Reactions produce a carboxylic acid metabolite

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Amino acid reactions

Several phase I reactions produce a carboxylic acid metabolite. Xenobiotic carboxylic acids can be metabolised before elimination by amino acid conjugation. Glycine; the most common conjugating amino acid forms ionic conjugates that are water soluble with aromatic, arylaliphatic and heterocyclic carboxylic acids. In these reactions, first the xenobiotic carboxylic acid is activated by ATP to form the AMP ester by the enzyme acyl synthetase. Then the AMP ester is converted to a Coenzyme-A thioester. Next, an amide or peptide bond is formed between the thioester and the amino group of glycine. The latter reaction is mediated by the enzyme acyl transferase. These reactions are shown in figure 1.

The amino acid conjugate produced is ionic and therefore water soluble, hence it is easily eliminated in the urine and bile. (1)

Glutathione conjugation

Glutathione is a protective compound in the body that removes potentially toxic electrophilic compounds and xenobiotics. Drugs are metabolised by phase I reactions to form strong elecrophiles that can react with glutathione to form conjugates that are not toxic. This phase II reaction differs from others since electrophiles are subject to conjugations rather than nucleophiles. The nucleophilic thiol group on the glutathione compound (figure 2) attacks elecrophiles (electrophilic carbons with leaving groups).

Compounds that can be conjugated to give thioether conjugates of glutathione:

  • Epoxides
  • Haloalkanes
  • Nitroalkanes
  • Alkenes
  • Aromatic halo- and nitro- compounds

Glutathione-S-transferases (GST) are enzymes which catalyse the reactions above. There are thirteen different human GST subunits which have been identified and they belong to five different classes. They are located in the cytosol of the liver, kidney and gut. The enzyme GST is thought to increase the ionisation of the thiol group of glutathione, leading to an increase in its nucleophilicity towards electrophiles.

Once formed, GSH conjugates may be excreted directly or more often they are further metabolised to N-acetylcysteine conjugates which can then be excreted via ‘phase III metabolism'.

Phase III Metabolism -

further modification and excretion

Before being excreted in the urine, most xenobiotics are made less toxic and more water soluble as polarity increases by metabolising enzymes in phase II reactions. In phase III metabolism water soluble compounds are excreted in the urine. However, some drug compounds are not metabolised and therefore are not excreted. These non-metabolised compounds are readily reabsorbed from the urine through the renal tubular membranes and into the plasma to be recirculated.

Some xenobiotic conjugates from phase II reactions are further metabolised during phase III metabolism reactions. Glutathione-S conjugates may be metabolised further by hydrolysis of the glutathione conjugate (GSR) at the y-glutamyl bond of the glutamate residues by y -glutamyl transferase (y -GT) followed by hydrolysis of glycine residues resulting in a cysteine conjugate containing a free amino group of the cysteine residue. This then undergoes N-acetylation to form mercapturic acid. The final products; mercapturic acids are S-derivatives of N-acetylcysteine synthesised from glutathione.

First-pass Metabolism

The metabolism of many drugs is dependent on the route of administation therefore orally administered drugs are subject to first pass metabolism and consequently their bioavailablity is reduced. This occurs as a result of the orally administered drugs entering the systemic circulation via the hepatic portal vein, so the drug is exposed to the intestinal wall and the liver, which is thought to be the main site of first-pass metabolism of orally administered drugs. Other possible sites are the gastrointestinal tract, blood, vascular endothelium and lungs.

First-pass Metabolism in the Liver

During first-pass metabolism, the cytochrome P450 enzymes family represent the most significant of the hepatic enzymes. It has been estimated that the endoplasmic reticulum of the liver contains approximately 25 000 nmol of cytochrome P450. Although there are several human P450 subfamilies and multiple individual isozymes within subfamilies, only five P450 enzymes are shown to be significant for the process of first-pass metabolism:

  • CYP1A2
  • CYP2C9
  • CYP2C19
  • CYP2D6
  • CYP3A4

Cytochrome P450 drug substrates are commonly highly extracted during first-pass metabolism. Examples of these drugs are; morphine, verapamil, propranolol, midazolam, lidocaine. Drugs that are highly extracted such as lidocaine have a low bioavailability when taken orally therefore they are not administered orally. CYP3A4 is the most commonly active isozyme against P450 drug substrates. This is possibly due to the enzyme's abundance and broad substrate specificity. Highly extracted substrates for conjugative, reductive or non-P450 oxidative enzymes are less common. These include labetalol, morphine, terbutaline, isoproterenol and pentoxifylline.

