Metabolism of Drugs


Metabolism of drugs is a complex and major process within the body, occurring primarily in the liver. The aim of metabolism is to make the drug more polar to enable excretion via the kidneys. The basic understanding of drug metabolism is paramount to ensure drug optimisation, maximum therapeutic benefits and a reduction in adverse effects.

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Essentially drug metabolism is broken down into two phases, Phase I and Phase II. Phase I is concerned with the biotransformation of compounds, and then transferred to Phase II. However, for some drugs this is the end of their metabolic journey in the body, as they produce more polar compounds which are readily excreted. Phase II reactions are where compounds are conjugated to produce more water soluble compounds for easy excretion. Phase I reactions are dominated by the Cytochrome-450 enzyme superfamily. These enzymes are found predominantly in the liver, which is the major site of drug metabolism. However, drug metabolism is not localised merely to the liver, there are other major sites at which this process occurs. Some of these sites include the skin, lungs, gastro-intestinal tract and the kidneys; close to all tissues have the ability to metabolise drugs due to the presence of metabolising enzymes. The most important enzymes are the cytomchrome-450 superfamily, which are abundant in most tissues.

Inactive drugs with the ability to reconvert to the active parent drug once metabolised to exert their therapeutic actions are defined as prodrugs. They are classified depending on the site of conversion and actions (gastrio-intestinal fluids, intracellular tissues or blood). This report gives different study examples of such prodrugs and how their metabolism differs within the body, compared to their active metabolites. Individual drug metabolism may be affected by variant factors, such as, age or sex. Drug metabolism can cause an increase in toxcity. The bioactivation of a parent compound can form electrophiles that bind to proteins and DNA. Some of this toxicity can occur in Phase I metabolism e.g. acetaminophen. However, in some circumstances toxicity occurs in Phase II e.g. zomepirac, polymorphism can also cause idiosyncracity of certain drugs to be toxic.

1.1 Phase I

Phase one, otherwise known as drug biotransformation pathway is generally broken into oxidation, reduction and hydrolysis. A reaction under this phase involves an addition of oxygen molecule aiming to improve the water solubility of drugs. As the result some metabolites from this phase can be extracted immediately if they are polar enough however at times a single addition of oxygen is not sufficient enough to overcome the lipophilicity of certain drugs and hence their metabolite from this phase has to be carried onto phase II for further reactions.

Major example of Oxidation:

Accounting for roughly 20 complex reactions the most important oxidative metabolic pathway dominating phase I is the cytochrome-P450 (CYP450) monooxygenase system processed by C-P450. Located primarily in the liver CYP450 was found to be present in all forms of organisms, including humans, plant and bacteria. It is important to note that the function of CYP450 goes beyond drug metabolism but it is also involved in metabolism of xenobiotics, fat soluble vitamin and synthesis of steroids. With substrate specificity of more than 1000 and its ability to produce activated metabolites such as epoxide are the underlying reason for its dominance and importance in drug discovery. The general mechanism the CYP450 monooxygenase oxidation is:

R + O2 + NADPH + H+ à ROH + H2O + NADP+ (fig 2)

From the above formula it can be this reaction is of NADPH (Nicotinamide adenine dinucleotide phosphate) and an oxygen molecule dependent. As mentioned above oxygen is important to increase the water solubility and in the same manner NADPH is also important for oxygen activation and source of electron. Also important for activation of oxygen is the presence of cystine amino acid located near the protein terminal carboxyl of CYP450. Among the 500 amino acid present in CYP450, cystine has proven to be most important as it activates the oxygen to a greater extend. This is due to the fact that it contains a thiol group as one of its ligand and it is the thiol which alerts the reactivity.

Highlighting the numerous intermediate structures involved as well as function of iron, oxygen and proton (Figure) shows the catalytic conversion required for cp450 oxidation reaction to place. The binding of the substrate with low spin ferric CYP450 enzyme induces a change in its active site. This will effects the stability of the water ligand and will displace it (shown in the diagram from a-b). Containing a high spin heme iron the enzyme and substrate form a ferric complex. The change in electronic state will result in the release and transfer of one electron from NADPH via electron transfer chain (reducing ferric heme iron to ferrous state) and thus reduction of the complex. The second electron is transferred when the complex reacts covalently with the oxygen forming a new ternanry complex. Initially the complex is an unstable oxy-P450(diagram d), however this is reduced to produce ferrous peroxide by a loss of an electron. This intermediate is short lived and undergoes protonation twice resulting in a release one water molecule. Out of the oxygen molecules released one in incorporated in this water molecule and the remaining into the substrate. Another method of forming the iron-oxo intermediate is via the peroxide shunt which elimited steps from C to F.

Some of the common addition of oxygen molecule reactions which CYP450 dependent are known as epoxidation (of double bond), N-hydroxylation, oxygen/nitrogen/ sulfur dealkylation, s-oxidation, dechlorination, oxidative desulfurisation and aromatic hydroxylation. Note they all follow the same principle of adding oxygen molecule to the substrate. The diagram below provides an example of how these reactions are processed:

Aromatic hydroxylation substrate mostly produces phenols such as that seen on figure 3. The production of Phenol can be either via a non enzymatic rearrangement or by Epoxide hydrolase and cytosolic dehydrogenase which will ultimately give rise a catechol. The position of hydroxylation depends greatly on the nature of the R- group attached to the ring; an electron withdrawing group will position the -OH group on the metha while the electron donating will position it on the para or ortha. Aromatic hydroxylation also involves a change in NIH shift, which involves the movement and shifting of the R group to an adjacent position during the oxidation. It is important to note that certain substrate for aromatic hydroxylation can also be oxidized via the aliphatic (C-H) hydroxylation. Under such condition the aliphatic C-H) hydroxylation will oxidize it. Aliphatic dehydrogenation can also occur involving electron transfer to the CYP450.

Currently more than 50 CYP-450 has been identified in human, however the bulk of drug metabolism is essentially carried by CYP1, CYP2 and CYP3 families, especially the CYP450-3A. The diagram on the right hand side clearly demonstrate just how much of drug metabolism is CYP450 3A responsibility in comparison to other, accounting for roughly 50%. Metabolism of drugs given orally are greatly determined by CYP450-3A primarily because this enzyme is present in both the liver and intestine and thus providing a barrier for all drugs before they can enter the systemic circulations, otherwise commonly known as ‘first pass effect’. Upon entering the drugs are taken up via passive diffusion and/or facilitated diffusion or active transport into the entercocyte where they can be metabolized by CYP450-3A. They can once again be metabolized by the very same enzyme when they enter the liver (hepatocyte) ,which unlike the intestine in order to reach the systemic circulation it is unavoidable. This family of enzymes are also known to be cause of many serious adverse effects as they are influenced by diet and drug components, hence drug-drug and drug-food interactions is an important factor.

Flavin monooxygenases

Similar to cytochrome p450 monooxygenases system,Flavin monooxygenasesalso plays a major role in metabolism of drugs, carcinogens and Nitrogen/ sulfur/ phosphorous containing compounds. Also oxygen and NAPDH dependent, Flavin monooxygenases has much broader substrate specificity than CYP450. Once they have become associated with substrate the flavin monooxygenases is activated into 4α-hyroperoxyflavin and unlike CYP450 the oxygen activation takes place without the need for substrate to bind to the intermediate. This pre-activated oxygen means that any compound binding to the intermediate is a substrate to be metabolized. The fact that this enzyme is able to remain stable and lacks any need for correct arrangement and disorientation of the substrate gives it ability to withhold all the energy required for the reaction to takes place and hence as soon as appropriate lipophilic substrate becomes available it starts the process immediately. Adverse side effects are rarely associated with these enzymes.

The binding of oxygen to the reduced flavin is processed via a non-radical nucleophilic displacement. The substrate is oxidized via a nucleophilic attack by the oxygen that is located at end of 4α-hyroperoxyflavin. This is then followed by cleavage of peroxide. The flavin monooxygenase catalytic cycle is finished once the original form of 4α-hyroperoxyflavin has been regained using NADPH, oxygen and hydrogen proton. Note the metabolite product can at any times undergo reduction back to its original parent form.

