**11.5. Phase II reactions**

Consist of synthetic/conjugation reactions in which an endogenous substance such as glucur‐ onic acid or glutathione combines with the functional group derived from phase I reactions to produce a highly polar drug conjugate. All tissues have some ability to carry out drug biotransformation reactions but the most important organs of biotransformation include; the liver, GIT, lungs, skin, and kidneys in that order and most phase II reactions result in a decrease in the pharmacological activity of the drug. The fact that the GIT and liver are the major sites of drug biotransformation means that drugs which are administered orally will be extensively bio-transformed before they eventually reach systemic circulation. This first-pass effect can severely limit the oral bio- availability of some drugs. In addition, intestinal micro-organisms are capable of catalyzing drug biotransformation reactions e.g. a glucuronide conjugate of a drug may be excreted through the intestine via the bile where gut bacteria may convert the conjugate back into free drug. The free drug is then reabsorbed and re-enters the liver via the portal vein where the conjugation process is repeated. This leads to a phenomenon known as entero-hepaticcirculation.

At sub-cellular level, enzymes of drug biotransformation are located in the endoplasmic reticulum, mitochondria, cytosol and lysosome. The major site of drug biotransformation within the hepatocytes and other cells is the membrane of the smooth endoplasmic reticulum. The smooth endoplasmic reticulum constitutes the microsome fraction during differential centrifugation of whole blood. The microsome fraction can be used to carry out many drug biotransformation reactions *in vitro.*

### **11.6. Mechanisms of phase I reactions**

#### *11.6.1. Oxidation*

Is the most important category of the microsomal drug oxidizing systems and requires partici‐ pation of two distinct proteins in endoplasmic reticulum; cytochromes P450 (which functions as a terminal oxidase) and cytochrome P450 reductase. The name Cyt450 is derived from the fact that the reduced form of this hemoprotein complexes with carbon monoxide to form a complex that has a unique absorption spectrum with a maximum at 450nm.Cytochrome P450 reductase serves to transfer reducing equivalent from NADPH to the cytochrome P450 oxidase:

The phospholipids of the endoplasmic reticulum are required for substrate binding, electron transfer, and facilitating the interaction between CytP450 and its reductase. However, cyto‐ chrome P450 does not catalyze all Oxidation reactions. The microsomal flavin– containing monooxygenases (FMOs) catalyze NADPH – dependent oxygenation of nucleophilic phos‐ phorous, nitrogen and sulfur atoms. These atoms are present in a wide variety of xenobiotics including the carbamate containing pesticides and therapeutic agents such as phenothiazines, ephedrine and N-methylamphetamine. Another important drug–oxidizing system is the

> prostaglandin synthetase

Many xenobiotics including phenytoin can be co-oxidized along with the above reduc‐ tion reaction. This pathway is of considerable toxicological importance as it often leads to generation of toxic reactive metabolites. Other enzymes that catalyze oxidation of xenobiotic include alcohol dehydrogenase, aldehyde dehydrogenase, xanthine oxidase

Some drugs with azo-linkages (RN=NR, e.g. prontosil) and nitrogen groups (RNO2, such as chloramphenicol) are transformed by reductive pathways. The Cyt P450 and NADPH–cyt P450reductase enzymes that catalyze oxidation reactions are also involved in reduction reactions for drugs containing quinine moieties. These transformation results in the formation of semiquinone free radicals illustrated in Figure 8. The free radicals that are generated cause oxidative stress, lipid peroxidation, DNA damage, and hence cytotoxicity. These effects are

Drugs containing ester functions (R1COOR2) such as procaine are hydrolyzed by a variety of non-specific esterases in liver, and plasma while drugs with amide bonds are hydrolyzed by amidases in the liver. The polypeptide drugs such as insulin and growth hormones are hydrolyzed by peptidases in the plasma and erythrocytes. The metabolites resulting from hydrolysis reactions are subjected to phase II biotransformation reactions before excretion in

The phase II reactions generally involve coupling of drug/drug metabolite with an endogenous substance to enhance their removal from the body. They require participation of specific

transferase enzymes and high energy activated endogenous substances.

particularly responsible for the antitumor property of a drug like doxorubicin.

prostaglandin H2

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prostaglandin synthetase–dependent co-oxidation.

and monoamine oxidase.

*11.6.2. Reduction*

*11.6.3. Hydrolysis*

the bile or urine.

**11.7. Mechanisms of phase II reactions**

Prostaglandin G2 reduced

DrugRH + O2+ NADPH + H<sup>+</sup><sup>→</sup> DrugROH (hydroxylated product) + H2O+ NADP<sup>+</sup>

The sequence of reactions that transform a drug to its hydroxylated product is shown below (Figure 7).

**Figure 7.** Phase 1 drug biotransformation reactions in the liver microsomal fraction in which the drug is converted to a more polar form.

The phospholipids of the endoplasmic reticulum are required for substrate binding, electron transfer, and facilitating the interaction between CytP450 and its reductase. However, cyto‐ chrome P450 does not catalyze all Oxidation reactions. The microsomal flavin– containing monooxygenases (FMOs) catalyze NADPH – dependent oxygenation of nucleophilic phos‐ phorous, nitrogen and sulfur atoms. These atoms are present in a wide variety of xenobiotics including the carbamate containing pesticides and therapeutic agents such as phenothiazines, ephedrine and N-methylamphetamine. Another important drug–oxidizing system is the prostaglandin synthetase–dependent co-oxidation.

#### Prostaglandin G2 reduced prostaglandin synthetase prostaglandin H2

Many xenobiotics including phenytoin can be co-oxidized along with the above reduc‐ tion reaction. This pathway is of considerable toxicological importance as it often leads to generation of toxic reactive metabolites. Other enzymes that catalyze oxidation of xenobiotic include alcohol dehydrogenase, aldehyde dehydrogenase, xanthine oxidase and monoamine oxidase.
