**4.2 Phase II reactions**

In phase II reactions, the activated xenobiotic metabolites are conjugated with charged species such as glutathione (GSH), sulfate, glycine or glycuronic acid and increased risk of early renal complications in type 2 diabetes mellitus (Banjoko & Akinlade 2010). These reactions are catalysed by substrate specific transferases which in total can metabolize almost any hydrophobic compound that contains nucleophilic or electrophilic group.

One of the most important of this group is the glutathione S-transferase (GSTs). The addition of large anionic groups such as glutathione detoxifies reactive electrophiles and produces more polar metabolites that cannot diffuse across membranes and may therefore be actively transported.

#### **4.2.1 Glutathione conjugation**

Glutathione is a tripeptide of glycine, cysteine and glutamic acid formed by the action of glutamylcysteine synthetase (glutathione synthetase).The enzyme glutathione transferase catalyses the conjugation of modified xenobiotic with glutathione. A large number of drugs are conjugated by glutathione during metabolism. Inhibitors of the enzyme include Buthione – S – Sulfoxine. Two types of reactions are common with glutathione. The first is displacement of halogen, sulfate, sulfonate or phosphonitro group. The second is the addition of glutathione to activated double bond or strained ring system. Some of the conjugation reactions include:

Pharmacogenetics: The Scientific Basis 189

This is a common minor pathway of xenobiotic biotransformation which generally decreases water solubility. Enzymes that catalyse the reactions are called methyltransferases and the co-factor is S-adenosylmethionine (SAM). In methylation, a methyl group (CH3) is transferred to O, N, S or C molecule on the substrates which include phenols, catecholamines and heavy metals like Hg, As and Se. There are several methyltransferases in human tissues examples of which are phenol – O – methyltransferases, catechol – O – methyltransferase, O-methyl transferase and S-methyl transferase. Genetic polymorpohism has been observed in thiopurine metabolism in a reaction catalysed by a member of this group of enzymes. High activity allele causes increased toxicity and low activity allele

This is the major route of biotransformation of aromatic amines and hydrazines. The reaction is catalysed by N – acetyl transferases (NAT) enzyme and the co factor acetylcoenzyme-A. The process generally causes a decrease in water solubility. Substrates of the enzyme include sulfanilamide, isoniazid, dapsone and caffeine. In humans three phenotypic forms have been identified and these are slow, intermediate and rapid acetylators (Evans 1999, Murphy 2001). Various mutations of the enzyme result in decreased enzyme activity or stability. Like every other entity exhibiting genetic polymorphism, there are various ethnic and tribal variations. For example, 70% of slow acetylator status was observed in Middle Eastern population, 50% in Caucasians and 25% in Asians (Hiratsuka 2006, Evans and Relling 1989). Drug toxicities in slow acetylators include nerve damage from dapsone and bladder cancer in cigarette smokers due to increased levels of hydroxylamines (Ohno

This is an alternative pathway to glucuronidation. Amino acid conjugation operate with two principles. The first is that carboxylic group (COOH) group of a substrate is conjugated with an amino (NH2) group of glycine, serine, glutamine requiring co enzyme-A activation. Notable example is the conjugation of benzoic acid with glycine to form hippuric acid. Benzoic acid is commonly used as a preservative in carbonated drinks. Alternatively aromatic NH2 or NHOH conjugate with COOH of serine proteins requiring ATP activation. This metabolic pathway demonstrate specie specificity in accepting amino acid. For example, in mammals, benzoic acid is conjugated by glycine whereas for the same substrate in birds, ornithine acts. Dogs and cats utilize taurine to conjugate bile acids while other non human primates utilize glutamine for conjugation. Metabolic activation of serine or proline results in N-esters of hydroxylamine which are unstable and may degrade to reactive

This pathway is important for the activation of many purine and pyrimidine antimetabolites

**4.2.4 Methylation** 

causes decreased efficacy.

**4.2.6 Amino acid conjugation** 

electrophile.

and Yamaguchi 2000, Evans 1999, Hiratsuka et al 2006).

**4.2.7 Ribonucleoside/nucleoside synthesis** 

used in cancer chemotherapy

**4.2.5 Acetylation** 


Distinct cytosolic and microsomal glutathione -S transferases have been identified. In all, four classes of soluble glutathione S transferase are known to exist. The enzyme also exhibit genetic polymorphism and overexpression of the enzyme leads to e.g. resistance of insects to DDT, corn to atrazine and cancer cells to chemotherapy. The enzyme also participates in reduction of hydroperoxides and prostaglandin metabolism. Inducers of the enzyme include 3-methylcholanthrene, phenobarbital, corticosteroids and antioxidants. GST exhibit specie specificity; for example, aflatoxin B1 is not carcinogenic in mice because it conjugates with glutathione very rapidly in them. Conjugates are excreted intact in bile or converted to mercapturic acid in kidney and excreted in urine in a reaction catalysed by glutamyl transpeptidase an aminopeptidase