Alcohol dehydrogenase and aldehyde dehydrogenase

These families of enzymes are both zinc containing NAD specific and catalyze the reversible oxidation of alcohol and aldehydes respectively. Grouped into 1-6 Alcohol dehydrogenase, are homodimer that exist in the soluble section of the tissue. It is involved in metabolism of some drugs such as cetirizine however it is more predominantly known as alcohol metabolism enzyme specifically ethanol, whether products of peroxides or that of exogenous (i.e administered drugs). It is important to note that although alcohol dehyrogenase is the main metabolic pathway for ethanol, however CYP2E1 also plays in its metabolism. CYP2E1 can be induced by ethanol resulting in adverse side effects between alcohol and with certain analgesics drugs. Alcohol dehydrogenase also metabolizes ethylene glycol and methanol. With a longer half life and rapid absorption from the gut, methanol can result in series of unpleasant side effects and metabolic acidosis, hence highlighting the importance of alcohol dehydrogenase. Similarly, aldehyde dehydrogenase catalysis the oxidation of aldehyde to its corresponding carboxylic acid. Class one of alcohol dehydrogenase plays a major role in detoxification of anti cancer drugs. Alcohol dehydrogenase is also involved in reduction pathway of aldehyde or ketone back to its pharmacologically active alcohol form.

Monoamine oxidase and diamine

Located in liver, intestine and kidney as few of its site, this membrane bound enzyme is divided into two classes in accordance to their substrates specificity, they are monoamines-A and monoamine-B. Responsible for metabolizing amines via deamination to aldehyde, these enzymes are flavin containing enzymes and within their cysteinyl residue the flavin is linked to the covalently bounded flavin via a thioether. Monoamine oxidase has several substrates, ranging from secondary to tertiary amines that have alky group smaller than methyl. The general mechanism for this enzyme is the two electron oxidation shown below:

R.CH2.NH2 + O2 + H2O à R.CHO + NH3 + H2O2 (fig 7)

As it can be seen this reaction requires oxygen to react and a hydrogen peroxide is produced as for every “one molecule of oxygen is absorbed for every molecule of substrate oxidized” (Principle of drug metabolism, 2007). Proportional to the rate of oxygen uptake this is commonly used to deduce the rate of reaction. Research has shown that monoamines-A is more commonly involved in oxidation of endogenous substrates such as noradrenalin while monoamine-B which is found mostly in platelets appears to catalyses exogenous substrates such as phenylethylamines. Their common substrate is dopamine. Inhibition of monoamine oxidase has long been of an interest for scientist in treatment of several of illness such as depression.

Present in liver, lungs and kidney as few of its locations diamine oxidase also catalyses the formation of aldehyde from histamine and diamines in the same manner.


This pathway of metabolism is enzymatically the least studied in phase I and yet it plays an important role in metabolism of disulfides and double bonds of for example progestational steroids as well as dehydroxylation of aliphatic and aromatic compounds. In general ketone containing xenobiotics are more readily metabolized and eliminated via this pathway in the mammalian tissue. This is due to the fact that the carbonyl group is very lipophilic, thus the lipophilicity will be reduced and elimination is ensured as ketone is converted to alcohol.

One of the major enzymes involved in this pathway is the NADPH cytochrome P450 reductase. Containing flavin adenine dinucleotide and flavin mononucleotide is an electron donor playing an important role in the metabolism of drugs such as chloramphenicol by reducing its nitro group.


As the name suggests this pathway uses water to cause a breakage of a bond. Major enzymes under this pathway are the amide and ester hydrolysis and hence amide and esters are the common substrates. Naturally esters are much easier targets to esterase hydrolysis than amides. A very common amide substrate is a local anesthetic, Lidocaine and an antiepileptic drug known as levetiracetam. Catalyzing ester and certain type of amides are the group of enzymes referred to as carboxylesterase. This enzyme hydrolysis choline like ester substrate and procaine. As a rule, the more lipophilic the amide the better it be accepted as a substrate for this enzyme and thus eliminated. Esters that are sterically hindered are however much harder and slower to be hydrolysed and will usually be eliminated unchanged at a high percentage such as that for atropine, eliminated 50% unchanged.

A very good example of esterase enzyme is the paraoxonase. The hydrolysis of substrate such as phenyl acetate and other acyl esters are catalyzed by this. For hydrolases and substrate to be involved in this pathway certain criterias are imperative for a fast reaction rate, these include having a electrophilic group a nucleophile that will attack the carbon attached to the oxygen resulting in a formation of tetrahedral orientation. The presence of a hydrogen donor to the improvers the leaving group abilities is the final requirement.

1.2 Phase II (Second part of drug metabolism):

Second part of drug metabolism, involves introduinh of new ionic chemicals on to the substrate (including the metabolites from phase I) in order to increase its water solubilyt for elimination. This phase is usually refered to as conjugation reaction and its products are generally inactive unlike those of phase 1. The following reaction are major conjugation of phase II.

Methylation is the transfer of methyl group to the substrate from cofactor s-adenosyl-L-methionine (fig 9). S-adenosyl-L-methione is an active intermediate that receives a transferred methyl group from methionine after its linkage with ATP in presence of adenosine transferase enzyme. It

is this methyl group that is ultimately transferred on to the substrate. S-adenosyl-L-methionine methyl group becomes attached to the sulfonium center marking “electrophilic character” (Principle of drug metabolism, 2007). Depending on the functional group present on the substrate Conjugation via methylation is broken down to nitrogen, oxygen and sulfate methylation.


O-merthylation is the most common reaction that occurs for substarte containing the organic (formally known as pyrocatechol compound, catechol moiety) hence why the enzyme responsible for this type of reaction is called catechol O-methyltransferase. This Magnesium dependent, found cyclic but also, less frequently, as a membrane bound enzyme, is found commonly in liver and kidney among other tissues. Common drug for this type reaction are L-DOPA, where generally the methyl is transferred on to the substrate in meta position and less commonly para, depending the substituent (R group) that is attached on the ring. According to ‘’Principle of drug metabolism” the rate of reactivity of O-methylation is decreased in accordance to size of the substituted group, the larger it is the slower the rate of reaction degree of acidity of the catechol group itself.


Naturally this reaction has substrate specificity of amine, involving however primary and seconday only. Unlike the above reaction, N-methylation consists of several enzymes, all of which are categorized in accordance to the specific type of amine substrate which they catalyze. Enzymes such as amine-N-Methyltransferase, nicotinamide-N-methyltransferase and histamine-N-methyltransferase are few examples. Despite the substrate specificity all the enzymes involved do however follow the same principle of transferring methyl fromcofactor s-adenosyl-L-methionine to the substrate.

With drug substrates such as captoril, reactions of N-methylation can be broken down into two distinct types as illustrated in Fig 11. Reactions that have a low pharmacological significant yield an ineffective n-methylation as the substrate and the product have a same electrical state thus the metabolites are usually less hydrophilic than parent. As it can be seen from fig 7a, in these

reactions one proton is exchange for a methyl group. On the other hand a more hydrophilic product and an effective reaction of detoxification is achieved with pyridine type (nitrogen atom) substrate. These substrate will result in a creation of positive change on the product (fig 7b) rather than an exchange process.

Sulfate and phosphate conjugation

Sulphate conjugation is one of the most important reactions in biotransformation of steroids, effecting its biological activates and decreasing its ability for its receptor. Nucleophilic hydroxyl groups such as alcohol and phenol, primary or seconday amine and drug containing a SO-3 group are the common substrates for this pathway. Generally sulphate are transferred via a membrane bound enzyme named sulfotransferase (located in golgi apparatus) from their cyclic cofactor 3′-phosphoadenosine 5′ (shown in fig 8 ) to substrate. 3′-phosphoadenosine 5′ is formed in a reaction between adenosine triphosphate and inorganic sulfate where the sulfate/phosphate group are bonded via a anhydride linkage which gives rise an exothermic reaction when broken, hence providing the energy for the reaction. In human there is two class, SULT 1A- 1E and SULT 2A-2B, each of which will have different specificity yet with overlaps. This enzyme acts on both endogenous as well as exogenous compounds as long as they possess an alcohol (less affinity with varying product stabilities) or phenol (products are stable arly sulfate esters with a high affinity). Substrates are generally of medium sized, highly ionized and hydrophilic, hence excreted easier via urine. The rate of this pathway is determined by the lipophilicity and nature of amino acid present on the substrate. Interestingly phenol is also of an interest for the Glucoronic conjugation pathway and are metabolized by this when they are at high concentration and 3′-phosphoadenosine 5′ becomes rate limiting. The sulfate conjugation will produce ester sulfate or sulfamide some of which will undergo further heterolytic reaction leading to electrophilic substrate and hence toxicity.

