Current Drug Metabolism - Volume 8, Issue 5, 2007

The topic of mechanism-based inactivation (MBI) of cytochrome P450 enzymes has been recently emerging as one of ever increasing importance. While our knowledge of some of the biochemistry and chemistry of the phenomenon of MBI of P450 enzymes has been with us for over three decades, the ramifications of MBI in drug discovery and clinical pharmacokinetics have been appreciated only more recently. While drug label warnings and contraindications due to P450 based drug-drug interactions (DDI) are numerous, the only drug known to be removed from the market due to an unfavorable drug-drug interaction profile (as a perpetrator of DDIs) was in fact a mechanism-based inactivator (mibefradil). Work done by Hall and co-workers over the past several years (Mayhew, et al., 2000; Wang, et al., 2004), as well as that of other eminent investigators has elevated the science such that we can now relate in vitro MBI to in vivo DDI. The importance of MBI in the development of new drugs has not gone unnoticed by government regulatory authorities. The FDA has included an assessment of MBI as an essential component to drug development in its recently released draft guidance document (FDA, 2006). Because of the importance of this topic, Current Drug Metabolism is publishing two back-to-back comprehensive reviews on MBI that were written by colleagues of mine at the research laboratories of Pfizer, Inc. In the first, the types of structures that can cause MBI of cytochrome P450 enzymes are described, as a useful reference for those involved in the chemical design of new drugs. The implications of MBI regarding DDI and pharmacokinetics are alluded to as a form of an introduction to the topic. In the second, the details are laid out regarding the mathematics behind how in vitro MBI data can be used in the development of drugs, for the prediction of DDI, and the design of clinical study strategies. We hope that the readers of Current Drug Metabolism find these two articles to be of value to their research efforts aimed toward the discovery and development of safe and effective new agents that will lack the property of causing DDI via mechanism-based inactivation of P450 enzymes. FDA (2006) Guidance for Industry. Drug Interaction Studies - Study Design, Data Analysis, and Implications for Dosing and Labeling http://www.fda.gov/cber/gdlns/interactstud.htm (accessed, March 27, 2007). Mayhew BS, Jones DR, Hall SD. (2000) An in vitro model for predicting in vivo inhibition of cytochrome P450 3A4 by metabolic intermediate complex formation. Drug Metab Dispos 28: 1031-1037. Wang YH, Jones DR, Hall SD. (2004) Prediction of cytochrome P450 3A inhibition by verapamil enantiomers and their metabolites. Drug Metab Dispos 32: 259-266.

Cytochrome P450 constitute a superfamily of heme-containing enzymes that catalyze the oxidative biotransformation of structurally diverse xenobiotics including drugs. Inhibition of P450 enzymes is by far the most common mechanism which can lead to DDIs. P450 inhibition can be categorized as reversible (competitive or non-competitive) or irreversible (mechanism-based inactivation). Mechanism-based P450 inactivation usually involves bioactivation of the xenobiotic to a reactive intermediate, which covalently modifies an active site amino acid residue and/or coordinates to the heme prosthetic group. Covalent modification of P450 enzymes can also lead to hapten formation and can in some cases trigger an autoimmune response resulting in toxicological consequences. Compared to reversible inhibition, irreversible inhibition more frequently results in unfavorable DDIs as the inactivated P450 enzyme has to be replaced by newly synthesized protein. For these reasons, most drug metabolism groups within pharmaceutical companies have well-established screening paradigms to assess mechanism-based inactivation of major human P450 enzymes by new chemical entities followed by indepth mechanistic studies to elucidate the mechanism of P450 inactivation when a positive finding is discerned. A deeper understanding of the process leading to enzyme inactivation by drug candidates can lead to rational chemical intervention strategies to circumvent the P450 inactivation/bioactivation liability. Apart from structure-activity relationship studies, methodology to predict the magnitude of in vivo metabolic DDIs using in vitro P450 inactivation data and predicted human pharmacokinetics of the candidate drug also exists and can be utilized to project the extent of clinical DDIs against P450 enzyme-specific substrates. In this review, a comprehensive analysis of the biochemical basis and known structure-activity relationships for P450 inactivation by xenobiotics is described. In addition, the current state-of-the-art of the methodology used in predicting the magnitude of DDIs using in vitro P450 inactivation data and human pharmacokinetic parameters is discussed in detail.