#### **4.2.2 Uridyl Diphosphate Glucuronyl transferase (UDPG transferase)**

The reaction of UDP Glucuronyl transferase results in the formation of O-, N-, S and Cglucuronides. Six forms of this enzyme have been identified in the liver. The cofactor for its reaction is UDP – glucuronic acid. Inducers include phenobarbital, indoles, 3 methyl cholanthrene and cigarette smoke. Some of its substrate are dextrophan, methalidone, morphine, p-nitrophenol, valproic acid, non steroidal anti-inflamatory drugs, bilirubin and steroid hormones. In Criggler Najjar syndrome; a severe form of bilirubinaemia, the enzyme is inactive hence inducers have no effect. However, in Gilbert's syndrome; a mild form of hyperbilirubinaemia, phenobarbital can increase the rate of bilirubin glucuronidation to normal functions . Other substrates of the enzymes include, morphine and chloramphenicol. Conjugates of UDPG transferase are excreted in bile and urine. An S-glucuronidase from the gut microflora cleaves the glucuronic acid, the glycone formed can be reabsorbed to undergo enterohepatic cycling. Other associated reactions include metabolic activation of 2, 6 dinitrotoluene by S-glucuronidase; whereby the latter removes glucuronic acid from Nglucuronide. The nitrogroup is then reduced by microbial N-reductase and the resultant hepatocarcinogen may be reabsorbed.

#### **4.2.3 Sulfation**

The sulfation process is catalysed by sulfotransferases which are widely distributed in the body. The co-factor for their reaction is 31 phosphoadenosine 5 phosphosulfate (PAPS). Their conjugation result in highly water soluble sulfate esters which are eliminated in urine and bile. Examples of substances for sulfation include phenols, catecholamines and hydroxylamines. Sulfation is a high affinity, low capacity pathway which is limited by low PAPS level. Acetaminophen is a drug that undergoes both sulfation and glucuronidation. At low doses, sulfation predominates but at high doses glucuronidation predominates. Four sulfotransferases in human liver cytosol have been identified to date. Aryl sulfatases in gut microflora remove sulfate groups in a sort of enterohepatic recycling. Sulfation decreases pharmacologic and toxic activities but can also cause activation of chemically unstable groups to carcinogens, for example hydroxylamine.

#### **4.2.4 Methylation**

188 Pharmacology

iii. Activation of trinitroglycine to oxidized glutathione (GSSG) dinitroglycerine and Nitric

Distinct cytosolic and microsomal glutathione -S transferases have been identified. In all, four classes of soluble glutathione S transferase are known to exist. The enzyme also exhibit genetic polymorphism and overexpression of the enzyme leads to e.g. resistance of insects to DDT, corn to atrazine and cancer cells to chemotherapy. The enzyme also participates in reduction of hydroperoxides and prostaglandin metabolism. Inducers of the enzyme include 3-methylcholanthrene, phenobarbital, corticosteroids and antioxidants. GST exhibit specie specificity; for example, aflatoxin B1 is not carcinogenic in mice because it conjugates with glutathione very rapidly in them. Conjugates are excreted intact in bile or converted to mercapturic acid in kidney and excreted in urine in a reaction catalysed by glutamyl

The reaction of UDP Glucuronyl transferase results in the formation of O-, N-, S and Cglucuronides. Six forms of this enzyme have been identified in the liver. The cofactor for its reaction is UDP – glucuronic acid. Inducers include phenobarbital, indoles, 3 methyl cholanthrene and cigarette smoke. Some of its substrate are dextrophan, methalidone, morphine, p-nitrophenol, valproic acid, non steroidal anti-inflamatory drugs, bilirubin and steroid hormones. In Criggler Najjar syndrome; a severe form of bilirubinaemia, the enzyme is inactive hence inducers have no effect. However, in Gilbert's syndrome; a mild form of hyperbilirubinaemia, phenobarbital can increase the rate of bilirubin glucuronidation to normal functions . Other substrates of the enzymes include, morphine and chloramphenicol. Conjugates of UDPG transferase are excreted in bile and urine. An S-glucuronidase from the gut microflora cleaves the glucuronic acid, the glycone formed can be reabsorbed to undergo enterohepatic cycling. Other associated reactions include metabolic activation of 2, 6 dinitrotoluene by S-glucuronidase; whereby the latter removes glucuronic acid from Nglucuronide. The nitrogroup is then reduced by microbial N-reductase and the resultant