Unlike the sulfate conjugation the phosphate conjugation is less common unless the drug in question is anticancer or antiviral. Catalyzed phosphotransferases.

conjugation The most important and major occurring metabolic pathway of phase II is the glucoronic conjugation, accounting for the largest share of conjugated metabolite in the urine. This pathway is important due to the fact there is a high availability of glucucronic acid, huge substrate specificity and the wide range of poorly reabsorbed metabolite. The glucoronic conjugation takes place as the glucoronic acid is transferred to the acceptor molecule from its cofactor uridine-5-diphosphh-alpha-glucoronic acid (fig 9 ) of which glucoroniuc acid is attached in 1 α configuration. However products produced are in β-configuartion. This is due to the nucleophilicity of the functional groups of the substrate. To be able to undergo this pathway of metabolism the functional group of drugs in question must have nucleophilic characteristics. Generally the drug that are at high affinity for this pathway is firstly phenol (paracetamol) and then alcohol (primary, secondary or tertiary) such a morphine. The transformation of the drugs involves a condensation reaction and hence release of water, while the conjugate replaces the hydrogen on the -OH group. Present in the ER uridine-5-diphosphae-alpha-D glucoronic acid is produced due to oxidation of carbon position six of UDP-α-D-glucose. Interaction of this co factor with the substrates is catalysed by one the two classes of UGT1 or UGT 2, present mostly in liver however still found in brain and lungs.

As this pathway produces a wide variety of procucts, work has been done to divide them into four groups of O/S/C/N glucoronides, with the o-glucoronides being the most important forming a reactive metabolite known as acyl-glucuronides. Generally drugs containing functional groups such as carboxylic acid, alcohol and phenol give rise more examples shown in fig 10.


Involving a transferring of an active acetyl linked via a thioester bridge to acetyl-coenzyme A (fig below) to a nucleophilic function group of substrate this metabolic pathway mainly occurs in liver involving amino groups of medium basic properties. One of the common drug metabolized by this pathway is the para-aminosalicly. Large group of enzymes known as acetyltransferase are enzymes involved in catalyzing this pathway, among these are the aromatic-hydroxylamine O-acetyltransferase and the arylamine N-acetyltransferase.

Interestingly, genetic polymerization of acetylation function has meant that the rate of reaction and occurrence of toxicity will differ in accordance to the polymers. Fast acetylation will have result in a fast conversion and elimination while slow acetylators will have the opposite effect and will lead to build of unconjugated compounds in the blood and hence leading to toxicity.

Conjugation with co-enzyme A

Commonly using this pathway are the carboxylic containing which are activated into an Intermediate and eventually forming a acetyl-CoA conjugate It is important to note that primary metabolites from this reaction do not show up in vivo and only in vitro, however some of its secondary and stable metabolites that have undergone further reactions do. A factor that seems to cause problems with this pathway is the occurrence of toxicity, rare but serious as it the conjugates interfere with normal endogenous pathway. A common example was seen with NSAID which have now been long removed from market.

Conjugation with amino acid

This metabolic pathway is the most important for carboxcylic drugs where they form conjugate with the most common amino acid, glycine. Products are non-toxic (with no exception) and more water soluble than their parent compound. The drugs first become activated to the co- enzyme A before forming an amide or peptide bond between its carboxylic group and amino acid. The enzymes that facilitate this reaction are those of N-acyl transferases, such as glutamine N-acyltransferase. Carboxylic substrate for this pathway are also of an competition for the glucoronic conjugation, at high concentration if drugs glucoronic conjugation is preferred due to high availability, while at low concentration conjugation with amino acid is used for the metabolism.

Conjugations with Glutathione

Conjugation with glutathione has a wide variety of substrate specificity; this is partly due to the fact that in vivo glutathione exists as in equilibrium between its oxidised and reduced form hence enabling it to accept a wider range of substrate. The reduced form of glutathione is able to act as a protecting agent as it removes free radicals while the oxidised form oxidizes peroxides. A thiol, the glutathione contains a tripeptide and with a pka of 9.0, allowing it to be an excellent nucleophile agents, due to the increase in the ionization due to the thiol group. As the result of these electrophilic groups are easily attacked, usually on the most electrophilic carbon (commonly sp3 or sp2 hybridised) that contains the functional group. Enzymes responsible for catalyzing these reactions are known as glutathione transferase, seven of which are found in human. They also serve an important role apart from catalysing as upon binding of the active side with the glutathione will results in a decrease in pka value and hence an increase in acidity (the thiol is deprotonated thiolate), thus enhancing the nucleophilic abilities.

Depending on the substrate in question the conjugation with glutathione can be divided into forms, nucleophilic substation or nucleophilic addition. During the nucleophilic addition, an addition followed by an elimination reaction occurs. Attack occur at the activate electron lacking CH2 group, which the glutathione substitutes as it becomes added on to the carbonyl as shown in fig 12. Nucleophilic substitution reaction is much more common with xenobiotic than drugs although it is seen with chloramphenicol, where its -CHCL2 becomes electrophilic due to a electron withdrawing group.

One of the most important conjugation in relation to glutathione is with epoxides giving rise to a protective mechanism of liver. The more chemically active epoxide undergo this reaction are attacked at carbon sp3 hybridised via nucleophilic addition. The metabolite will lose a water molecule via dehydration catalyzed by acid giving rise to a GSH aromatic conjugate. As a final metabolite a mercapturic acid (a condensation reaction exerted by urine) as shown in (fig below) is formed via a series reactions including cleavage and n-acetylation .

2.1 Metabolism in the liver

When a drug can be cleaved by enzymes or biochemically transformed, this is referred to as drug metabolism. The main site of drug metabolism within the body occurs in the liver, however, this is not the only site in which metabolism of drugs occurs, this will be discussed later. The liver ensures drugs are subjected to attack by various metabolic enzymes; the main purpose of these enzymes is to convert a non-polar drug into more polar molecules, thereby increasing elimination via the kidneys. The polar molecules formed are known as metabolites, these lose a certain degree of activity compared to the original drug. Metabolic enzymes, cytochrome P450 enzymes enable the addition of a polar compound to particular drugs, making them now polar and more water-soluble. On the other hand, some drugs may become activated and then have the desired effect within the body, these are referred to as pro-drugs; and will be considered in greater detail later.

Drug metabolism is split into two stages known as Phase I reaction and Phase II reaction, both of which have been discussed earlier. Certain oral drugs undergo a first pass effect in the liver, thereby reducing bioavailablity of the drug. This can lead to numerous problems, such as, individual variation that can then lead to unpredictable drug action, and a marked increase in metabolism of the drug. These problems related to the first pass effect may hinder the desired therapeutic effects from being fully achieved. Many drugs undergo first pass metabolism, previously seen as a disadvantage, but now due to a greater understanding of hepatic metabolism it can be used advantageously, for example Naproxcinod. Naproxcinod is related to naproxen, which will be discussed below, we will also be examining the metabolism of propanolol.

Naproxcinod is derived from the non-steroidal anti-inflammatory drug (NSAID), Naproxen. First we will examine the metabolism of Naproxen (6-methoxy-a-methyl-2-naphthyl acetic acid). Naproxen is a widely used NSAID, possible of blocking both cyclo-oxygenase isoforms 1 and 2, therefore making it a non-selective inhibitor of these isoforms. Rheumatoid arthritis and osteoarthritis are the main reason for use of naproxen, which is administered orally as the S-enantiomer.

