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Tab. 7.2 Reactions performed by the cytochrome P450 system.
Reaction Product Typical example
Aromatic hydroxylation Aliphatic hydroxylation N-dealkylation O-dealkylation S-dealkylation N-oxidation S-oxidation Alcohol oxidation Phenyl to phenol Methyl to carbinol Tertiary to secondary amine Ether to alcohol Thioether to thiol Pyridine to pyridine N-oxide Sulphoxide to sulphone Alcohol to carboxylic acid Phenytoin Ibuprofen Lidocaine Naproxen 6-methylthiopurine Voriconazole Omeprazole Losartan
7.2 Cytochrome P450 77
Fig. 7.1 Heteroatom oxidation of drugs by cytochrome P450 leading to heteroatom oxides or dealkylation products.
substrate to a carbon and oxidation of this function to form the unstable carbinol and ultimately heteroatom dealkylation. A possible reaction sequence is illustrated as Figure 7.1.
Many of the investigations into the enzymology of cytochrome P450 over the previous 20 years have focused on the pathway that generates this reactive species as illustrated in Figure 7.2 particularly the donation of electrons and protons to yield the (FeO)3+ substrate complex which is the oxidizing species. As part of the cycle substrate binds to the enzyme as an initial step before the addition of electrons and molecular oxygen. The final stage of the cycle is the actual attack of the (FeO)3+ species on the substrate.
Fig. 7.2 Cytochrome P450 cycle showing the key stages of substrate interaction.
The critical points of the cycle involving substrate-enzyme interactions are illustrated in Figure 7.2 and explored below:
a) The initial binding of the substrate to the CYP which causes a change in the spin state of the haem iron eventually resulting in the formation of the (FeO)3+-sub-strate complex. This is obviously a key substrate-protein interaction and depends on the actual 3D-structure of the substrate and the topography of the active site.
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However, it cannot be assumed that this initial binding is the same as the final conformation that the protein and substrate adopt during actual substrate attack.
b)The final stages of the cycle, when the geometry and chemical reactivity of this complex determine the structure of the metabolite produced.
Analysis of the literature indicates that three major forms of CYPs are involved in the metabolism of pharmaceuticals in man: CYP2D6, CYP2C9 and CYP3A4, CYP1A2, CYP2C19 and CYP2E1 are also involved, but this involvement is much less extensive. The catalytic selectivity of the major CYPs has been reviewed .
Catalytic Selectivity of CYP2D6
Substrates for CYP2D6 include tricyclic antidepressants, p-blockers, class 1 anti-arrhythmics. In brief, the structural similarities of many of the substrates and inhibitors in terms of position of hydroxylation, overall structure (aryl-alkylamine) and physicochemistry (ionized nitrogen at physiological pH), have allowed template models such as that illustrated as Figure 7.3 to be constructed.
Fig. 7.3 Template model for CYP2D6 with Y the site of oxidation, a is the distance from Y to a heteroatom which is positively charged (normally 5-7 A).
All the template models produced have the same common features of a basic nitrogen atom at a distance of 5-7 A from the site of metabolism which is in general on or near a planar aromatic system. It is currently believed that aspartic acid residue 301 provides the carboxylate residues which binds the basic nitrogen of the substrates.
With CYP2D6 therefore the catalytic selectivity relies heavily on a substrate-pro-tein interaction. The relative strength of the proposed ion pair association between the basic nitrogen and the active site of aspartic acid means that the affinity for substrates will be high. This is borne out by the enzyme having lower Km and Ki values than other CYPs. Thus CYP2D6 is often a major enzyme in drug oxidation despite its low abundance in human liver. This statement is particularly true for low concentrations or doses of drugs, the low Km values rendering the enzyme easily saturable
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(see Section 2.12). An example of this is the antiarrythmic compound propafenone (Figure 7.4)  which is converted to 5-hydroxy propafenone by CYP2D6.
The dependence on CYP2D6 metabolism and the relatively high clinical dose (see Figure 7.5) mean that the metabolism is readily saturable over a narrow clinical dose range, so that small increases in dose can lead to disproportionate increases in plasma concentration, and a resultant steep dose-response curve.
Fig. 7.5 Relationship between plasma concentration and dose of propafenone, a CYP2D6 substrate.
CYP2D6 is also problematic in drug therapy since the enzyme is absent in about
7 % of Caucasians due to genetic polymorphism. In these 7 % (poor metabolizers) clearance of CYP2D6 substrates such as propafenone (Figure 7.4)  are markedly lower and can lead to side-effects in these subsets of the population. This correlation of enhanced side-effects in poor metabolizers (lacking CYP2D6) compared to extensive metabolizers (active CYP2D6) has been made for propafenone. The example of betaxolol  shows how knowledge of the properties that bestow pharmacological activity can be combined with metabolism concepts to produce a molecule with improved performance. Cardioselectivity for р-adrenoceptor agents can be conferred by substitution in the para position of the phenoxy-propanolamine skeleton. The para position or methoxyethyl substituents (e.g. metoprolol) in this position are the major sites of metabolism for these compounds. This reaction is catalysed by CYP2D6, and the efficiency of the enzyme means that metoprolol shows high clearance and resultant low bioavailability and short half-life. Manoury et al.  designed the series of compounds leading to betaxolol on the hypothesis that bulky stable substituents in the para position (Figure 7.6) would be resistant to metabolism and also cardioselec-tive.