduced forms of glutathione, FAD, FMN, and NAD[P]).

Functional groups can be either reduced or oxidized. Eg: 1. aldehydes (RCHO-reduced to an alcohol (RCH2OH) or oxidized

to a carboxylic acid (RCOOH) 2. sulfoxides (R1SOR2) can be reduced to a sulfide (R1SR2) or oxidized to a sulfone (R1SO2R2). Enzymes, alcohol dehydrogenase, aldehyde oxidase, and cytochrome P450, can catalyze both reductive and oxidative reactions depending on the substrate or conditions. In the case of halogenated hydrocarbons, such as halothane, dehalogenation can proceed by an oxidative or reductive pathway, both of which are catalyzed by the same enzyme (namely, cytochrome P450). In azo-reduction, nitro-reduction, and the reduction of certain alkenes, the reaction is largely catalyzed by intestinal microflora

REDUCTION :
the acceptance of one or more electron(s) or their equivalent from another substrate.

Reductive reactions, which usually involve addition of hydrogen to the drug molecule, occur less frequently than the oxidative reactions. Biotransformation by reduction is also capable of generating polar functional groups such as hydroxy and amino groups, which can undergo further biotransformation.

Many reductive reactions are exact opposite of the oxidative reactions (reversible reactions) catalysed either by the same enzyme (true reversible reaction) or by different enzymes (apparent reversible reactions). Such reversible reactions usually lead to conversion of inactive metabolite into active drug, thereby delaying drug removal from the body.

Reduction
Azo- and Nitro-Reduction Carbonyl Reduction Disulfide Reduction Sulfoxide and N-Oxide Reduction

Quinone Reduction
Dihydropyrimidine Dehydrogenase Dehalogenation DehydroxylationCYP B5 and AldehydeOxidase Aldehyde OxidaseReductive Reactions

AZO REDUCTION During azo-reduction, the nitrogennitrogen double bond is sequentially reduced and cleaved to produce two primary amines, a reaction requiring four reducing equivalents Eg: Prontosil Reduction of prontosil is of historical interest. Treatment of streptococcal and pneumococcal infections with prontosil marked the beginning of specific antibacterial chemotherapy.(Domagk) Subsequently, it was discovered that the active drug was not prontosil but its metabolite, sulfanilamide (para-aminobenzene sulfonamide), a product of azo-reduction.

NITRO-REDUCTION requires six reducing equivalents which are consumed in three sequential reactions Azo- and nitro-reduction reactions are generally catalyzed by intestinal microflora. However, under low oxygen tension, the reactions can be catalyzed by liver microsomal cytochrome P450 and NAD(P)Hquinone oxidoreductase (NQO1, a cytosolic flavoprotein, known as DT-diaphorase) and, in the case of nitroaromatics, by cytosolic aldehyde oxidase. The anaerobic environment of the lower gastrointestinal tract is well suited for azo- and nitro-reduction, which is why intestinal microflora contribute significantly to these reactions. The reduction of quinic acid to benzoic acid is another example of a reductive reaction catalyzed by gut microflora Nitro-reduction by intestinal microflora- play an important role in the toxicity of several nitroaromatic compounds including 2,6dinitrotoluene, which is hepatotumorigenic to male rats.

R1 and R2 are almost always aromatic

Usually only seen when the NO2 functional group is attached directly to an aromatic ring and are rare
Nitro reduction is carried out by NADPH-dependent microsomal and soluble nitroreductases (hepatic)

NADPH dependent multicomponent hepatic microsomal reductase system reduces the azo
Bacterial reductases in intestine can reduce both nitro and azo
O H2N S O O N H2 H2N S O N H2 H2N

N N NH2
H N O

+
NH2

NH2

Prontosil

Sulfanilamide

1,2,3-Triaminobenzene

O
O2 N N Cl

O S N H N

O2N O N N O

O NNa

HO O OH

N N

Clonazepam

Sulfasalazine

Dantrolene

 Nitro-reduction by intestinal microflora also plays an important role in the biotransformation of musk xylene (1,3,5-trinitro-2- tbutyl-4,6-dimethylbenzene).