This particular drug is well absorbed by the body and is metabolised in vivo to form various metabolites, the major metabolites being naproxen-b-1-O-acylglucuronide (naproxen-AGLU) and desmethyl-naproxen (DM-naproxen).

Naproxen is conjugated in a Phase II reaction with glucuronic acid to form an acyl glucuronide (Diagram 2), with the intermediate being DM-naproxen. Usually conjugation reactions produce inactive metabolites, however with glucuronic acid the metabolite formed can occasionally become active. This reaction is facilitated by the superfamily UDP-glucuronosyl transferase (UGT) enzymes. The major UGT isoforms found in the liver are: 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7 2B10, 2B15, 2B17 and 2B28. The isoform 2A1 is found mainly in the nasal epithelium, while 1A7, 1A8 and 1A10 are only localised to the gastro-intestinal tract. UGT acts as a catalyst enabling glucuronic acid to bind to naproxen at the carboxylic acid group via covalent bonding.

It has been found that all UGT isoforms contribute to the conversion of naproxen to its metabolite naproxen-AGLU, except UGT-1A4, 2B4, 2B15, and 2B171. This reaction produces a highly polar glucuronic acid molecule bound to naproxen. Its main mode of elimination is through the urine. The next major metabolite of naproxen is, DM-naproxen. Demethylation of naproxen forms DM-naproxen, via removal of a single methyl group, as shown in Diagram 3. An unstable metabolite is formed during this process, however it is hydrolysed immediately to DM-naproxen. The enzymes involved in this reaction are cytochrome P450 1A2 and 2C9 from Phase I.

Once DM-naproxen has formed it is glucuronidated with the help of UGT enzymes 1A1, 1A3, 1A6, 1A9 and 2B7 and converted to its acyl glucuronide. UGT-2B7 is a high affinity enzyme and so has a high activity in this process, as does UGT-1A6. UGT-1A4, 2B15 and 2B17 do not contribute to the acyl glucuronidation process1. DM-naproxen is also converted to phenolic glucuronide; this is formed by the UGT enzymes 1A1 and 1A9. Enzymes UGT 1A3, 1A6 and 2B7 appear to play no part in this reaction. UGT 2B7 works well in glucuronidating the carboxylic acid moiety in particular drugs; however it is unable to glucuronidate the phenolic group, so for this reason is not involved in forming phenolic glucuronide.

The aim of hepatic metabolism is to ensure metabolites are made more water-soluble hence easily excreted. All metabolites formed from naproxen are water soluble and easily eliminated from the body. However, there are two metabolites that have been found to be far more water soluble, these are naproxen-AGLU and acyl glucuronide2. Huq (2006) explains this is due to the high solvation energy of both metabolites compared to naproxen and its other metabolites.

Metabolites of Naproxen:

Naproxen is a widely prescribed NSAID and works extraordinarily well; however there are several undesirable adverse effects, which precipitate after an extended period of use, such as increase in blood pressure. A new drug has been derived from naproxen without this effect, Naproxcinod. From Diagram 19 it is possible to see that the hydrogen on the OH moiety has been substituted by a nitrobutyl ester thereby forming naproxcinod. Naproxcinod is a new class of drug called cyclo-oxygenase inhibiting nitric oxide donators (CINODs)6. It is capable of producing the same type of anti-inflammatory effect as naproxen, by inhibiting both COX-1 and COX-2. However, an added effect of this new class of drug is its ability to release nitric oxide after metabolism. Nitric oxide is a vasodilator, and so prevents the usual increased blood pressure associated with NSAIDs.

Naproxcinod is a newly developed drug and is still undergoing the late stages of Phase III clinical trials. However, it is confirmed that Naproxcinod undergoes a great deal of first pass metabolism and is metabolised to naproxen in the liver and gastro-intestinal tract, while also a NO donating moiety is cleaved. Naproxen is the main metabolite of Naproxcinod and is produced via hydrolysis as seen in Diagram 20 below.

Propranolol, 1-isopropylamino-3- (1-naphthoxy)-2-propranolol, along with the two above drugs is extensively metabolised in the liver. Propranolol is a non-selective b-adrenoceptor blocker used for hypertension, angina pectoris and cardiac arrhythmias. It is found in either its R-enantiomer or S-enantiomer, due to the asymmetric carbon on the side chain. The S-enantiomer is approximately 100 times more active than the R-enantiomer on b-adrenoceptors5. Propranolol is usually given orally as a racemate, and undergoes first pass metabolism in the liver to enable elimination.

First pass metabolism is extensive in the liver, with propranolol being metabolised mainly by CYP2C19 and CYP2D63. CYP2D6 is a major enzyme involved in catalysing oxidation reactions for many drugs, and is heavily involved in the metabolism of propranolol. The reactions that occur have been found to be naphthalene oxidation at position 4, and 5, to form 4-hydroxy propranolol (4-OH PL) and 5-hydroxy propranolol (5-OH PL) 5 as shown in Diagram 22. Oxidation at the side chain generates the formation of N-deisopropyl propranolol (NDP), glucuronidation also occurs. Propranolol can undergo glucuronidation to form glucuronide; it is suspected to act as a storage fund for propranolol, as it can be deconjugated in the systemic system back to propranolol4. Naphthalene ring oxidation reactions are governed by the action of CYP2D6, whilst CYP1A2 is involved in side chain oxidation, N-deisopropylation. The main metabolites of propranolol have been found to be, 4-OH PL and naphthyl acetic acid.

Due to the enantiomerisation of propranolol it has been found that the oxidation rates for R (+) propranolol appears to be greater than the S (-) enantiomer in humans. All the oxidation reactions of propranolol exhibit biphasic kinetics5, meaning that there are two phases within the one phase. For example the reactions may have a low Km phase and have a high Km phase within the same system. The enzyme CYP2D6 is capable of catalysing the low Km phase of the naphthalene ring oxidation. The R (+) propranolol has a greater affinity for the enzyme compared to the S (-) enantiomer.

It is a given that the liver as described in detail above is the major site of drug metabolism in the body, via CYP enzymes to aid elimination; but every biological tissue has some ability to metabolise drugs. This is due to the presence of the same metabolising enzymes as the liver mostly cytochrome P450. Some of these other sites of metabolism include the lungs, gastric intestinal tract, the skin and kidneys; this is because each tissue has its own particular profile of P450 enzymes which determines their metabolic actions. They can be responsible for localized drug target reactions; without exerting the same level of effects on the systemic system; or metabolism in these areas can account for just a minor part of their overall biotransformation which occurs elsewhere mainly the liver. We shall be looking into a few of these metabolic tissues and some of the drugs which are metabolised there.

2.2 Metabolism in the lungs:

The pulmonary tissue in the lungs can up-take, retain, metabolise and delay the release of many drugs; the preferred structure of the drugs acting upon the lungs include basic amines with a pKa >8; but they are not limited to this. The lungs play a role in the metabolism of xenobiotics; these are chemicals found in an organism which are not naturally present there. Most drugs are xenobiotics but they also refer to toxins and pollution; taken in by the body from the surrounding environment. The uptake of drugs in the lungs can help with localised diseases especially within the bronchi, as this route of administration can help to deliver the therapeutic agent to the target area while at the same time reducing the distribution of the medication to other areas of the body. The liver exhibits as expected a higher amount and rate of drug metabolism compared to that of the pulmonary tissue in vitro testing; but in vivo the blood flow, distribution and systemic circulation could affect this, causing the pulmonary tissue being the main site of drug metabolism for some drugs.

Aerosols are the most common administration techniques for getting pharmaceutics into the pulmonary tissue. Like every other site of metabolism; the lungs are no different in respect to which enzymes are involved in the majority of metabolism of drugs; the cytochrome P-450 super family. Both bronchial and bronchiolar epithelium expresses some of these enzymes and with sensitive techniques it has been possible to determine which ones; (figure one shows the CYP expressed in the lungs).

Fig. 231Human CYP involved in xenobiotic metabolism. The human CYP superfamily comprises ca. 50 genes, according to the mapped human genome. Genes are classified into families and subfamilies according to the degree of nucleotide and amino acid sequence homology. Italic typed indicates the predominant enzymes in human lung.

