Atypical Kinetic Profiles Research Articles

Biotransformation of phenytoin in the electrochemically-driven CYP2C19 system

The possibility of the detection of atypical kinetic profiles of drug biotransformation using electrochemical systems based on immobilized cytochromes P450 with phenytoin hydroxylation by cytochrome P450 2C19 (CYP2C19) as an example was evaluated for the first time. For this purpose, we developed an electrochemical system, where one of the electrodes was modified by didodecyldimethylammonium bromide (DDAB) and was used as an electron donor for reduction of heme iron ion of the immobilized CYP2C19 and initiation of the catalytic reaction, while the second electrode was not modified and served for an electrochemical quantitation of 4-hydroxyphenytoin, which is a metabolite of antiepileptic drug phenytoin, by its oxidation peak. It was revealed that the dependence of the rate of 4-hydroxyphenytoin formation on phenytoin concentration is described by the equation for two enzymes or two binding sites indicating the existing of high- and low-affinity forms of the enzyme. The atypical kinetics and the kinetic parameters of CYP2C19-mediated phenytoin hydroxylation in the electrochemical system correlate to the same characteristics obtained by other authors in an alternative enzymatic system. Our results demonstrate the possibility of electrochemical systems based on cytochromes P450 to be applied for the detection of atypical kinetic profiles of drug metabolism.

Fundamentals of Enzyme Kinetics: Michaelis-Menten and Non-Michaelis-Type (Atypical) Enzyme Kinetics.

This chapter will provide a general introduction to the kinetics of enzyme-catalyzed reactions, including a general discussion of catalysts, reaction rates, and binding constants. This section will be followed by a discussion of various types of enzyme kinetics observed in drug metabolism reactions. A large number of enzymatic reactions can be adequately described by Michaelis-Menten kinetics. The Michaelis-Menten equation represents a rectangular hyperbola, with a y-asymptote at the Vmax value. However, in other cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231-242, 2004). Several cytochromes P450 (CYPs) have large active sites that enable binding of multiple molecules (Yano et al., J Biol Chem 279:38091-38094, 2004; Wester et al., J Biol Chem 279:35630-35637, 2004). Thus, atypical kinetics are not uncommon in in vitro drug metabolism studies.

Enzyme kinetics: the whole picture reveals hidden meanings.

The methodology adopted by Michaelis and Menten in 1913 is still routinely used to characterize the catalytic power and selectivity of enzymes. These kinetic measurements must be performed soon after the purified enzyme is mixed with a large excess of substrate. Other time scales and solution compositions are no less physiologically relevant, but fall outside the range of applicability of the classical formalism. Here we show that the complete picture of an enzyme's mode of function is critically obscured by the limited scope of conventional kinetic analysis, even in the simplest case of a single active site without inhibition. This picture is now unveiled in a mathematically closed form that remains valid over the reaction time for all combinations of enzyme/substrate concentrations and rate constants. Algebraic simplicity is maintained in the new formalism when stationary reaction phases are considered. By achieving this century-old objective, the otherwise hidden role of the reversible binding step is revealed and atypical kinetic profiles are explained. Most singular kinetic behaviors are identified in a critical region of conditions that coincide with typical cell conditions. Because it is not covered by the Michaelis-Menten model, the critical region has been missed until now by low- and high-throughput screenings of new drugs. New possibilities are therefore raised for novel and once-promising inhibitors to therapeutically target enzymes.

Glucuronidation of the Red Clover Isoflavone Irilone by Liver Microsomes from Different Species and Human UDP-Glucuronosyltransferases

Red clover (Trifolium pratense L.) is used as a source for isoflavone (IF) dietary supplements. In this study, we focused on the red clover IF irilone (IRI), because of its reported comparatively high bioavailability. Because the conjugative metabolism plays a key role in the elimination of IF, we investigated the species-specific differences and glucuronidation kinetics of IRI using different liver microsomes as well as the recombinant UDP-glucuronosyltransferases (UGTs) 1A1, 1A7, 1A8, 1A9, 1A10, and 2B15. Both possible monoglucuronides, the IRI-O-4'-monoglucuronide (IRI-G4') and the IRI-O-5-monoglucuronide (IRI-G5), were detected. Human liver microsomes (HLM) as well as rat liver microsomes predominantly formed IRI-G5, whereas for porcine liver microsomes, IRI-G4' prevailed. HLM showed an apparent V(max) value of 0.43 nmol/min · mg and an apparent K(m) value of 9.8 μM for the formation of IRI-G5 and a V(max) of 0.35 nmol/min · mg and a K(m) of 64.7 μM in the case of IRI-G4'. Formation of both glucuronides was best fit using the substrate inhibition equation. The glucuronidation of IRI by UGTs led to values for the intrinsic clearance varying between 4 and 100 ml/min · mg, with UGT1A7 showing the lowest and UGT1A10 the highest IRI conversion rate. The results indicate that IRI undergoes an efficient glucuronidation, presumably in the intestine and liver, following atypical kinetic profiles.

Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites

UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration-dependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.

