Oxidation of Caffeine by CYP1A2: Isotope Effects and Metabolic Switching

Abstract

ABSTRACT:Caffeine (1,3,7-trimethylxanthine) has previously been shown to undergo metabolic switching in vivo when the N-1 or the N-7 methyl groups were trideuteromethylated [Horning et al. (1976) Proceedings of the Second International Conference on Stable Isotopes, pp 41-54]. We have examined the effect of replacing the N-3 methyl group with a trideuteromethyl group. The corresponding isotope effects can then be used to distinguish the kinetic mechanism by which four primary metabolites can be formed from one substrate by one cytochrome P450 (P450). We have synthesized 3-CD 3 -caffeine and 3-CD 3 -7-CD 3 -caffeine as well as trideuteromethylated analogs of each of the in vitro metabolites formed by cytochrome P4501A2. The observed competitive isotope effects for the metabolites, which do not result from deuterium abstraction (theobromine, theophylline), demonstrate that the nondissociative mechanism applies to caffeine metabolism by cytochrome P4501A2. Thus, there must be equilibration of the kinetically distinguishable activated P450-substrate complexes at rates competitive with hydrogen abstraction. The true isotope effects for the N-3 demethylation of caffeine were derived from the ratios of the amount of paraxanthine relative to the amount of theobromine or theophylline. The resultant ratios indicate that these isotope effects are essentially intrinsic. Observation of the isotope effects on N-3 demethylation was facilitated by branching to the minor in vitro metabolites as well as water formation. Product release is not rate-limiting for this system. Cytochrome P4501A2 (CYP1A2) is estimated to be responsible for 90% of the primary metabolism of caffeine in humans (Tassaneeyakul et al., 1994) and is believed to be largely responsible for formation of the major metabolite, paraxanthine (PX), in vivo. The four in vitro metabolites formed by human CYP1A2 are PX (80%), theobromine (TB; 11%), theophylline (TP; 4%) and 1,3,7-trimethyluric acid (TMU; 1%) ( Fig. 1; Gu et al., 1992). Presumably, multiple binding orientations exist within the active site of CYP1A2 since the entire periphery of caffeine is available for metabolism.Experimental evidence suggests that when metabolically susceptible hydrogen(s) are replaced with deuterium atoms, there can be a decrease in the oxidation rate at the labeled site with no concomitant change in the overall extent of metabolism (Harada et al., 1984;Atkins and Sligar, 1986;Jones et al., 1986). Thus, the net effect of deuteration is an increased rate of formation of one or all of the alternate metabolites, a phenomenon known as "isotopically sensitive branching" or "metabolic switching." On the basis of studies that exhibited classical metabolic switching (Harada et al., 1984), it was hypothesized that switching occurs at the level of the activated P450 or "EOS complex," and not earlier in the catalytic cycle. Theoretical studies have supported this concept (Korzekwa et al., 1989;Nelson and Trager, 2003). Hence, deuterium-induced metabolic switching indicates that isomeric EOS co...

Determination of temporal changes in hepatic drug‐oxidizing capacity using plasma metabolite/caffeine ratios in non‐pregnant and pregnant goats

“…In previous studies on sheep and hair goats, it was determined that CF was metabolized to TB, PX and TP (Uney et al, 2011; Uney & Traş, 2011). Also, previous studies have shown that the primary metabolite of CF is TP in sheep (Danielson & Golsteyn, 1996; Uney & Traş, 2011), PX and TP in hairy goats (Uney et al, 2011), and PX in humans (Berthou et al, 1992) and cattle (Danielson & Golsteyn, 1996). Similarly, it is thought that the primary metabolite of CF could be TP that TB and PX could be produced at approximately the same rate at 10 h after administration in Saanen goats.…”

“…It is the most preferred probe drug in determining the hepatic drug‐oxidizing (metabolic) capacity because it has many features and criteria that an ideal probe drug should carry (Kalow & Tang, 1993). Studies on the determination of the hepatic drug‐oxidizing capacity using CF have been reported in sheep (Uney & Traş, 2011), goat (Uney et al, 2011; Uney & Traş, 2011) and cattle (Danielson & Golsteyn, 1996). However, no information has been found on temporal changes in pregnant goats.…”

Determination of 7α-OH cholesterol by LC–MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs

“…Another source of interference signal might come from to 7β-OH cholesterol, an auto-oxidation products of the cholesterol itself [13, 18, 32]. Although many articles reported that the formation of 7β-OH cholesterol during incubation in an undetectable level [9, 20], one experiment was nevertheless performed to eliminate the potential interference. Cholesterol was incubated in two inactive systems for 1 hour, the boiled microsome with NADPH regenerating system and the normal microsome without NADPH regenerating system.…”

“…All microsomes were prepared by standard differential ultracentrifugation [19] with small modification [20]. All the procedures below were done above ice bath in the cold room.…”

Deuterium in drug discovery: progress, opportunities and challenges

“…The changes in metabolic pathways are not always predictable and therefore are measured by in vitro and/or in vivo metabolism studies. For example, caffeine deuteration at one methyl group at a time attenuates the oxidation at the deuterated position, whereas it induces significant metabolic switching at the non-deuterated soft spots 34 , 35 . Only per-deuteration of all the three methyl groups ( d 9 -caffeine) (Table 1 ) can effectively reduce N-dealkylation reactions and improve the PK profile 36 , 37 , as judged by a fourfold higher exposure to d 9 -caffeine and a fivefold to tenfold reduced exposure to downstream metabolites relative to unlabelled caffeine in a phase I trial 38 .…”