Reduction of one or both of the nitro groups is required for musk xylene to induce (as well as markedly inhibit) liver microsomal cytochrome P450 (namely,CYP2B) in rodents

CARBONYL REDUCTIONSDRs and AKRs A variety of xenobiotics contain a carbonyl function (RCHO and R1COR2) that undergoes reduction in vivo. The reduction of aldehydes to primary alcohols and of ketones to secondary alcohols is generally catalyzed in mammals by NAD(P)H-dependent reductases belonging to one of two superfamilies, the aldoketo reductases (AKRs), and the short-chain dehydrogenases/reductases (SDRs)

AKRs Members of a superfamily of cytosolic enzymes that reduce both xenobiotic and endobiotic compounds, as their alternative names imply Members of the AKR superfamily can function as dihydrodiol dehydrogenases and oxidize the trans-dihydrodiols of various polycyclic aromatic hydrocarbon oxiranes (formed by epoxide hydrolase) to the corresponding ortho-quinones One of the AKRs, namely, AKR7A (also known as aflatoxin aldehyde reductase) is one of the many enzymes induced following activation of Nrf2 by oxidative stress, exposure to electrophiles, or depletion of glutathione. Reduction of aldehydes to alcohols can be catalyzed by alcohol dehydrogenase, for the conversion of the sedative-hypnotic, chloral hydrate, to trichloroethanol.

NAD(P)H-dependent reductases
Aldoketo reductases (AKRs): Cytosolic enzymes that reduce both xenobiotic and endobiotic compounds, Function as dihydrodiol dehydrogenases and oxidize the trans-dihydrodiols of various polycyclic aromatic hydrocarbon oxiranes (formed by epoxide hydrolase)

 Alcohol dehydrogenases belong to the medium chain dehydrogenases/reductases (MDRs). They typically convert alcohols to aldehydes, for oxidative reactions In the case of chloral hydrate, the reverse reaction is favored by the presence of the trichloromethyl group, which is a strong electron-withdrawing group

Medium chain dehydrogenases/reductases (MDRs) : convert alcohols to aldehydes Eg: alcohol dehydrogenases

SDR CARBONYL REDUCTASES Monomeric, NADPHdependent enzymes present in erythrocytes and both the cytosolic and microsomal fraction of the liver, kidney, brain, and many other tissues. The major circulating metabolite of the antipsychotic drug, haloperidol, is a secondary alcohol formed by carbonyl reductases in the blood and liver. Other xenobiotics that are reduced by carbonyl reductases include pentoxifylline , acetohexamide, daunorubicin, doxorubicin, loxoprofen, menadione, 4-nitroacetophenone, timiperone, and Rwarfarin Liver cytosol and microsomes contain different forms of carbonyl reductase, and these can differ in the degree to which they stereoselectively reduce ketones to secondary alcohols. Eg: keto-reduction of pentoxifylline produces two enantiomeric secondary alcohols: one with the R-configuration (which is known as lisofylline) and one with the S-configuration

 Many of the xenobiotics reduced by AKRs and also reduced by SDRs, and in most cases the relative contribution of individual carbonyl-reducing enzymes is not known. Genetic polymorphisms of AKRs or SDRs have not been shown to impact the disposition or safety of carbonyl-containing drugs There appear to be no reports of drugdrug interactions involving the inhibition or induction of AKRs or SDRs

DISULFIDE REDUCTION Some disulfides are reduced and cleaved to their sulfhydryl components, as shown in Fig. 6-13 for the alcohol deterrent, disulfiram . Disulfide reduction by glutathione is a three-step process, the last of which is catalyzed by glutathione reductase. The first steps can be catalyzed by glutathione transferase, or they can occur nonenzymatically