The sulfation process is catalysed by sulfotransferases which are widely distributed in the body. The co-factor for their reaction is 31 phosphoadenosine 5 phosphosulfate (PAPS). Their conjugation result in highly water soluble sulfate esters which are eliminated in urine and bile. Examples of substances for sulfation include phenols, catecholamines and hydroxylamines. Sulfation is a high affinity, low capacity pathway which is limited by low PAPS level. Acetaminophen is a drug that undergoes both sulfation and glucuronidation. At low doses, sulfation predominates but at high doses glucuronidation predominates. Four sulfotransferases in human liver cytosol have been identified to date. Aryl sulfatases in gut microflora remove sulfate groups in a sort of enterohepatic recycling. Sulfation decreases pharmacologic and toxic activities but can also cause activation of chemically unstable

i. N – acetylbenzoquinoneimine, an activated metabolic of acetaminophen.

**4.2.2 Uridyl Diphosphate Glucuronyl transferase (UDPG transferase)** 

ii. O – demethylation of organophosphates

oxide (NO) a vasodilator.

transpeptidase an aminopeptidase

hepatocarcinogen may be reabsorbed.

groups to carcinogens, for example hydroxylamine.

**4.2.3 Sulfation** 

This is a common minor pathway of xenobiotic biotransformation which generally decreases water solubility. Enzymes that catalyse the reactions are called methyltransferases and the co-factor is S-adenosylmethionine (SAM). In methylation, a methyl group (CH3) is transferred to O, N, S or C molecule on the substrates which include phenols, catecholamines and heavy metals like Hg, As and Se. There are several methyltransferases in human tissues examples of which are phenol – O – methyltransferases, catechol – O – methyltransferase, O-methyl transferase and S-methyl transferase. Genetic polymorpohism has been observed in thiopurine metabolism in a reaction catalysed by a member of this group of enzymes. High activity allele causes increased toxicity and low activity allele causes decreased efficacy.

#### **4.2.5 Acetylation**

This is the major route of biotransformation of aromatic amines and hydrazines. The reaction is catalysed by N – acetyl transferases (NAT) enzyme and the co factor acetylcoenzyme-A. The process generally causes a decrease in water solubility. Substrates of the enzyme include sulfanilamide, isoniazid, dapsone and caffeine. In humans three phenotypic forms have been identified and these are slow, intermediate and rapid acetylators (Evans 1999, Murphy 2001). Various mutations of the enzyme result in decreased enzyme activity or stability. Like every other entity exhibiting genetic polymorphism, there are various ethnic and tribal variations. For example, 70% of slow acetylator status was observed in Middle Eastern population, 50% in Caucasians and 25% in Asians (Hiratsuka 2006, Evans and Relling 1989). Drug toxicities in slow acetylators include nerve damage from dapsone and bladder cancer in cigarette smokers due to increased levels of hydroxylamines (Ohno and Yamaguchi 2000, Evans 1999, Hiratsuka et al 2006).

#### **4.2.6 Amino acid conjugation**

This is an alternative pathway to glucuronidation. Amino acid conjugation operate with two principles. The first is that carboxylic group (COOH) group of a substrate is conjugated with an amino (NH2) group of glycine, serine, glutamine requiring co enzyme-A activation. Notable example is the conjugation of benzoic acid with glycine to form hippuric acid. Benzoic acid is commonly used as a preservative in carbonated drinks. Alternatively aromatic NH2 or NHOH conjugate with COOH of serine proteins requiring ATP activation. This metabolic pathway demonstrate specie specificity in accepting amino acid. For example, in mammals, benzoic acid is conjugated by glycine whereas for the same substrate in birds, ornithine acts. Dogs and cats utilize taurine to conjugate bile acids while other non human primates utilize glutamine for conjugation. Metabolic activation of serine or proline results in N-esters of hydroxylamine which are unstable and may degrade to reactive electrophile.

#### **4.2.7 Ribonucleoside/nucleoside synthesis**

This pathway is important for the activation of many purine and pyrimidine antimetabolites used in cancer chemotherapy

Pharmacogenetics: The Scientific Basis 191

group. CYP3A4, CYP2D6, CYP2C9, CYP219, CYP2B6 and CYP1A2 play the most critical role and account for more than 90% of drugs metabolized by P450 ( Evans & Relling 1999). These enzymes have proven genetic polymorphism with associated drug responses (Hiratsuka et al 2002, Wong et al 2005, McAlpine et al 2011) and racial variations (Meyer 2004 & Suarez – Kurtz 2005). As discussed earlier, genetic polymorphism can manifest at both pharmacokinetic and pharmacodynamic levels whereby many genetic variants of respective enzymes, membrane transporters, receptors and ion channels have been