This commentary discusses the approaches to, and key considerations in the in vitro-in vivo extrapolation of drug-drug interactions (DDI) resulting from mechanism-based inactivation (MBI) of cytochrome P450 (CYP) enzymes and clinical pharmacologic implications. In vitro kinetic assessment and prediction of DDI produced via reversible inhibition and MBI rely on operationally and conceptually distinct approaches. DDI risk assessment for inactivators requires estimation of maximal inactivation rate (kinact) and inactivator potency (KI) in vitro, that need to be considered in context of the biological turnover rate of the enzyme (kdeg) and clinical exposures of the inactivator (I), respectively, to predict interaction magnitude. Risk assessment cannot be performed by a simple comparison of inactivator potency against in vivo exposure since inactivation is both concentration and time-dependent. MBI contour plots tracking combinations of I:KI and kinact:kdeg resulting in identical fold-reductions in intrinsic clearance are proposed as a useful framework for DDI risk assessment. Additionally, substrate-specific factors like fraction of the total clearance of the object drug via the enzyme being inactivated (fm(CYP)) and the bioavailability fraction across the intestine for CYP3A substrates (FG) are important determinants of interaction magnitude. Sensitivity analysis of predicted DDI magnitude to uncertainty in input parameters is recommended to inform confidence in predictions. The time course of reversal of DDI resulting from CYP inactivation is determined by the half-life of the enzyme which is an important consideration in the design and interpretation of clinical DDI studies with inactivators.

In this article approaches to predict human pharmacokinetics (PK) are discussed and the capability of the exemplified methodologies to estimate individual PK parameters and therapeutic dose for a set of marketed oral drugs has been assessed. For a set of 63 drugs where the minimum efficacious concentration (MEC) and human PK were known, the clinical dose was shown to be well predicted or in some cases over-estimated using a simple one-compartment oral PK model. For a subset of these drugs, in vitro potency against the primary human targets was gathered, and compared to the observed MEC. When corrected for plasma protein binding, the MEC of the majority of compounds was ≤ 3 fold over the respective in vitro target potency value. A series of in vitro and in vivo experiments were conducted to predict the human PK parameters. Metabolic clearance was generally predicted well from human hepatocytes. Interestingly, for this compound set, allometry or glomerular filtration rate (GFR) ratio methods appeared to be applicable for renal CL even where CLrenal > GFR. For ∼90% of compounds studied, the predicted CL using in vitro-in vivo (IVIV) extrapolation together with a CLrenal estimate, where appropriate, was within 2-fold of that observed clinically. Encouragingly volume of distribution at steady state (Vss) estimated in preclinical species (rat and dog) when corrected for plasma protein binding, predicted human Vss successfully on the majority of occasions - 73% of compounds within 2-fold. In this laboratory, absorption estimated from oral rat PK studies was lower than the observed human absorption for most drugs, even when solubility and permeability appeared not to be limiting. Preliminary data indicate absorption in the dog may be more representative of human for compounds absorbed via the transcellular pathway. Using predicted PK and MEC values estimated from in vitro potency assays there was a good correlation between predicted and observed dose. This analysis suggests that for oral therapies, human PK parameters and clinical dose can be estimated from a consideration of data obtained from in vitro screens using human derived material and in vivo animal studies. The benefits and limitations of this holistic approach to PK and dose prediction within the drug discovery process are exemplified and discussed.