SULFOXIDE AND N-OXIDE REDUCTION

Thioredoxin-dependent enzymes in liver and kidney cytosolreduce sulfoxides, which themselves may be formed by cytochrome P450 or flavin monooxygenases It has been suggested that recycling through these counteracting enzyme systems may prolong the half-life of certain xenobiotics. Sulindac is a sulfoxide that undergoes reduction to a sulfide, which is excreted in bile and reabsorbed from the intestine . This enterohepatic cycling prolongs the duration of action of the drug such that this nonsteroidal anti-inflammatory drug (NSAID) need only be taken twice daily.

 Sulfoxide reduction may also occur nonenzymatically at an appreciable rate, as in the case of the proton pump inhibitor rabeprazole Diethyldithiocarbamate methyl ester, a metabolite of disulfiram, is oxidized to a sulfine, which is reduced o the parent methyl ester by glutathione. Just as sulfoxide reduction can reverse the effect of sulfoxidation, so the reduction of N-oxides can reverse the Noxygenationof amines, which is catalyzed by flavin monooxygenases and cytochrome P450. Under reduced oxygen tension, reduction of the Noxides of imipramine, tiaramide, indicine, and N,N-dimethylaniline can be catalyzed by mitochondrial and/or microsomal enzymes in the presence of NADH or NADPH The NADPH-dependent reduction of N-oxides in liver microsomes appears to be catalyzed by cytochrome P450 although in some cases NADPH-cytochrome P450 reductase may play an important role.

 As a class, N-oxides are not inherently toxic compounds. However, certain aromatic and aliphatic N-oxides have been exploited as bioreductive drugs (also known as DNA affinic drugs) for the treatment of certain cancers and infectious diseases In these cases, N-oxides have been used as prodrugs that are converted to cytotoxic or DNA-binding drugs under hypoxic conditions. The fact that N-oxides of certain drugs are converted to toxic metabolites under hypoxic conditions is the basis for their selective toxicity to certain solid tumors (namely, those that are hypoxic and, hence, resistant to radiotherapy) and anaerobic bacteria. For example, tirapazamine (SR 4233) is a benzotriazine di-N-oxide that is preferentially toxic to hypoxic cells, such as those present in solid tumors, apparently due to its rapid activation by one-electron reduction of the N-oxide to an oxidizing nitroxide radical This reaction is catalyzed by cytochrome P450 and NADPH-cytochrome P450 reductase

QUINONE REDUCTION Bioreductive alkylating agents, which include such drugs as mitomycins, anthracyclins, and aziridinylbenzoquinones represent another class of anticancer agents that require activation by reduction. These bioactivation also involves a two-electron reduction reaction, which is largely catalyzed by NQO, NQO1 and NQO2 Quinones can be reduced to hydroquinones by two closely related, cytosolic flavoproteins- NQO1 and NQO2. NQO1 : NAD(P)Hquinone oxidoreductase-1, (DT-diaphorase) NQO2 : NAD(P)H-quinone oxidoreductase-2,(NRH-quinone oxidoreductase) because it prefers the unusual electron donor dihydronicotinamide riboside (NMR) over NAD(P)H. Physiological role in the metabolism of vitamin K hydroquinone Although closely related enzymes ,NQO1andNQO2 have different substrate specificities, and they can be distinguished on the basis of their differential inhibition by dicoumarol and quercetin (which are selective inhibitors of NQO1 and NQO2, respectively)