The highest levels of drug metabolising enzymes are present in the Clara cells, then the alveolar epithelial cells and then pulmonary alveoli macrophage. The exact cellular location of the CYP enzymes is not really known; but it is recognised that CYP1A1 and CYP1B1 are mostly predominant in the bronchial epithelium and alveolar cells. There has also be evidence of phase II enzymes including Glutathione s-transferases (GST’s) UDP-glucuronyltransferase (UGT’s) and epoxide hydrolase presence in pulmonary tissue.

“Inhaled medications have been available for many years for the treatment of lung diseases and are widely accepted as being the optimal route of administration of first-line therapy for asthma and chronic obstructive pulmonary diseases”2

Theophylline even though it is known to be mainly metabolised in the liver; has had its metabolites found in pulmonary tissue. Theophylline is used to treat chronic obstructive pulmonary disease (COPD) and acute severe asthma via tablets and capsules; so even though it is not directly inhaled like many lung metabolised drugs, the systemic distribution of it can cause it the be metabolised in pulmonary tissue; this is due to theophylline being distributed in the lung at the same concentration as in the blood. The enzyme responsible for this metabolism has been under disputed; some believe that CYP2A13 via 8-hydroxylation is responsible but other believe it is CYP1A2 and CYP2E1 are involved; all these enzymes are found in pulmonary tissue, but what is agreed on is the major metabolite produced; 1,3 di-methyluric acid (figure 24).

On the other hand a drug which is inhaled and also metabolised in the lungs is beclomethasone, esterase is present in high concentrations in alveolar, and acts by hydrolysing beclomethasone dipropionate to its monopropionate and beclomethasone. The lungs seem to be less able to continue on after phase I to phase II biotransformation; a section of the body which is able to do that more readily is the gastrointestinal tract.

2.3 metabolisms in the gastrointestinal tract:

When drugs are administered orally such as tablets they undergo metabolism in the liver and gastrointestinal (GI) tract before they reach the plasma and systemic circulation; this is described as first pass metabolism. Metabolism can occur in the cells of the GI tract, there is evidence of the presence of the metabolising enzymes; CYP 1A1, 2D6, 2E1, 3A subfamily, epoxide hydrolase, UGT and GST. The most common of these being CYP2E1 and CYP3A; most drugs are metabolised by them in the GI tract. All these enzymes have been found in various positions along the GI tract; including ileum, small intestine, large intestine and colon.

Some of the many drugs which undergo a degree of metabolised in the intestine via CYP3A4 include; nifedipine via ring oxidation, cyclosporine via oxidation and diltiazem via N-demethylation. I will now concentrate on midazolam which is also as no surprise metabolised via CYP3A4 in the intestine to cause the first pass metabolism effect. The evidence for its metabolism via CYP3A enzyme is that there are known interactions between CYP3A inhibiting substances such as grapefruit juice and clarithromycin on midazolam. Midazolam is a sedative used in pre-operation period to reduce anxiety, reduce pain during the procedure and to enhance the actions of the anaesthetic agents used. It undergoes oxidation at the most exposed part of the ring to form its major metabolite hydroxyl-midazolam; which then goes on to phase II biotransformation via glucuronidation with UGT’s of the hydroxyl functional group; to the metabolite hydroxyl-midazolam glucuronide (Fig 25).

The other two minor metabolites are 4-hydroxy midazolam and 1,4-dihydroxy midazolam which consists of 3% and 1% of the dose received (fig 26).

Midazolam is interesting in the way it converts into an open ring form under acid conditions via the opening of the 4,5-double bond, but only the closed ring form is absorbed into the systemic circulation; therefore it converts back into the closed ring form to be absorbed. At a pH 5-8 60% is closed ring and 40% open ring in an equilibrium.

The gastrointestinal tract has also been found to be involved in the metabolism of non-steroidal anti-inflammatory drugs (NSAIDs); as all NSAIDs already contain a carbonyl functional group they do not go under phase I biotransformation but goes straight to phase II conjugation with glucuronic acid; they are then excreted as acylglucuronides in urine. The enzymes responsible for this glucuronidation are UGT1A10, UGT1A9, UGT1A3 (known to metabolise ketoprofen) and UGT2B7 (known to metabolite ketoprofen and naproxen). The site which the majority of metabolism of NSAIDs occurs is in the small intestine; while none has been found to occur in the duodenum and stomach. The NSAID which was found to be highly biotransformed in the small intestine was etodolac by the enzymes UGT1A10, 1A9 and 2B7 (Fig 27).

Metabolism of drugs by the gastrointestinal tract especially by the CYP3A enzymes in the intestine is a major influence in the dose of medication given orally. This is due to the first pass metabolism which reduces its bioavailability; therefore medication given orally it always a higher dose than their parental counterpart.

2.4 metabolism in the Skin

The skin is the body’s largest organ with a surface area of around 1.5-2.0m3 and is in direct contact with a wide variety of xenobiotics. It was once thought to be an inactive barrier to protect the body but it is now known to contain many metabolizing enzymes; thus has been used by the pharmaceutical industry to administer topically medication. Cytochrome P-450 is the most important drug metabolising enzyme in mammalian skin, the exact position and abundance of cytochrome P-450 enzymes is not known; but some experimental techniques have determined the presence of 13 of the cytochrome P-450 enzymes (as shown in Fig.28)

The presence of these enzymes can both be a help and a hindrance; as they can metabolise active compound into inactive ones but they can also cause inactive ones into active ones (some of which can be carcinogenic).

Tretinoin is a topically applied cream or gel used to treat acne and psoriasis; it is the acid form of vitamin A (all-trans retinoic acid). It helps to develop epithelial tissue and treat skin lesions from Kaposi’s sarcoma; this drug is of importance to us due to its metabolism by the skin once applied. It is rapidly metabolised via oxidisation in phase I from retinoic acid to the inactive all-trans-4-hydroxy-retinoic acid metabolite, due to hydroxylation at C-4 (fig 29). The alcohol group attaches onto the 4th carbon in the ring.

The exact CYP enzymes which cause the metabolism of retinoic acid is not known; but there is evidence that suggests this reaction is due to CYP1A1 and CYP1A2 enzymes; as retinoic acid is a potential CYP1A1 substrate. Others suggest that it is down to the CYP3A4, 2C8 and 2E enzymes. The other metabolites which are produced due to oxidation include; all-trans- 5,6-epoxyretinoic acid (fig 30), 4-oxoretinoic acid and 3,4-didehydroretonic acid; all these metabolites are inactive in the body.

Phase II glucuronidation of all-tans-4-hydroxyretinoic acid causes formation of more metabolites ready for the body to excrete including 4-oxo trans retinoic acid glucuronide, via conjugation with glucuronide (as shown in figure 31 below).

There is some exciting new progression in the world of topical medication; a gel based HIV protection is under stage II clinical trials in Africa. Its active ingredient is tenofovir and is a nucleotide that binds to the reverse transcriptase enzyme; which is how the human immunodeficiency virus replicates. These types of gels have been manufactured as an extra precaution to condoms and if they work will help to prevent the HIV spreading and infecting more people.

2.5 Metabolism in the Kidneys

From the journal ‘Drug metabolism enzyme expression and activity in primary cultures of human proximal tubular cells’3; it tells us that there are eleven phase I cytochrome P-450 metabolizing enzymes expressed in the proximal tubular cells in the kidney (Fig.32).

Drugs and other xenobiotics can be metabolized in the kidneys two ways; one involved the liver already metabolising the administered drug into metabolites which are then further biotransformed by enzymes in the kidney (this is called relay metabolism), the other is the sole metabolism of drugs by the kidneys. An example of relay metabolism is hexachlorobutadiene which is transformed in the liver into its glutathione conjugate; which then travels to the kidney and the β-lyase enzyme there cleaves its conjugate; causing the free drug to be released into the renal tubule cells and excreted in urine.