Genetic variations have been observed particularly with CYP2D6, CYP2C9 aand CYP2C19 genotypes (Ingelman – Sundberg 1999, Hiratsuka 2006) and therefore will be further

i. **CYP2D6:** With regards to this CYP subtype**,** 5 – 10% of Caucasians are poor metabolizers and have little enzyme activity and there is a distinct racial diversity in the frequency of the classes. About 50 genetic polymorphisms of CYP2D6 have been reported. The popular ones are CYP2D6\*3, CYP2D6\*4 and CYP2D6\*5. More than 90% of PMs in Caucasians are ascribable to these three genetic polymorphism (Daly et al 1996,& Suzuki et al 2011). In blacks, the common variant is CYP2D\*17 (Evans 1989) ii. **CYP2C9:** is involved in the metabolism of an epileptic agent; phenytoin and an anticoagulant; warfarin. To date, 12 CYP2C9 variants have been reported. For example in cases with phenytoin, oral clearance decreased to one quarter in subjects with homozygous polymorphism for CYP2CP\*3 (Kidd et al 1999 , Scodo et al 2002, Linder et al 2009). Many studies focused on CYP2C9 polymorphism to link variability with warfarin therapy. However only about 10% of dosage variation can be attributed to CYP2C9 polymorphism. It is thought that environmental and genetic factors can influence warfarin response therefore dosage is individualized based on sex, age, vitatmin K intake, and disease states. Warfarin dosing can be challenging because of its narrow therapeutic index and the serious risk of bleeding in overdosage. Warfarin exerts its anticoagulant effects by inhibiting hepatic vitamin K epoxide reductase; an enzyme involved in the vitamin K epoxide reductase complex sub unit 1 (VKORC1). The gene that encodes this enzyme has been identified and is believed to contribute to the variability in warfarin response (Scodo et al 2002, Aquilante et al 2006, Linder et al

iii. **The CYP2C19:** enzyme metabolizes many drugs including the proton pump inhibitor; citalopram (lelexa) diazepam (valium) and imipramine (toranil). More than 16 variants of CYP2C19 associated with deficient, reduced, normal or increased activity have been identified. The most common genotypic variants for poor metabolizers are CYP2C19\*2 and CYP2C19\*3. The CYP2C19\*17 variant is associated with ultrarapid metabolizers and seems to be common in Swedes (18%), Ethiopians (18%) and Chinese (4%). (Sum et al 2006). The proton pump inhibitor omeprazole (prilosec) is primarily metabolized by CYP2C19 to its inactive metabolite 5 – hydroxyl-omeprazole. Individuals who are CYP2C19 poor metabolizers can have five fold higher blood concentrations of omeprazole and experience superior acid suppression and higher cure rate than the rest

dectected ( Wiesler et al 2008, Phipps – Green et al 2010 & Bouamar et al 2011)

**5.1 Pharmacokinetic related genes** 

2009, Guengerich 2001).

elucidated.

**5.1.1 Genes of phase i reaction enzymes** 

#### **4.3 Phase III reactions**

Phase III reactions can be described as a stage of further modification and excretion. Although many authors do not regard this phase as a distinct phase, current knowledge of efflux transporters tend to support the categorization. A common example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid) conjugates (Boyland and Chassaud 1969). In this scenario, glutamate and glycine residing in the glutathione molecule are removed by gamma-glutamyltranspeptidase and dipeptidases. Finally, the cysteine residue in the conjugate is acetylated .The conjugates and their metabolites can then be excreted from cells in phase III of their metabolism with anionic groups acting as affinity taps for a variety of membrane transporters of the multidrug resistance protein (MRP) family (Homolya et al 2003). These proteins are members of the family of ATP-binding cassette transporters and can facilitate the ATP dependent transport of a large varieties of hydrophobic ions (Konig et al 1999) and thus act to remove phase II products to the extracellular medium, where they may be further metabolized and excreted (Commandeur et al 1995).

Since the discovery of permeability glycoprotein (P – glycoprotein) complex; an initial member of the ATP binding cassette (ABC) family of drug transporters by Juliano and Ling in 1976, (Juliano and Ling 1976) research into this group of proteins has been gaining wide interests. Some of the membrane transporters confer on the cells the ability to be resistant not only to the selective agent but also to a broad spectrum of structurally and functionally distinct antibiotics and alkaloids.This phenotypic character is referred to as multiple drug resistance (MDR). The MDR genotype/phenotype relationship is complex with over 18 ABC genes associated with human disease (Dean and Annilo 2005).

In addition to the ABC transporters, other important drug/xenobiotic transporters include the organic cation transporters of the SLC 22A super family and the organic anion transporting peptides of the SLC21 superfamily (Hagenbuch 2010). It is expected that with the growing interests in this phase of drug metabolism, investigations on the transcriptional regulatory control of this important transport system in target organs such as the liver, kidney and central nervous system will become intense in the next few decades (Omiecinski 2011).
