Untitled Document

Drug Metabolism by Cytochrome P450s

Figure 2: Cytochrome P450 3A4 at 2.3 angstrom resolution with the heme group in dark gray

Image acquired from the Protein Data Bank file 1CPT.pdb

Cytochrome P450s are a group of enzymes present throughout the body, with the highest concentrations and/or diverse isozymes found in the liver and the intestinal walls. The P450 monooxygenases catalyze metabolism of numerous compounds-everything from biotransformation of natural endogenous compounds like steroids and cholesterol, to detoxification of exogenous compounds such as drugs and pollutants (Dahan et al, 2003). This family of enzymes is responsible for triggering chemical reactions required to metabolize many different drugs. P450s play a key role in determining both the intensity and duration of a drug's action.

All P450 enzymes are heme-containing proteins. The heme iron is usually in the ferric state (FeIII) but is reduced to the ferrous state (FeII) upon activity. The basic reaction catalyzed by cytochrome P450s is:

One atom of oxygen is often incorporated as a substrate hydroxyl group (RH) and the other, oxygen, is reduced to water with reducing equivalents derived from NADPH. During catalysis, it is believed that cytochrome P450s bind directly to substrates and molecular oxygen, and does not interact directly with either NADPH or NADH.

Figure 3: The Catalytic Cycle of P450 enzymes (Schultz-Utermoehl et al, 2000)

Figure 4: A detailed diagram of the reactions specifically carried out by the P450 isozyme CYP3A4 (Göller et al, 1997)

Figure 4 shows a more comprehensive diagram illustrating the enzymatic processes of CYP3A4. A shows the enzyme in its native conformation before binding substrate. B shows the complex formation of CYP3A4 with the alkane. C shows the reduction of the iron from a +3 state to a +2 state. D1 and D2 shows the addition of a diatomic oxygen molecule to the complex. E shows a further reduction reaction changing the overall charge on the complex from -1 to -2. Steps F and G are two protonation reactions. Finally, before steps H1 and H2, water is eliminated from the complex. The final oxidation step that recovers the original enzyme is shown in steps H1 and H2. The processed substrate is released as ROH. With the addition of water to the enzyme, it returns to its initial form A.

Of all the cytochrome P450s, CYP3A4 is the most abundant member in the body and is responsible for metabolizing approximately 60% of all drugs taken (Dahan et al, 2003).

Grapefruit-more specifically compounds in grapefruit that have so far been identified-inhibits the activity of CYP3A4. This means that when grapefruit or grapefruit juice is consumed, a compound within the grapefruit disrupts the intestinal CYP3A4 enzyme's ability to metabolize a drug. There are two possible scenarios for a specific drug. First, if it is the unmetabolized form of the drug that is active, then higher levels of the drug may enter the bloodstream since CYP3A4 is no longer able to metabolize the active drug into its inactive metabolite. Second, if it is the metabolized form of the drug that is active, then lower amounts of the drug will enter the body since CYP3A4 will be unable to react with the inactive drug and convert it to its active metabolite. The first situation is equivalent to a drug overdose, while the second situation is equivalent to an under dosing of the drug. Either of these situations can be potentially dangerous.

Because CYP3A4 has such an important role in drug metabolism, many researchers have conducted studies to determine how the structure of the enzyme is related to its function.

Figure 5: Conformational changes observed in CYP3A4 with and without substrate bound (Wester et al, 2003)

Mutagenesis studies have indicated that the labeled G and H helices seen in Figure 5 section a may play a role in substrate entry. It has also been proposed that the movement of the G and F helix (as seen in the superimposed images of substrate bound versus no substrate bound in Figure 5) causes the conformational changes in the enzyme allowing it to interact with its substrate.

Normally, CYP3A4 catalyzes the oxidation of drugs in the small intestine. However, it has been found that four hours after the consumption of grapefruit juice, there is significant reduction of drug presystemic metabolism. A corresponding decrease of 47% in enterocyte (intestinal cell) CYP3A4 protein levels has been observed through immunoblot analysis of duodenal cells before and after drinking grapefruit juice.

Figure 6: Immunoblot assay in enterocytes for levels of CYP3A4 before and after consumption of grapefruit juice (Dahan et al, 2003)

Pre grapefruit consumption levels show a strong band for both CYP3A4 concentrations in the intestine. However, after consumption of grapefruit juice, post CYP3A4 levels show a marked decline.

Other studies have indicated that mRNA levels of CYP3A4 remain unchanged after grapefruit juice consumption.

Figure 7: mRNA electroporesis gel versus Western blot of CYP3A4 levels in patients before and after consuming grapefruit juice (Lown et al, 1997)

Figure 7 shows the results of an experiment in which several patients were analyzed for levels of CYP3A4 mRNA as well as protein before and after consumption of grapefruit juice. The amount of CYP3A4 mRNA remains constant in each patient. However, the Western blot shows that CYP3A4 protein in patients after consumption of grapefruit juice does change a great deal. In fact, the amount of actual CYP 3A4 protein in the patients' intestinal cells goes down drastically.

Because mRNA levels remain constant, the mechanism of CYP3A4 inhibition by grapefruit juice is probably post-translational. Thus, grapefruit juice components most likely responsible for the effect cause degradation of the enzyme in some way.

Because new protein must be synthesized by the cell to replace degraded CYP3A4, it can take up to 72 hours for CYP3A4 protein levels to recover in intestinal cells. The length of time is dependent on the rate of new CYP3A4 enzyme synthesis as determined by transcription and translation in the enterocyte.