One metabolic pathway which occurs in the kidneys is N-dealkylation due to cytochrome P-450; this is the oxidation of an active carbon and thus simultaneous breakup of a molecule. It takes place in drugs which contain an amine group attached to an alkyl one such as Aminopyrine; and is thought to be due to CYP2E1. Aminopyrine produces metabolites including 4-acetylaminoantipyrine, 4-aminoantipyrine, and 4-monomethylamine antipyrine (fig 33). It is believed that this happens in the cortex of the kidney but the research for this theory is minimal.

Aminopyrine is a white crystalline substance used in preparations to produce an analgesic and antipyretic effect in patients; it can have to side effect of agranulocytosis and if this occurs the medication needs to be stopped immediately.

Cyclosporine A is an immunosuppressive medication used commonly to reduce the rejection rates of bone marrow transplants. It is a large molecule (C62H111N11O12) which is known be metabolised in the liver and kidney; by CYP3A5 and 3A4. The major metabolites are AM1, AM9 and AM4N (this is shown in figure 34). CYP3A4 oxidises cyclosporine at many positions and CYP3A5 attacks at amino acid 9.

The liver remains to be the most active metabolic organ the body possesses but even so chemical and pharmaceutical companies are beginning to properly understand and use the other sites of drug metabolism; this means many new drugs are aimed to be metabolised at these other sites to aid administration, bioavailability of the drug, to target specific tissues and help to exclude systemic interference.

The mechanisms described above are all for drugs which are in their active form upon administration; but there are a set of drugs which are inactive when administered then once metabolised are active; and carry out their role in the body; Pro-drugs.

3.1 Prodrugs

In most cases drugs are metabolised in vivo into inactive metabolites, which are then excreted in the urine without having any pharmacological effect. On the other hand there are some inactive drugs that are activated once metabolised in the body such drugs are known as prodrugs. They differ from drugs by devoiding intrinsic pharmacological activity. They are designed for the purpose of attributing to absorption, distribution, metabolism and to improve poorly soluble drugs bioavailability.

3.2 Classification

There are several forms of prodrugs, and are classified into two major types and subtypes A, B (table 1). Their subdivision is based upon whether the prodrugs’ site of conversion into their active forms is the site for their therapeutic actions. TypeI, those that are converted intracellularly, and type II extracellularly converted. Sometimes a drug may have a therapeutic target at the same site it was converted, in that case it belongs to both TypeI A and B. an example of such prodrug is HMG Co-A reductase inhibitors.

Type of prodrug

Conversion site


Tissue location of conversion

Type I


Type IA

Therapeutic target tissue/cells

Type IB

Metabolic tissues (liver, lung, etc.)

Type II


Type IIA

GI fluid

Type IIB

Systemic circulation

Table 1

Prodrugs can be classified differently based on their chemical arrangements. Thus they are divided into four major types:

  • Carrier-linked prodrug as the name states, it means that the active agent (promoiety) is linked to a vehicle that is then metabolised via hydrolysis, oxidation or reduction. (e.g. Prontosil)
  • Bio-precursors prodrugs that do not contain a promoiety but activated once metabolised.
  • Macro-molecular prodrugs such as a PEG macromolecular carrier (e.g. polyethyleneglycol)
  • Drug-antibody conjugates an example of a carrier being an antibody raised against tumour cells


Type 1A

Zidovudine (ZDV) is one of the first line therapy Anti-HIV drugs to treat HIV positives. Zidovudine is a synthetic nucleoside prodrug that like other nucleoside analogues requires being intracelluraly phosphorylated in order to exhibit its actions as well as all anti-HIV nucleoside analogues. ZDV prodrug has three metabolising pathways (figure 1) in which phosphorylation process is the one that results in the active 5΄-tri-phosphate metabolite (ZDV-TP). Within the lymphocytes there are three cellular kinases responsible for phosphorylation of ZDV. At first thymidine kinase enzyme is responsible for the conversion of ZDV into monophosphate ZDV, into diphosphate ZDV via thimidylate kinase and finally triphosphate ZDV, the active metabolite, via the enzyme pyrimidine nucleoside diphosphate kinase enzyme.

However, Zidovudine is predominantly metabolised into an inactive metabolite 3′-azido-3′-deoxy-5′ B-D-glucopyranuronosyl-thymidine (5′-glucuronyl Zidovudine; GZDV), Fig. 2, which is excreted in urine. This metabolic pathway is known as glucuronidation and possibly mediated via UDP-glucuronyl transferease (UDPGT) enzymes.

And finally the third possible metabolism pathway that ZDV may undergo is via reduction of the azido group (N3) on the ribose ring. This results in the formation of 3′-amino-3′-deoxythymidine (AMT) via both CYP450 isoenzymes and NADPH-CYP450 reductase in human liver. (Fig. 37). The resulting metabolite is very important because of its alleged cytotoxicity.

3.2.2 Type1 B

Establishment of sulfenamide derivatives of Carbamazepine (CBZ), prodrugs came to hand to deliver the drug intravenously given that the drug is poorly soluble in water and cannot be prepared in a parental form. The use of co-solvent or surfactants to overcome those properties was not a successful try-out due to some formulation toxicities and resulted allergic reactions by the drug; therefore the prodrugs utilization was an improved synthetic concept. Once the prodrug is administered it reconvert to parent CBZ drug via displacement reaction of sulfide bond on the sulfenamide prodrug to NH acidic group (CBZ), Fig 4. At the outset the sulfur atom is bivalent and positively protonized; therefore it is prone to a nucleophiles attack by the free thiols (cysteine, glutathione, etc.) in vivo. This results in the NS single bond being cleaved and formation of NH-acidic drug (CBZ). As the drug is released, rapidly, they exert its therapeutic anti-epileptic actions.

N-cysteamine-CBZ prodrug, Fig. 5, is an example of sulfenamide derivative that is chemically modified to release CBZ throughout the sulfenamide prodrug strategy.

The prodrugs’ resulting active metabolites are transported by p-glycoprotein across the blood brain barrier; therefore its site of action is not within its site of conversion thus it’s categorized within type IB prodrugs. Other examples of this type of prodrugs are Captopril, Heroin, Milsodomine, Primidone, Phenacetin and Paliperidone.

3.2.3 Type 2 A

Some prodrugs are converted extracellularly within the Gastro intestinal fluids to release the active drug . Lisdexamfetamine dimesylate (LDX) is an example of this prodrug class that is absorbed from the GI tract and metabolised thereafter in the small intestine. LDX are prodrugs of Phenethylamine that is a psychoactive drug to treat attention deficit or hyperactivity disorder in children and adults. LDX prodrug consists of a natural amino acid Lysine coupled to Dextro-Amphetamine in a covalent bond. The amino acid exerts the drug therapeutic actions once cleaved from the prodrug via trypsin or peptidase enzymes, Fig. 6. Trypsin enzyme is found in the GI fluids to hydrolyse the peptide bond of the amino acid at its carboxyl side

In conflict there are some studies suggest that the site of conversion occurs within the blood therefore it is a type II B prodrug, which is backed by LDX concentration versus time profile. ‘’After oral administration of LDX, its plasma concentration peaks rapidly (Tmax, -1 hour). Simultaneously, d-amphetamine concentrations rise slowly, and peak levels are observed 3.5 hours post dose. These observations suggest that the contribution of the gastrointestinal tract to the enzymatic conversion of LDX is low and that the majority of conversion occurs in the systemic circulation”.

3.2.4 Type 2 B

Bacampicillin ester prodrug of Ampicillin is ideal for enhancing the drugs bioavailability and the rate of absorption. The ester group makes the prodrug prone to hydrolysis by esterases within the intestinal wall releasing the active Ampicillin with acetaldehyde and CO2 to exert its bactericidal properties. Some studies show that following oral ingestion of Bacampicillin a high bioavailability of the drug resulted within the lymph interstitial fluid and that it is rapidly bio-transformed without any unchanged Bacampicillin left within the serum or urine.