We have analyzed several members of drug-metabolizing enzymes (DMEs) and other polymorphisms in genes implicated in tumor aggressivity regarding possible links between specific genetic variability in systemic drug bioavailability and toxicity in breast cancer patients treated with adjuvant anthracycline-based treatment. PCR-RFLP and sequencing analyses technique were used for evaluating fourteen previously identified polymorphisms in 94 patients. GSTP1A>G and MTHFR 1298A>C genotypes remained as significant predictors in a multivariate logistic regression analysis. GSTP1 polymorphism was linked to haematological GIII-IV toxicity (P = 0.044, HR= 6.4, 95% CI = 1.05 to 39. Increased and significant HR was obtained for MTHFR-1298 AC+CC group when non-haematological toxicities GIII-IV toxicities were evaluated (HR = 24; 95% CI = 2.3 to 254), P = 0.008. Our results suggest that GSTP1 and MTHFR genotypes may be consider relevant and independent factors of toxicity in adjuvant anthracycline- based treatment of breast cancer..

Trimethylaminuria (fish odor syndrome) is a metabolic disorder characterized by the inability to convert malodorous dietaryderived trimethylamine (TMA) to odorless TMA N-oxide by the flavin-containing monooxygenase 3 (FMO3). Mutations of the FMO3 gene were investigated in Japanese trimethylaminuria that showed low FMO3 metabolic capacity. Novel polymorphisms in the FMO3 gene causing stop codons at Cys197, Trp388, Gln470 or Arg500 of FMO3 were discovered in self-reported trimethylaminuria Japanese volunteers. Different metabolic capacities of FMO3 were observed for Asn114Ser, Thr201Lys, Arg205Cys or Met260Val FMO3 variants in addition to common Glu158Lys, Val257Met, and Glu308Gly FMO3. Estimated allelic frequencies for these novel mutated FMO3 genes for the Japanese population examined was ∼1-4 % in this Japanese cohort. Recombinant Arg500stop (94% of the whole FMO3 structure) and several missense FMO3 variants showed no detectable activity and different effects on N- and S-oxygenation activities, respectively. The family members of Japanese probands who were heterozygous for these nonsense mutants generally showed moderate TMA N-oxygenation metabolic capacity, suggesting that heterozygotes for the nonsense mutations will exhibit trimethylaminuria symptoms only if they have, on the other chromosome, a mutation that substantially impairs enzyme activity. In addition, other causal factors for decreased FMO3 metabolic capacity such as liver damage or menstruation and treatment with copper chlorophyllin are also included in this minireview. The present article provides fundamental information for the importance of future investigations of the human FMO3 gene associated with trimethylaminuria (fish odor syndrome).

The aim of the present study was to develop and improve methods for phenotyping of CYP2E1, an important enzyme in the biotransformation of many industrial chemicals, therapeutic drugs and endogenous substances. The possibility to measure CYP2E1 activity in lymphocytes by using p-nitrophenol as a substrate and CYP2E1 protein levels by flow cytometry were studied in vitro. Further, the conventional chlorzoxazone method for in vivo phenotyping was studied by adjusting the dose to body weight in 10 healthy volunteers. Finally, the possibility to obtain the chlorzoxazone metabolic ratio in saliva samples was investigated. No CYP2E1 protein in lymphocytes was detected by using flow cytometry. Some enzyme activity was found in the experiments with p-nitrophenol, however, it could not be verified that it was catalyzed by CYP2E1. Chlorzoxazone and 6-hydroxychlorzoxazone were not detectable in saliva samples. The present in vivo experiments, combined with our previous data (in total 356 experiments in 50 subjects) show that the metabolic ratio increases with decreasing absorbed dose, expressed as the sum of chlorzoxazone and 6-hydroxychlorzoxazone in plasma at 2 h. The increase becomes pronounced at sum concentrations below 100 μM. In conclusion, chlorzoxazone metabolism in vivo remains the only available method for CYP2E1 phenotyping. The administered dose as well as the absorption of the probe influences the chlorzoxazone ratio. We suggest that a dose of 10 mg chlorzoxazone per kg body weight is used to estimate the CYP2E1 phenotype. Further, metabolic ratios should be disregarded if the sum of plasma chlorzoxazone and 6-hydroxychlorzoxazone is below 100 μM (blood sampled after 2 h).