Amiodarone Analog-Dependent Effects on CYP2C9-Mediated Metabolism and Kinetic Profiles

CYP2C9 substrates can exhibit both hyperbolic and atypical kinetic profiles, and their metabolism can be activated or inhibited depending on the effector studied. CYP2C9 genetic variants can also affect both substrate turnover and kinetic profile. The present study assessed whether analogs of the effector amiodarone differentially altered the atypical kinetic profile of the substrate naproxen and whether this effect was genotype-dependent. Amiodarone, desethylamiodarone, benzbromarone, and its dimethyl analog (benz(meth)arone) were incubated with naproxen and either CYP2C9.1 or CYP2C9.3. Amiodarone activated naproxen demethylation at lower concentrations, regardless of the CYP2C9 allele, and inhibited metabolism at higher concentrations without altering the kinetic profile. Desethylamiodarone was a potent inhibitor of naproxen demethylation, irrespective of the CYP2C9 allele. Benzbromarone altered naproxen demethylation kinetics from a biphasic profile to that of a hyperbolic form in CYP2C9.1 and CYP2C9.3, resulting in inhibition and activation, respectively. In contrast, benz(meth)arone activated naproxen demethylation in both CYP2C9.1 and CYP2C9.3. In addition, the kinetic profile of naproxen demethylation became more hyperbolic at lower concentrations of benz(meth)arone and then reverted back to biphasic as the benz(meth)arone was increased further. Equilibrium binding and multiple-ligand docking studies were used to propose how such similar compounds exerted very different effects on naproxen metabolism. In summary, effectors of CYP2C9 metabolism can alter not only the degree of substrate turnover (activation or inhibition) but also the kinetic profile of metabolism of CYP2C9 substrates through effects on substrate binding and orientation. In addition, these kinetics effects are concentration- and genotype-dependent.

Atypical Cytochrome P450 Kinetics

The Michaelis-Menten model is commonly used to estimate a drug's potential in vivo hepatic clearance based on in vitro data obtained during drug discovery and development. This paradigm assumes that the drug obeys 'typical' enzyme kinetics and thus can be described by this model. However, it is increasingly being recognised that a number of drugs metabolised not only by the cytochrome P450 enzymes but also by other enzymes and transporters can exhibit atypical kinetic profiles, and thus are not accurately modeled with the Michaelis-Menten model. Application of an incorrect model can then lead to mis-estimation of in vitro intrinsic clearance and thus affect the prediction of in vivo clearance. This review discusses several types of atypical kinetic profiles that may be observed, including examples of homotropic cooperativity (i.e. sigmoidal kinetics, biphasic kinetics and substrate inhibition kinetics) as well as heterotropic cooperativity (i.e. activation). Application of the incorrect kinetic model may profoundly affect estimations of intrinsic clearance. For example, incorrectly applying the Michaelis-Menten model to a kinetic profile exhibiting substrate inhibition kinetics will result in an underestimation of Km (Michaelis-Menten constant) and V(max) (maximal velocity), whereas application of the Michaelis-Menten model to sigmoidal kinetic data typically results in an overestimation of Km and V(max) at the lower substrate concentrations that are typically therapeutically relevant. One must also be careful of potential artefactual causes of atypical kinetic profiles, such as enzyme activation by solvents, buffer dependent kinetic profiles, or altered kinetic parameter estimates due to nonspecific binding of the substrate to proteins. Despite a plethora of data on the effects of atypical kinetic profiles in vitro, only modest effects have been noted in vivo (with the exception of substrate dependent inhibition). Thus, the clinical relevance of these phenomena remains inconclusive.

Modeling Kinetic Data from In Vitro Drug Metabolism Enzyme Experiments

Modeling of in vitro enzyme kinetic data derived from drug metabolism experiments can greatly facilitate the drug development process because estimation of kinetic parameters can facilitate decision making regarding whether to continue development of a compound. From this information, predictions can be made regarding the “metabolic stability” of a compound and even the in vivo intrinsic clearance of the drug. Many drugs exhibit typical Michaelis–Menten‐type kinetics in vitro that result in a hyperbolic kinetic profile from which Km and Vm can be readily estimated. However, it is increasingly being recognized that many drug compounds exhibit “atypical” enzyme kinetics in vitro, requiring use of more complex kinetic models for data fitting and parameter estimation. These atypical kinetic profiles may include sigmoidal kinetics (autoactivation), biphasic kinetics, substrate inhibition kinetics, and heterotropic cooperativity (activation). This article briefly summarizes the types of equations necessary to adequately model both typical and atypical kinetic profiles in order to facilitate correct estimation of the relevant kinetic parameters.

Atypical enzyme kinetics: their effect on in vitro-in vivo pharmacokinetic predictions and drug interactions.

The most common method for estimating a drug's in vivo clearance from in vitro data has involved using the classical Michaelis-Menten model to describe the observed in vitro data and from this estimate intrinsic clearance. This process assumes that the drug obeys standard enzyme kinetics that can be described by this model. However, the observation of atypical enzyme kinetics, particularly involving cytochrome P450 enzymes, has become relatively common, and occurrences have been reported with virtually all of the cytochrome P450 isoforms. Since predictions of a drug's in vivo clearance are made based on kinetic parameters observed during in vitro experiments (in vitro estimated intrinsic clearance), mis-identification of the drug's kinetic profile can lead to inaccurate predictions of intrinsic clearance. In addition to these potential ramifications of in vitro-in vivo predictions, information is becoming available concerning the in vivo implications of drugs that exhibit atypical kinetic profiles. This review will discuss the various types of atypical kinetic profiles that may be observed during in vitro experiments, hypotheses for why they may occur, examples of how a drug's intrinsic clearance may be mis-estimated when atypical kinetics are incorrectly assessed, and the potential ramifications of these issues for prediction of drug interactions. Furthermore, potential, artifactual causes of atypical kinetic phenomena will be described.

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.

Atypical kinetic profiles in drug metabolism reactions.

The cytochrome P450 enzymes are involved in the biotransformation of both xenobiotic and endobiotic hydrophobic compounds, implicated in the bioactivation of certain procarcinogens (e.g., benzo[ a ]pyrene), and responsible for many metabolism-based drug-drug interactions ([Wrighton and Stevens, 1992