4. Factors influencing drug metabolism

4.1 Age

There is no clear pattern for the age influence on drug metabolism, due to other possible factors which may have a more powerful effect on the individual. Physiological changes continue with aging effecting the drug absorption and renal drug clearance; for example chlomethiazole which shows a high bioavailability within the elderly, therefore they require a lower dose; an example of their aging is a decrease in the rate of gastric emptying and gastro intestinal motility. The metabolism changes that occur with ageing is also due to the liver enzymes being diminished or changes to liver size and blood flow. Clinical studies show that there is a decrease in the drug metabolism which involves oxidation but there are no significant effects on the phase II metabolism. Phase I metabolism may be impaired with age specifically for esterases enzymes actions.

4.2 Sex

Pre-clinical studies have proven that female rats required half the dose of Barbiturates than male rats to induce sleep. This was said to be associated with CYP3A4 induction in females, therefore female rats are less able to metabolise this drugs, and have slower metabolising rate than male ones hence higher bioavailability of the drug.

4.3 Disease states and drug metabolism

Physiological disorders or diseases of the GI tract without doubt affect the drug stability and absorption if it undergoes first-pass metabolism 80%; it causes a reduction in first pass metabolism thus lower doses are needed. A patient with GI tract diseases of disorders who have the same dose as a healthy patient might experience toxicity, as it takes the drug longer to be metabolised.

4.4 Diet

Food has an effect on raising the GI pH and providing a viscous environment, which reduces absorption of weakly basic drug and vice versa for a weakly acidic one. Some enzymatic metabolised drugs are degraded by pepsin in the GI which is secreted in response to food; pepsin reducing the drug bioavailability. Nutrient-drug interactions may reduce, delay, increase, accelerate absorption, or have no effect on hepatic metabolism of drugs. Grapefruit has the tendency to increase CYP3A metabolized drugs bioavailability as discussed above; as grapefruit inhibits this CYP450 intestinal enzyme hence less enzymes are available to break down the drugs. An example of such drug-nutrient interaction is between Terfenadine antihistamines and grapefruit or verapamil, ciclosporin.

5.1 Toxicity:

Drug metabolism can lead to either a decrease or an increase in the toxicity of the parent compound. This is mainly reliant on the biological potency of the drug as well as the metabolite. There are five different ways in which drugs can cause toxicity, on-target toxicity, un-related off target, hypersensitivity and immunological reactions, idiosyncratic drug reactions and the bioactivation to reactive metabolites. It is the latter mechanism which involves drug metabolism and has been reviewed here in greater detail.

Bioactivation is considered to be the process by which drug metabolism leads to an increase in toxicity. Drugs are bioactivated to reactive metabolites that bind covalently (irreversibly) to proteins and DNA (adducts). This binding of reactive metabolites to macromolecules triggers a series of biochemical changes and cell death.

Although cytochrome P450 has been described to work by metabolising drugs in a protective mechanism (allowing elimination) as described previously, they are also the most imperative enzyme involved in this toxicology. CYP’s can cause metabolic activation of drugs into reactive, toxic metabolites. Two features of CYP’s are toxicologically important, the genetic variability (e.g. polymorphisms) and the regulation/ induction potential.

The table below shows examples of cases when hepatic metabolism of drugs leads to toxification of the parent compound:


Metabolic Pathway


2- Acetylaminofluorene

N-Hydroxylation and Sulphation












Acetylation and Hydrolysis

Hepatic necrosis




Table 2

Two chemical reactions are involved in bioactivation. The first involves the reaction between the electrophile (formed by the drug) and a nucleophile (present in cells) and the second involves free radical propagation. Most studies attempting to study mechanisms of bioactivation are carried out in vitro and it is not always possible to apply these results in vivo.

Trichloroethylene (TCE) has been used as an anaesthetic in the past and was praised to be an anaesthetic revolution. P450 oxidation of TCE produces a sedative product named choral by carrying out a 1,2-chloride migration. Another minor oxidation product (TCE oxide) rearranges to produce glyoxal chloride. This then reacts with protein lysines by two possible pathways (outlined in the diagram) to produce either a 1- or 2- carbon conjugate. The glyoxal chloride also reacts to form a third product.

With the use of mass spectrometry it is possible to observe a reaction with the electrophile and the ACTH peptide. The binding can then be analysed and the amount of covalent binding was estimated according to the integration peaks. It was found that treating the TCE oxide with an altered ACTH (containing a mild base), removed a large amount of the adducts. This indicates more ester bonds were present than lysine amide bonds. An additional finding of studies carried out on this drug, demonstrate that the rearrangement product (oxalyl chloride) reacts with phosphate rather than protein, this is stable and can be isolated (half life= 100 minutes).

The major oxidation system in neutrophils is the combination of NADPH oxidase; this creates superoxide and in due course hydrogen peroxide and myeloperoxidase. Myeloperoxidase is then oxidised by hydrogen peroxide and in turn oxides the chloride to HOCl. This system forms reactive metabolites of drugs known to be involved in Agranulocytosis (neutrophil being the target of toxicity).

Clozapineis an antipsychotic, with a high incidence of Agranulocytosis/ liver toxicity. It is metabolised by the liver to a reactive metabolite. The reactive intermediate activated by neutrophils will covalently bind to neutrophils. HOCl can also carry out this oxidation (mass spectrum can show the intermediate). The compound formed is a nitrenium ion and has a delocalised positive charge. The ion is a carbene, which means singlet and triplet states are present. Singlet nitrenium ions react quickly with nucleophiles. This metabolite can be trapped by glutathione and adducts are formed at 6 and 9 positions. Patients taking this drug require a blood cell count weekly for the first months of use.

Diclofenac, a 2-arylacetic acid, NSAID drug has been investigated for cytotoxicity that is related to drug metabolism by cytochrome P-450. Covalent binding seems to due to P450-created and glucuronide reactive metabolite. Binding mediated by P450 is due to the formation of quinonimines, which seem to play an important role in the toxicity. The diagram below shows how the reactive quinonimine intermediate can be formed by further CYP oxidiation which then leads to destructive drug-protein adducts or reactions with glutathione (producing glutathione adducts).

Paracetamolis a well documented case of when drug metabolism can go wrong. Paracetamol hepatotoxicity is one of the most common causes of acute liver failure. The metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is formed when cytochrome P450 enzymes CYP2E1 and CYP3A4 convert the drug NAPQI. This then leads to an accumulation of NAPQI, which undergoes conjugation with glutathione. Conjugation exhausts glutathione (antioxidant) which leads to cell damage and death. An antidote (N-acetylcysteine or NAC) acts as a precursor for glutathione aiding the body to regenerate glutathione.

An in detail document has looked at in this section for acetaminophen’s contribution to toxicity and it considers factors such as oxidative stress. The formation of NAPQI was found to be formed by a two-electron oxidation. When a toxic dose of acetaminophen is taken, the detoxification of NAPQI by glutathione (phase II metabolism) to form a conjugate is reduced as GSH is depleted by up to 90%; the mechanism is shown in detail (figure 43), the binding with cysteine groups in proteins to form an acetaminophen-protein adduct can be seen:

A possible mechanism of cell death caused by acetaminophen protein adducts, is that covalent binding to critical cellular proteins results in the loss of function/ death of the cell- it is most likely that these are mitochondrial proteins (therefore a loss of energy control). This is one protein that forms adducts with acetaminophen is N-10-formyltetrahydrofolate dehydrogenase. Studies with mice, showed a decrease of 20% at 2 hours in activity of this enzyme (1- carbon metabolism and oxidisation of formaldehyde to carbon dioxide) when acetaminophen was introduced.

Oxidative stress is also believed to be another mechanism in the development of acetaminophen toxicity. Fenton-type mechanisms would take place in which an increase in superoxide formation leads to hydrogen peroxide and peroxidation reactions. When NAPQI is formed, GSH concentrations are low and GSH peroxide (the peroxide detoxification enzyme) is expected to be inhibited. Additionally when NAPQI is formed by P450, a superoxide ion is formed and dismutated to form hydrogen peroxide. Another suggested mechanism is that peroxidation of acetaminophen to the semiquinone free radical leads to redox cycling between acetaminophen and semiquinone. This can also lead to an increase in superoxide and toxicity.

