Phase I Enzyme Systems

6.2.1. Esterase/Amidase Enzymes General Esterase activity can be found in many mammalian tissues and in blood9-11 and can be the result of catalysis by a number of distinct enzyme families, including carboxylesterases, paraoxonase, and cholinesterases. The esterase enzymes generally have wide substrate specificities and are capable of the hydrolysis of a wide range of hydrolytic biotransformations, although certain members such as cholinesterase have highly specialized functions. Substrates for biotransformations mediated by esterase enzymes include a wide variety of ester- or amide-containing compounds and also include carbamates and thioesters. Several recent papers have covered substrate preferences for different esterases and have demonstrated some structure-metabolism relationships.12,13 The apparent differences in substrate preference found between rodents and humans should be an important consideration when designing ester-based prodrugs. The most important family of esterase enzymes is the carboxylesterase family (EC, and members of this family are responsible for the hydrolysis of a variety of drugs.12 These enzymes are localized to the endoplasmic reticulum and are strongly inhibited by sulfonyl or phosphonyl fluorides. The enzymes employ a Ser-His-Glu triad as their catalytic domain. Improved nomenclature has recently been suggested.12 The major forms of the enzymes are designated hCE-1, hCE-2, and hCE-3.11,14 These enzymes have important differences in substrate recognition that can lead to dramatic interspecies differences in enzyme activity. The carboxylesterase enzymes are responsible for the hydrolysis of the majority of prodrug esters. An important substrate for hCE-1/hCE-2 is CPT-11,15,16 which is a carbamate derivative of SN-38, a molecule much more active against the target topoisomerase enzyme. The hydrolysis to SN-38 is thought to be crucial for maximum efficacy, and one hypothesis for a poor patient response to this therapy is low conversion due to polymorphic variants of carboxylesterase enzymes.17,18

Paraoxonase enzymes clearly belong to a distinct enzyme family.11,19 They are not serine or cysteine proteases but instead use divalent metal ion for catalysis. The enzymes have limited substrate specificity but do hydrolyze a variety of aromatic and aliphatic lactones, including several statin lactones.20 Tissue Distribution Although the highest esterase activity is normally found in liver, the intestine is also high in this activity. The carboxylase most abundant in human intestinal tissue is designated hCE-2.11 This enzyme is found in human liver, but hCE-1 is the most abundant liver carboxylesterse enzyme.11 In general, human blood/plasma has lower overall esterase activity relative to rodent plasma.10

6.2.2. Cytochrome P450 Enzymes General The cytochrome P450 (CYP) enzymes are a large family of related enzymes expressed predominantly in the liver, but found in many tissues, that play a role in the metabolic clearance of well over 50% of drugs. Various aspects of CYP enzymology, substrate specificity, and clinical importance have been the subject of recent reviews.21-24 The enzymes have broad and somewhat overlapping substrate specificity. Although over 50 human CYP enzymes have been characterized, only 5 seem to be are responsible for the majority of drug metabolism: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Of these, by far the most important enzyme is CYP3A4. This enzyme is found in the greatest quantity (>30% of total liver CYP enzyme)25 and has the broadest range of known substrates.24,26,27

The mechanism of action of the enzymes is a complex multistep process that leads to the biotransformation of substrate, most often to an oxidized product.28-30 The process of oxidation involves high-energy intermediates and often involves the generation of reactive electrophilic intermediates at the enzyme active site that are sometimes released and can react with cellular components.31-35 This process is thought to contribute to the acute or idiosyncratic toxicity displayed by some compounds.32-34