The gut is also an important organ involved in pre-systemic metabolism. Metabolism here for drugs with high first-pass metabolism leads to a reduced bioavailability. Some metabolizing enzymes such as CYP3A4 is found at a higher level in enterocytes than in the liver. Recent findings state that gut wall metabolism is the major cause of low bioavailability of certain drugs.

Intestinal First-pass Metabolism

Various drug metabolizing enzymes found in the liver are also found within the epithelium of the gastrointestinal tract. These include cytochromes P450, glucuronosyl transferases, sulfotransferases, N-acetyl transferase, glutathione S-transferases, esterases, epoxide hydrolase and alcohol dehydrogenase. The small intestine contains high amounts of three cytochrome P450 enzymes; CYP3A, CYP2D6 and CYP2C. Unlike the liver which has a relatively uniform distribution of P450enzymes, the distribution of P450 enzymes is not uniform along the small intestine and villi. Proximal mucosal P450 content is normally higher than distal mucosa P450 content.

Therefore it has been established that protein level and catalytic activity of drug-metabolizing enzymes in the small intestine are generally lower than those in the liver. This has been demonstrated by comparison of cytochrome P450 enzymes in the liver and the small intestine. The extent of first-pass metabolism can result from interindividual variability:

  1. Genetic variation
  2. Induction or inhibition of metabolic enzymes
  3. Food increases liver blood flow. This can increase the bioavailablity of some drugs by increasing the amount of drug presented to the liver to an amount that is above the threshold for complete hepatic extraction
  4. Drugs that increase liver blood flow (similar effects to food) and drugs that reduce liver blood flow
  5. Non- linear first pass kinetics, i.e. dose
  6. Liver disease increases the bioavailability of some drugs with extensive first-pass metabolism

To avoid first pass metabolism a drug can be administered sublingual and buccal routes. These routes lead to drugs being absorbed by the oral mucosa. During sublingual administration the drug is put under the tongue where it dissolves in salivary secretions. An example of a sublingual drug is nitroglycerine. During buccal administration the drug is positioned between the teeth and the mucous membrane of the cheek. Both of these routes avoid destruction by the GI fluids and first pass effect of the liver. Drugs may also be administered via other routes to avoid first-pass metabolism, for example; rectal, inhalation, transdermal, intravenous.

Prodrugs

Prodrugs can be used to improve oral delivery of poorly water-soluble drugs. Many drugs require metabolic activation in order to exert their pharmacological action; these are described as pro-drugs. Metabolic activation is usually linked to phase I metabolism enzymes. Prodrugs can be used to target a drug to its specific site of action; an example of this is the drug used in Parkinson's disease levodopa; the inactive form which is metabolised in the neurone by the enzyme dopa decarboxylase to the active form; dopamine. Dopamine does not cross the blood-brain barrier so it is given as the levodopa precursor which is lipophilic so it can cross the barrier and then metabolized in vivo to dopamine.

Another example of the use of prodrugs is the pharmacological activation of a pro-drug to mercaptopurine which is a chemotherapeutic agent used in the treatment of leukaemia. When mercaptopurine is administered, its clinical usefulness is restricted because of its rapid biotransformation by xanthine oxidase to an inactive metabolite 6-thiouric acid (azathioprine). Therefore larger doses have to be given as it has a low bioavailability, this leads to toxicity. By administering mercaptopurine as its cysteine conjugate, the limitations can be overcome. This ionic form of the pro-drug conjugate is selectively taken up by the renal organic anion transport system. The kidney B-lyase enzyme system then cleaves the prodrug conjugate to give the active mercaptopurine in the kidney (figure 5). (8)(9)

The table below shows some examples of prodrugs, their metabolic conversion and the active form of the drug produced:

Prodrug

Clinical Use

Metabolic Conversion

Active Drug/ Metabolite

Azathioprine

Immunosuppressant

Thio-ether hydrolysis

Mercaptopurine

Glyceryl triacetate

Antifungal

Ester hydrolysis

Acetic acid

Levodopa

Antiparkinsonian

Decarboxylation

Dopamine

Prednisone

Anti-inflammatory

Keto reduction

Prednisolone

Primidone

Anti-epileptic

Oxidation

Phenobarbitone

Proguanil

Antimalarial

Cyclization

Cycloguanil

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