Nitrotyrosine occurs in centrilobular cells of the liver of acetaminophen treated mice and analysis of the cells found that nitration occurred in the same cells that contain acetaminophen adducts and development of necrosis. It is believed that nitrotyosine is formed by a reaction between peroxynitrite and tyrosine. Nitration of tyrosine is a biomarker of peroxynitrite formation. It is formed in a reaction between nitric oxide and superoxide. It was found that NO synthesis increased with acetaminophen toxicity. Peroxynitrite not only leads to nitration of tyrosine but it is an oxidant that will attack biological targets.

Tyrosine nitration of acetaminophen was investigated using inhibitors of nitric oxide synthetase in mice with deficient iNOS. This experiment showed that acetaminophen caused a 3-fold increase in oxidative stress (lipid peroxidation) in the iNOS knockout mice group but no increase in the wild-type mice. Wild-type mice toxicity was therefore followed by tyrosine nitration, whereas in the iNOS knockout mice the toxicity was followed by oxidative stress.

In general the excess levels of superoxide were formed in acetaminophen toxicity. When NO was present (e.g. wild-type mice), the superoxide reacts to form peroxynitrite, this nitrates the proteins. When NO is absent the superperoxide leads to peroxidation. This study signifies the importance of NO in the disposition of superoxide which leads to oxidative stress.

The role of oxidative stress in acetaminophen toxicity

Oxidativestress is caused by an imbalance between the formation of reactive oxygen and a biological system’s ability to readily detoxify the reactive intermediates. Free radicals formed will interact with other molecules within cells and cause oxidative damage to proteins, membranes and genes. Oxidative stress is an important component of toxicity caused by most chemicals activated to electrophiles. H2O2 and O2.-are formed in uncoupled reaction that are catalysed by microsomes and purified P450’s. Inducing mammalian P450 has been shown to lead to oxidative damage of the cell.

However CYP’s are not the only enzymes involved in the bioactivation. It has been found that Phase II enzymes are also involved in catalysing bioactivation of drugs in certain settings, even though they are usually linked with detoxification reactions, such as conjugation and hydrolysis.

Three examples include the role of epoxide hydrolase, glutathione transferase and sulphonylating enzymes. Epoxide hyrdolase normally functions by detoxifying epoxides to trans- dihydrodiols; however an example of when this has lead to activation is with benzo[α]pyrene drugs. (+)benzo[a]pyrene -7,8 dihydrodiol-9,10 epoxide is formed which covalently binds to DNA making it a procarcinogen. Glutathione transferases have been found to convert ethylene dibromide (pesticide) to a toxic eposulphonium ion by conjugating these compounds with reduced glutathione to facilitate dissolution in aqueous media. The sulphonation of certain compounds (aryl hydroxylamines and benzylic alcohols) may produce reactive, toxic nitrenium ions and carbocations respectively).

However one of the most known examples of phase II related bioactivation leading to toxicity is that of glucuronidation of morphine. Glucuronidation conjugation utilizes UDP-glucuronosyltransferases to form much more water soluble compounds; however morphine is cleared to two isomeric glucuronides- morphine-3- and 6-glucuronide (M3G and M6G). The minor metabolite M6G is more potent than morphine as an analgesic yet the M3G major metabolite antagonises morphine analgesia. Although the minor metabolite will contribute to clinical efficacy during chronic morphine therapy, it has been suggested that accumulation of Morphine-6-glucuronide might cause toxicity and hence renal impairment.

Glucuronidation can cause toxic species by rearranging functional groups to form reactive electrophilic intermediates. Drugs such as zomepirac have been found to experience this problem. Its acyl glucuronide conjugate covalently binds via nucleophilic displacement of the glucuronic acid moiety, thus a non-hydrolysable conjugate is formed which is an electrophilic species that binds to free tyrosine, cysteine thiols or the lysine residue of a protein. Many other acidic drugs are metabolised to acyl glucuronides such as Ibuprofen and Valporic acid, yet the glucuronidation lead to safe products being formed. A study on rats was conducted to demonstrate the toxicity of zomepirac-1-O-acyl-glucuronide (the metabolised form of Zomepirac). The ability of Zomepirac (ZP) to become bioactivated to reactive metabolites that transacylate glutathione to form ZP-S-acyl-glutathione thioester (ZP-SG) in vitro and in vivo were examined. ZP was incubated with rat hepatocytes and the ZP-SG was detected by a liquid chromatography mass spectrometry technique. ZP-SG was initially developing rapidly then decreased in a linear fahion after 60 minutes of incubation. ZP-SG was found to be unstable as it underwent a rapid hydrolysis (half life =0.8 minutes). ZP was also administered to a Sprague-Dawley rat (100mg/Kg), bile was then collected for ZP-SG and a large amount was found excreted after 6 hours. Collectively this study shows ZP is metabolically activated to a reactive acylating derivative such as zomepirac-1-O-acyl-glucuronide, that transacylate glutathione forming ZP-SG. Zomepirac was withdrawn from the market after such studies demonstrated its metabolism related toxicity. The diagram below demonstrates ZP’s activation which leads to detrimental covalent bonding with proteins.

A structure activity relationship can be observed for acyl glucuronide rearrangement, covalent bonding and toxicity. Acyl glucuronides unsubstituted at the α position appear to be the most reactive and give the highest covalent bonding (due to a lack of steric hindrance for rearrangement and nucleophilic attack). With increasing substitution reactivity decreases (an exception to this is Valproic acid).

Therefore although glucuronidation is usually considered detoxifying there are cases when glucuronide conjugates are associated with creating toxicities. Two classes of glucuronides are involved:

  1. Glucuronides may have effects by non-covalent interactions such as morphine 6-O-glucuronide or retinoid and D-ring of estrogens glucuronides.
  2. Electrophilic chemical reactivity such as N-O-glucuronides of hydroxamic acids and acyl glucuronides of carboxylic acids.

Glucuronides can therefore have important effects which include arylamine-induced carcinogenesis as well as toxicities associated with morphine analgesia and carboxylic acid drugs.

In a similar method to UDP-glucuronosyltransferase (UGT) families, Sulphotransferases are enzymes that will catalyse metabolic clearance by conjugation of drugs; however certain products of phase II metabolism (e.g. unstable sulphate conjugates) are genotoxic.


As mentioned previously there is an implication of genetic variation in drug metabolism. Certain toxicological effects of drugs are exaggerated due to the heritable deficiency in P450 enzyme. Idiosyncratic toxicity of drugs can be due to genetic polymorphism. Troglitazone is used in the treatment of insulin-resistant diabetes mellitus. However there is a rare case of hepatotoxicty with some patients. CYP3A4 catalyses the production of chemically reactive forms of Troglitazone metabolites (glutathione conjugates). It has been reported that epoxide metabolites are formed. Patients with a null genotype of GSTT1 and GSTM1 had the highest cases of hepatotoxicity. This study clearly demonstrates that for each individual the chemistry behind drug toxicity can differ.

Most mechanisms involved in the protein adduct-induced toxicity are largely undefined and experiments carried out, have been on animal systems and extrapolated to humans. Pharmacokinetic (PBPK) models are now being used. The approach focuses on the physical aspects of distribution which is established in different species (blood flow, tissue extraction). It is important to identify the main pathways involved in bioactivation and detoxification, and characterising in terms of quantitative parameters.

Predicting toxicity is very important in drug development. Many chemicals are inert unless converted to metabolites by P450 or other enzymes. Reactive electrophiles are produced this way and acetaminophen remains a chief example.

6.1 Conclusion

Drug metabolism is a very specific and complex biological activity, which involved many different chemical processes including; hydrolysis, oxidation, n-dealkylation, glucuronidation, N-demethylation. These occur primarily in the liver but other tissues have the ability to complete these processes, including the lungs, kidney, skin and gastrointestinal tract. Pro drugs are an important part of the pharmaceutical industry they provide an increases bioavailability of some drugs and enable others to be given parentally. Further investigation into this area of chemistry and pharmaceutics can help to develop more potent and increased bioavailability drugs. First pass metabolism of drugs was previously seen as a disadvantage but now can be used advantageously as seen from drugs such as, naproxcinod, zidovudine, carbamazepine, and LDX. In other words, prodrugs are now the way forward to prevent previously seen problems with first pass effect metabolism.

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