The variability in expression and activity of the CYP enzymes is a major concern in modern clinical pharmacology. This variability is due to a combination of genetic variation, environmental factors, and inhibition or induction by drugs or xenobiotics. Drugs that are cleared via metabolism by a single CYP enzyme often have significant variability in exposure that can have significant consequences for the efficacy and toxicology of the compound. This is especially problematic for drugs that are cleared primarily by CYP3A4 and CYP2D6. CYP3A4 expression is controlled by genetic factors,36 and the enzyme is inhibited and/or induced by an extensive list of pharmaceutical agents, so new drugs that rely on this enzyme for a large percentage of their clearance will likely be the subject of multiple drug-drug interactions.24 CYP2D6 is polymorphically expressed in the human population, and between 1% and approximately 10% of the population are functionally deficient in this enzyme activity and can be defined as poor metabolizers.37 There are also significant populations which overexpress this enzyme.37 Drugs that rely on CYP2D6 for a large portion of their metabolism will likely have widely variable pharmocokinetics due to these factors. Other CYP enzymes either have polymorphic distribution or are subject to induction or inhibition, but the magnitude of the interactions is not as important as those seen with CYP2D6 and CYP3A4. CYP Enzymes in the Intestine Characterization of the CYP content of the human intestine has been challenging, and the exact complement of enzymes present remains unclear.38-40 It is clear that the major drug-metabolizing enzymes present are CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The major enzyme present in intestinal tissue is CYP3A4, and the activity of intestinal microsomes for a variety of substrates has been shown to be in good agreement with activity in liver. The importance of intestinal CYP oxidation to the first-pass metabolism of drugs has been clearly shown with experiments performed during the anhepatic phase of liver transplantation. These experiments have shown that intestinal oxidation is responsible for significant first-pass elimination of cyclosporin and midazolam.41,42 The content of CYP3A4 in the intestine has been shown to be quite variable among subjects and contributes to interpatient variability found for CYP3A4 substrates.42

One aspect of intestinal metabolism that must be considered is the relatively small amount of enzyme present relative to liver and the potential for higher drug concentrations during enterocyte transit. This makes the total enzymatic capacity of the intestine lower and more susceptible to saturation than the liver, especially for high-dose drugs.1 Also, the potential for concentration difference may lead to alterations in the enzyme responsible for metabolism in the two organs, as hypothesized for the intestinal and liver metabolism of NE-100.43 There is growing evidence, however, that the P-gp system works in concert with CYP3A4 to effectively limit substrate concentration to the enzyme and minimize the poten-

tial for saturation.

One area that has received a considerable amount of recent attention is the inhibition of intestinal CYP enzymes by components of grapefruit juice.48 This effect has been shown to produce substantial increases in the plasma concentrations of a wide variety of CYP3A substrates. CYP Enzymes in the Liver The liver contains the highest quantity of CYP enzymes of any organ and has an impressive capacity to metabolize drugs and xenobiotics. The CYP enzymes of the liver constitute the most important barrier to the entry of drug-like molecules into the systemic circulation. All major CYP2D6 enzymes are present in liver in quantities that are relatively high compared to other organs. The abundance of the major CYP enzymes in the average human liver has been reported to be CYP1A2 (13%), CYP2A6 (4.0%), CYP2B6 (0.2%), CYP2C9/CYP2C19 (18%), CYP2D6 (1.5%), CYP2E1 (6.6%), and CYP3A4/5 (29%).25

6.2.3. Flavin-Containing Monoxygenase Enzymes

The flavin-containing monoxygenase enzymes (FMOs) are expressed in the endo-plasmic reticulum and are capable of carrying out a variety of oxidative biotransformations. Various aspects of this enzyme family have recently been reviewed.49,50 Typical reactions are the oxidation of nitrogen or sulfur heteroatoms. The major FMO enzyme expressed in humans is FMO3 and is quite abundant in adult liver.51 The enzyme has been shown to metabolize a large number of xenobiotics and drugs in vitro and in vivo, including drugs such as cimitidine, tamoxifen, itopride, and sulindac. There seems to be a high level of interindividual variability in the expression of FMO3, but the exact understanding of the magnitude of the variability is complicated because the enzyme can be degraded during isolation. The variability is thought to be due largely to genetic polymorphisms in the FMO3 gene.51,52 A rare genetic defect in the FMO3 gene has been shown to be the cause of trimethylaminuria.53,54 FMO1 is expressed in intestine and kidney, but the significance of this enzyme for drug metabolism has not been well established.51 The FMO enzymes are not thought to be inducible by small molecules, and no examples of drug-drug interactions due to inhibition of the enzyme have been described.51

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