 Drugs or drug candidates that are activated by NQO to anticancer agents include the aziridinylbenzoquinone diaziquone, the anthraquinone mitoxantrone, the indolquinones mitomycin C and EO9 (an analog of mitomycin C that is more rapidly reduced by NQO1), and the anthracycline antibiotics daunorubicin and doxorubicin Bioreductive alkylating agents are reduced by NQO1 to generate semiquinone free radicals and other reactive intermediates that undergo nucleophilic additions with DNA, resulting in single-strand DNA breaks. The reason such drugs are preferentially toxic to tumor cells is that tumor cells, especially those in solid tumors, are hypoxic, and hypoxia induces the synthesis of NQO1 (by a mechanism that involves the Activator Protein 1 [AP-1] and Nuclear Factor-B [NFB] response elements in the 5-promoter region of the NQO1 gene). Therefore, tumor cells often express high levels of NQO1, which predisposes them to the toxic effects of quinone-reductive anticancer drugs like mitomycin C

 NQO1 is inducible up to tenfold by two classes of inducers, which have been categorized as bifunctional and monofunctional inducers The bifunctional inducers: -naphthoflavone, benzo[a]pyrene, 3methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), which induce both oxidative enzymes (suchas the cytochrome P450 enzyme CYP1A1) and conjugating enzymes (such as glutathione transferase and UDP-glucuronosyltransferase). The monofunctional inducers: tend to induce conjugating and other non-CYP enzymes (although in mice, monofunctional inducers can induce CYP2C55 and 2U1, as well as aldehyde oxidase). These inducers signal through two distinct mechanisms involving the XRE (xenobiotic-response element) involving the ARE (antioxidant response element), which is also known as the EpRE (electrophilic response element). (Response elements are short sequences of DNA, often located in the 5-promoter region of a gene, that bind the transcription factors that control gene expression.)

 Among the monofunctional inducers that apparently increase NQO1 via ARE is sulforaphane, an ingredient of broccoli that may be responsible for the anticarcinogenic effects of this cruciferous vegetable Isothiocyanates (which are also present at high levels in cruciferous vegetables) likely exert their chemopreventive effects largely through induction of the detoxifying enzymes under the control of ARE, namely, glutathione transferase (GSTA1), microsomal epoxide hydrolase, aldo keto reductase (AKR7A, also known as aflatoxin aldehyde reductase), NAD(P)H-quinone oxidoreductase (NQO1, also known as DT-diaphorase), glutamate-cysteine ligase (GCL), as well as genes involved in apoptosis

DIHYDROPYRIMIDINE DEHYDROGENASE (DPD)

An NADPH-requiring, homodimeric protein (Mr 210 kDa) containing FMN/FAD an liver cytosold an ironsulfur cluster in each subunit. Location:, where it catalyzes the reduction of 5-fluorouracil and related pyrimidines. Sorivudine is converted in part by gut flora to (E)-5-(2-bromovinyl) uracil (BVU), which lacks antiviral activity but which is converted by DPD to a metabolite that binds covalently to the enzyme- Resulting in irreversible inactivation(suicidal inactivation) of DPD Marked inhibition of 5-fluorouracil metabolism, which increases blood levels of 5- fluorouracil to toxic lethal levels Genetic polymorphisms that result in a partial or complete loss of DPD activity 5-fluorouracil lethality has been documented in rare individuals who are completely deficient in DPD (one individual in about 10,000). Assessing an individuals DPD genotype (by analyzing DNA for allelic variants) or phenotyping (by measuringDPDactivity in peripheral blood mononuclear cells or PBMCs) is advocated prior to 5-fluorouracil therapy so that the dosage of this anticancer drug can be adjusted on an individual basis.

Dehalogenation Three mechanisms for removing halogens (F, Cl, Br, and I) from aliphatic xenobiotics 1. The first, -reductive dehalogenation, involves replacement of a halogen with hydrogen, as shown below

2. oxidative dehalogenation-a halogen and hydrogen on the same carbon atom are replaced with oxygen. Depending on the structure of the haloalkane, oxidative dehalogenation leads to the formation of an acylhalide or aldehyde

3.

