Cytochrome P450 Enzymes

CYPs are a superfamily of heme-thiolate monooxygenases that were originally characterized by their absorption spectrum at 450 nm in the presence of carbon monoxide. These enzymes are located on the endoplasmic reticulum and present predominantly in the liver, but significant quantities are also found in the small intestine, kidney, and skin (15-17).

This family of enzymes oxidizes a wide variety of endogenous and exogenous substrates, including xenobiotics. The term xenobiotic refers to molecules foreign to the body and includes pollutants, pesticides, and drugs. The importance of CYPs in pharmacology arises from their participation in the oxidative (phase I) metabolism of a wide number of medications (18). The CYP monooxygenase catalytic activity can be described as RH + NADPH + H+ + O2 ^ ROH + NADP + H2O, in which RH is the drug molecule and ROH is a more hydrophilic metabolite of the drug (19). CYPs biotransform drugs to more polar metabolites to enhance renal excretion. Typically, the metabolism results in less bioactivity although some medications, such as cyclophosphamide (20), undergo biotransformation to pharmacologically active agents. In brief, CYP-mediated metabolism is an important determinant of the eventual disposition of most drugs (21-25).

There are a particularly large number of such examples for phase I metabolism of drugs by multiple CYPs. The antiepileptic hydantoin is N-demethylated by CYP2B6 (26) and C4'-hydroxylated by CYP2C18 (18) and CYP2C19 (27). The tricyclic antidepressant imipramine is C2-hydroxylated by CYP2D6 (28)

and N-demethylated by CYP1A2 and CYP2C18 (29). The analgesic codeine is O-demethylated by CYP2D6 (30) and N-demethylated by CYP3A4 (31). The ability to selectively inhibit a particular metabolic pathway can be used creatively to enhance the pharmacological disposition of a drug. For example, a strategy to inhibit the CYP3A4-mediated N-demethylation of codeine to norcodeine is likely to redirect its metabolism to CYP2D6, which O-demethylates it to the highly active metabolite morphine (32).

1.1.1. The CYP3A Family

The CYP3A family is the most abundant CYP in the human liver, representing about 40% of the total specific content of CYP. The entire CYP3A family is located at chromosome 7q21.1. The human CYP3A family includes CYP3A4 and its allelic variant CYP3A3, as well as CYP3A5 and CYP3A7 (33). CYP3A7 is considered a fetal enzyme, and its expression is not observed in the liver after birth, but expression has been observed in the endometrium and placenta (34). CYP3A5 is expressed in only 10-30% of livers but is expressed in 80% of all human kidneys.

The major isoform CYP3A4 is expressed in human liver, small intestine, and skin. This 57-kDa enzyme has also been referred to as nifedipine oxidase, NF-25, P450-PCN1, or polypeptide 4. This versatile enzyme can catalyze the following types of reactions: O-demethylation, oxidative deamination, N-hydroxyla-tion, aliphatic oxidation, and N-dealkylation (35,36).

Enormous interindividual variations have been reported in both enzyme content and activity in the liver and small intestines. The specific content of CYP3A4 in the liver has been reported to vary by a factor of 20-fold, and the enzyme activity, as determined by the erythromycin breath test, has been reported to vary by a factor of 10-fold (37). The CYP3A4 content of the small intestine has been reported to vary by a factor of 10- to 49-fold (38-40). These large differences in CYP3A4 activity, in turn, are likely responsible for interindividual variations in therapeutic efficacy and disposition of a variety of drugs (25).

A variety of factors, including genetic polymorphisms, influence CYP3A4 activity. Two well-defined genetic polymorphisms are reported: (1) 12 known alternative coding variants, each containing defined alterations in amino acid sequence identified as CYP3A4*1 (wild type) through CYP3A4*13; (2) an A-to-G transition located 295 bp upstream of the coding region, A(-292)G, representing a genetic variant within the CYP3A4 promoter (nifedipine-specific element) that has been associated with lower CYP3A4 expression (41) and higher cancer risk (42, 43). Various xenobiotics also influence CYP3A4 activity. Drugs that inhibit CYP3A4 activity include clotrimazole, ethinylestradiol, gestodene, ketaconazole, miconazole, cimetidine, and troleandomycin. On the other hand, carbamazepine, dexamethasone, phénobarbital, phenytoin, rifampin, sulfadimidine, sulfinpyrazone, troleandomycin, and paclitaxel are reported to elevate CYP3A4 activity (44). Overall, the importance of CYP3A4 is underlined by the fact that it contributes to the metabolism, and eventual disposition, of more than half of all medications (23).

1.1.2. Rat CYP3A2

Because the studies described in this chapter were conducted in rats, it is important to highlight important differences between human and rat CYP3A families. The rat CYP3A family includes 3A1 and 3A2, which are functionally equivalent to human isoforms 3A3 and 3A4, respectively. CYP3A1 is difficult to detect in a normal, untreated rat (45) whereas 3A2 constitutes approx 25% of the total specific content of CYP in male rat liver (46). CYP3A2 is constitu-tively expressed in neonates of either sex but is present only in males on maturation. Hence, the use of male rats was employed in the current studies. The lack of the CYP3A2 expression in females may be related to the continuous female secretion pattern of growth hormone, which generally suppresses the expression of CYP3A family (47-49).

AVI-4472, the antisense oligomer used in the studies presented in this chapter, targets the AUG translation initiation region of the rat CYP3A2 mRNA.

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