Polyphenolic compounds are abundant in the human diet and gram quantities are ingested daily. The consumption of polyphenols is expected to rise due to the use of dietary supplements and public health initiatives promoting the consumption of more fruits and vegetables. It is known that these dietary polyphenols are extensively metabolized. Many of these compounds are therefore are expected to compete with other substrates of Phases I, II, III enzymes and transporters. In addition, some dietary polyphenols may induce certain drug metabolizing enzymes and affect the metabolism of important therapeutic agents. This review will discuss 1) the metabolism of dietary polyphenols using green tea polyphenols (catechins) as an example, 2) inhibition of drug metabolism by polyphenols, and 3) induction of drug metabolizing enzymes by dietary polyphenols. The potential consequences of these effects on drug metabolism will also be discussed.

To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. Consequently, “injectable-to-oral-conversions” are highly on demand. Apart from a poor membrane uptake, however, an extensive presystemic metabolism of orally given peptide drugs is responsible for a comparatively very poor oral bioavailability. This presystemic metabolism in the gastrointestinal tract is based on luminally secreted enzymes (I) including pepsins, trypsin, chymotrypsin, elastase and carboxypeptidase A/B, on brush border membrane bound enzymes (II) including various carboxypeptidases and aminopeptidases and on cytosolic enzymes (III). In addition, thiol-disulphide exchange reactions between orally administered peptide drugs and sulfhydryl bearing components of the gastrointestinal juice are responsible for a presystemic metabolism. Strategies to avoid a presystemic metabolism in the gastrointestinal tract are on the one hand based on chemical modifications of peptide drugs in order to make them more stable towards an enzymatic attack. On the other hand various formulation techniques are applied in order to protect therapeutic peptides, being incorporated in appropriate carrier systems. They include liposomes, nano-/microparticles and matrix tablets comprising various auxiliary agents such as enzyme inhibitors and multifunctional polymers. Within this review an overview about “the enemy's strength” and the current strategies to avoid a presystemic metabolism of orally administered peptides is provided.

Calcium ion is an essential structural component of the skeleton. There is growing evidence for the importance of nutrition in the maintenance of bones and joints health. Nutritional imbalance combined with endocrine abnormalities may be involved in osteoporosis. For example, essential fatty acids and their metabolites were reported to have beneficial action in osteoporosis. The mechanism by which fatty acids prevent osteoporosis may involve inhibition of pro-inflammatory cytokines, which are known to have a major role in osteoporosis through induction of oxidative stress which had adverse effects on the skeleton. Other risk factors for osteoporosis, such as smoking, hypertension and diabetes mellitus are also associated with increased oxidative stress and free radicals levels. When bone fracture occurs, a remarkable yield of free radicals is generated by the damaged tissues. However, controlled production of free radicals by normally functioning osteoclasts could accelerate destruction of calcified tissues and assist bone remodeling. Enhanced osteoclastic activity observed in bone disorders may have been responsible for increased production of reactive oxygen species [ROS] in the form of superoxide, which is evident by increased levels of serum malondialdehyde [MDA] levels. One of the most damaging effects of ROS is lipid peroxidation, the end product of which is MDA which also served as a measure of osteoclastic activity. Inhibition of the antioxidant enzymes activities, such as superoxide dismutase and glutathione peroxidase, was found to increase superoxide production by the osteoclasts which represented by increased levels of MDA. Therefore, oxidative stress is an important mediator of bone loss since deficiency of antioxidant vitamins has been found to be more common in the elderly osteoporotic patients. It is concluded from this review that increased free radical production overwhelms the natural antioxidants defense mechanisms, subjecting individuals to hyperoxidant stress and thus leading to osteoporosis. In addition, administration of antioxidants might protect bones from osteoporosis and also might help in the acceleration of healing of fractured bones.