Elimination of two halogens on adjacent carbon atoms to form a carboncarbon double bond- A variation on this third mechanism is dehydrohalogenation, in which a halogen and hydrogen on adjacent carbon atoms are eliminated to form a carboncarbon double bond.
Dehalogenation reactions leading to double bond formation are catalyzed by cytochrome P450 and glutathione transferase. These reactions play an important role in the biotransformation and metabolic activation of several halogenated alkanes, as the following examples illustrat

DehydroxylationCytochrome b5 and Aldehyde Oxidase

In the presence of NADH and NADH-cytochrome b5 reductase (CYB5R3), the microsomal hemoprotein cytochrome b5 (CYB5A) can catalyze the N-dehydroxylation of various amidoximes, for the amidoxime metabolite of the antimicrobial prodrug DB289 Because these carcinogenic arylhydroxylamines can be further activated by glucuronidation, sulfonation, or acetylation in various tissues, reduction by NADH-cytochrome b5 reductase and cytochrome b5 represents a competing detoxication pathway. Dehydroxylation reaction catalyzed by aldehyde oxidase, an enzyme that can catalyze both reductive and oxidative reaction

Dehydroxylation by cytochrome b5 and aldehyde oxidase.

ALDEHYDE OXIDASE Reductive Reactions Aldehyde oxidase is a cytosolic molybdozyme that catalyzes the oxidation of some xenobiotics and the reduction of others In contrast to the large number of drugs that are known to be (or suspected of being) oxidized by aldehyde oxidase in vivo, only a few drugs are known to be (or suspected of being) reduced by aldehyde oxidase in vivo, including nitrofurazone, zonisamide, and ziprasidone.

Short chainDehydrogenases/reductases (SDRs):

Erythrocytic.cytosolic and microsomal carbonyl reductase, Reduction of a wide variety of carbonylcontaining xenobiotics (other species express more than two carbonyl reductases).

SULFOXIDE REDUCTASE
Thioredoxin-dependent enzymes in liver and kidney cytosol Sulfoxide and N-Oxide Reduction: Reduce sulfoxides, which themselves may be formed by cytochrome P450 or flavin monooxygenases Eg: Sulindac is a sulfoxide that undergoes reduction to a sulfide, which is excreted in bile and reabsorbed from the intestine. Reduction may also occur nonenzymatically at an appreciable rate, as in the case of the proton pump inhibitor rabeprazole

XANTHINE OXIDOREDUCTASE (XOR)

Xanthine dehydrogenase (XD) and Xanthine oxidase (XO); Cytosol- Highest levels in heart, brain, liver, skeletal muscle, pancreas, small intestine, colon, and placenta Two forms of the same enzyme that differ in the electron acceptor. XD: the final electron acceptor is NAD+ (dehydrogenase activity), XO : the final electron acceptor is oxygen (oxidase activity). XD is converted to XO by oxidation of cysteine residues (Cys993 and Cys1326 of the human enzyme) and/or proteolytic cleavage. Under normal physiologic conditions, XD is the predominant form of the enzyme found in vivo. However, during tissue processing, the dehydrogenase form tends to be converted to oxidase form

XOR Ischemia/Hypoxia: XO levels increase- XOR gene transcription, XD XO. XO contributes to oxidative stress and lipid peroxidation because the oxidase activity of XO involves the reduction of molecular oxygen, which can lead to the formation of reactive oxygen species LPS, a bacterial endotoxin that triggers an acute inflammatory response, increases XO activity both by inducing XOR transcription and by converting XD to XO.

XOR First-pass elimination of purine derivatives (e.g., 6mercaptopurine and 2,6-dithiopurine), limits the therapeutic effects of Certain prodrugs are activated by xanthine oxidase. Eg: antiviral prodrugs 6-deoxyacyclovir and 2_-fluoroarabinodideoxypurine, which are relatively well absorbed after oral dosing, are oxidized by xanthine oxidase to their respective active forms, acyclovir and 2--fluoroarabino-dideoxyinosine, which are otherwise poorly absorbed Catalyzes, the sequential oxidation of hypoxanthine to xanthine and uric acid

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