Biotransformation

Lipophilicity, a desirable drug characteristic for absorption and distribution across biological membranes, is a hindrance to elimination. To prevent accumulation of xenobiotics, the body chemically alters lipophilic compounds to more water soluble products. The sum of all the processes that convert lipophilic substances to more hydrophilic metabolites is termed biotransformation. These biochemical processes are usually enzymatic and are commonly divided into Phase I and Phase II reactions.9 Phase I reactions generally expose or introduce a polar group to the parent drug, thereby increasing its water solubility. These reactions are oxidative or hydrolytic in nature and include N- and O-dealkylation, aliphatic and aromatic hydroxylation, N- and S-oxidation, and deamination. These reactions usually result in loss of pharmacological activity, although there are numerous examples of enhanced activity. Indeed, formation of a Phase I product is desirable in the case of administration of prodrugs.

Phase II reactions are conjugation reactions and involve covalent bonding of functional groups with endogenous compounds. Highly water soluble conjugates are formed by combination of the drug or metabolite with glucuronic acid, sulfate, glutathione, amino acids, or acetate. Again, these products are generally pharmacologically inactive or less active than the parent compound. An exception is the metabolite, morphine-6-glucuronide. In this case, glucuronidation at the 6-position increases the affinity of morphine for binding at the mu receptor and results in equivalent or enhanced pharmacological activity.10

The enzymes that catalyze the biotransformation of drugs are found mainly in the liver. This is not surprising considering the primary function of the liver is to handle compounds absorbed from the G.I. tract. In addition, the liver receives all the blood perfusing the splanchnic area. Therefore, this organ has developed a high capacity to remove substances from blood, and store, transform, and/or release substances into the general circulation. In its primary role of biotransformation, the liver acts as a homogenous unit, with all parenchymal cells or hepatocytes exhibiting enzymatic activity. In tissues involved in extrahepatic biotransformation processes, typically only one or two cell types are used. Many organs have demonstrated activity towards foreign compounds but the major extrahepatic tissues are those involved in the absorption or excretion of chemicals. These include the kidney, lung, intestine, skin, and testes. The main cells containing biotransformation enzymes in these organs are the proximal tubular cells, clara cells, mucosa lining cells, epithelial cells, and seminiferous tubules, respectively.

3.1.2.1 Phase I Enzymes

Phase I enzymes are located primarily in the endoplasmic reticulum of cells. These enzymes are membrane bound within a lipoprotein matrix and are referred to as microsomal enzymes. This is in reference to the subcellular fraction isolated by differential centrifugation of a liver homogenate. The two most important enzyme systems involved in Phase I biotransformation reactions are the cytochrome P-450 system and the mixed function amine oxidase. With the advances in recombinant DNA technology, eight major mammalian gene families of hepatic and extrahepatic cytochrome P-450 have been identified.2 A comprehensive discussion of the cytochrome P-450 system is beyond the scope of this chapter and the reader is referred to a number of reviews.11-13 Briefly, this system is comprised of two coupled enzymes: NADPH-cytochrome P-450 reductase and a heme containing enzyme, cytochrome P-450. This complex is associated with another cytochrome, cytochrome b5 with a reductase enzyme. In reactions catalyzed by cytochrome P-450, the substrate combines with the oxidized form of cytochrome P-450 (Fe3+) to form a complex. This complex accepts an electron from NADPH which reduces the iron in the cytochrome P-450 heme moiety to Fe2+. This reduced substrate-cytochrome P450 complex then combines with molecular oxygen which in turn accepts another electron from NADPH. In some cases, the second electron is provided by NADH via cytochrome b5. Both electrons are transferred to molecular oxygen, resulting in a highly reactive and unstable species. One atom of the unstable oxygen molecule is transferred to the substrate and the other is reduced to water. The substrate then dissociates as a result, regenerating the oxidized form of cytochrome P-450.

3.1.2.2 Phase II Enzymes

Many of the Phase II enzymes are located in the cytosol or supernatant fraction after differential centrifugation of a liver homogenate. These reactions are biosynthetic and therefore require energy. This is accomplished by transforming the substrate or cofactors to high energy intermediates. One of the major Phase II reactions is glucuronidation. The resultant glucu-

ronides are eliminated in the bile or urine. The enzyme, uridine diphosphate (UDP) glucuronosyltransferase is located in the endoplasmic reticulum. This enzyme catalyzes the reaction between UDP-glucuronic acid and the functional group of the substrate. The location of this enzyme means that it has direct access to the products of Phase I enzymatic reactions. Another important conjugation reaction in humans is sulfation of hydroxyl groups. The sulfotransferases are a group of soluble enzymes, classified as aryl, hydroxysteroid, estrone, and bile salt sulfotransferases. Their primary function is the transfer of inorganic sulfate to the hydroxyl moiety of phenol or aliphatic alcohols.

Another important family of enzymes is the glutathione -S-transferases which are located in both the cytoplasm and endoplasmic reticulum of cells. The activity of the cytosolic transferase is 5 to 40 times greater than the endoplasmic enzyme. These transferase enzymes catalyze the reaction between the sulfhydryl group of the tripeptide glutathione with substances containing electrophilic carbon atoms. The glutathione conjugates are cleaved to cysteine derivatives, primarily in the kidney. These derivatives are then acetylated resulting in mercapturic acid conjugates which are excreted in the urine.

Many factors affect the rate at which a drug is biotransformed. One of the important factors is obviously the concentration of the drug at the site of action of biotransforming enzymes. Physicochemical properties of the drug, such as lipophilicity, are important, in addition to dose and route of administration. Certain physiological, pharmacological, and environmental factors may also affect the rate of biotransformation of a compound. Numerous variables affect biotransformation including sex, age, genetic polymorphisms, time of day or circadian rhythms, nutritional status, enzyme induction or inhibition, hepatic injury, and disease states.

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