Showing papers in "Expert Opinion on Drug Metabolism & Toxicology in 2006"

Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction.

Abstract: Animal models are commonly used in the preclinical development of new drugs to predict the metabolic behaviour of new compounds in humans. It is, however, important to realise that humans differ from animals with regards to isoform composition, expression and catalytic activities of drug-metabolising enzymes. In this review the authors describe similarities and differences in this respect among the different species, including man. This may be helpful for drug researchers to choose the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. The authors focus on CYPs, which are the main enzymes involved in numerous oxidative reactions and often play a critical role in the metabolism and pharmacokinetics of xenobiotics. In addition, induction and inhibition of CYPs are compared among species. The authors conclude that CYP2E1 shows no large differences between species, and extrapolation between species appears to hold quite well. In contrast, the species-specific isoforms of CYP1A, -2C, -2D and -3A show appreciable interspecies differences in terms of catalytic activity and some caution should be applied when extrapolating metabolism data from animal models to humans.

Zebrafish assays for drug toxicity screening

Abstract: Zebrafish are vertebrate organisms that are of growing interest for preclinical drug discovery applications. Zebrafish embryos develop most of the major organ systems present in mammals, including the cardiovascular, nervous and digestive systems, in < 1 week. Additional characteristics that make them advantageous for compound screening are their small size, transparency and ability to absorb compounds through the water. Furthermore, gene function analysis with antisense technology is now routine procedure. Thus, it is relatively simple to assess whether compounds or gene knockdowns cause toxic effects in zebrafish. Assays are being developed to exploit the unique characteristics of zebrafish for pharmacological toxicology. This review discusses assays that may be used to assess invivo toxicity and provides examples of compounds known to be toxic to humans that have been demonstrated to function similarly in zebrafish.

Structure and function of the MRP2 (ABCC2) protein and its role in drug disposition

Abstract: The multi-drug resistance protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter playing an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, glucuronate and sulfate, which are collectively known as phase II products of biotransformation. In addition, MRP2 can also transport uncharged compounds in cotransport with glutathione, and thus can modulate the pharmacokinetics of many drugs. The other way around, its expression and activity are also altered by certain drugs and disease states. Unlike other members of the MRP/ABCC family, MRP2 is specifically expressed on the apical membrane domain of polarised cells as hepatocytes, renal proximal tubular cells, enterocytes and syncytiotrophoblasts of the placenta. Several naturally occurring mutations leading to the absence of functional MRP2 protein from the apical membrane have been described causing the human Dubin-Johnson syndrome associated with conjugated hyperbilirubinaemia. Experimental mutation studies have revealed critical amino acids for substrate binding in the MRP2 molecule. This review is, therefore, focused on the structure and function of MRP2, the substrates transported and the clinical relevance of MRP2.

Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies.

Abstract: Gaining knowledge on the metabolism of a drug, the enzymes involved and its inhibition or induction potential is a necessary step in pharmaceutical development of new compounds. Primary human hepatocytes are considered a cellular model of reference, as they express the majority of drug-metabolising enzymes, respond to enzyme inducers and are capable of generating in vitro a metabolic profile similar to what is found in vivo. However, hepatocytes show phenotypic instability and have a restricted accessibility. Different alternatives have been explored in the past recent years to overcome the limitations of primary hepatocytes. These include immortalisation of adult or fetal human hepatic cells by means of transforming tumour virus genes, oncogenes, conditionally immortalised hepatocytes, and cell fusion. New strategies are currently being used to upregulate the expression of drug-metabolising enzymes in cell lines or to derive hepatocytes from progenitor cells. This paper reviews the features of liver-derived cell lines, their suitability for drug metabolism studies as well as the state-of-the-art of the strategies pursued in order to generate metabolically competent hepatic cell lines.

Luminogenic cytochrome P450 assays.

Abstract: Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-GloTM (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

The road map to oral bioavailability: an industrial perspective

Abstract: Optimisation of oral bioavailability is a continuing challenge for the pharmaceutical and biotechnology industries. The number of potential drug candidates requiring invivo evaluation has significantly increased with the advent of combinatorial chemistry. In addition, drug discovery programmes are increasingly forced into more lipophilic and lower solubility chemical space. To aid in the use of invitro and insilico tools as well as reduce the number of invivo studies required, a team-based discussion tool is proposed that provides a road map to guide the selection of profiling assays that should be considered when optimising oral bioavailability. This road map divides the factors that contribute to poor oral bioavailability into two interrelated categories: absorption and metabolism. This road map provides an interface for cross discipline discussions and a systematic approach to the experimentation that drives the drug discovery process towards a common goal acceptable oral bioavailability using minimal ...

Preclinical formulations for discovery and toxicology: physicochemical challenges

Abstract: Formulations play a key role in assessing the biological properties of a molecule during drug discovery. Maximising exposure is the primary objective in early animal experimentation, so that the pharmacokinetics, pharmacodynamics and toxicological signals can be put into context with the biological response to specific targets. Consistency in the exposure is also a key aspect, and effective formulation and drug delivery strategies are important to achieve this. Diversity in the physiology between various animal species, routes of administration and limitations posed by specific pharmacological models make formulation development that much more challenging. Poor physicochemical properties of compounds in the early stages need to be kept under consideration while screening for formulation vehicles. This review captures the various challenges posed at different stages of drug discovery for formulation of a compound to dose in animals. Approaches to formulations for various routes of administration are discussed. Limitations posed by the goals for various animal studies such as early efficacy studies, pharmacokinetic studies and toxicology studies are identified and some strategies are proposed. Physicochemical characterisations that are needed to select formulation vehicles as well as to identify potential issues are suggested.

Role of innate immunity in acetaminophen-induced hepatotoxicity

Abstract: Acetaminophen (APAP) hepatotoxicity is currently the single most important cause of acute liver failure in the US, and is associated with a significant number of deaths. The toxic response to APAP is triggered by a highly reactive metabolite N-acetyl-p-benzoquinone-imine. Following the hepatocellular initiation events, such as glutathione depletion and covalent binding, intracellular stress simultaneously activates signal transduction and transcription factor pathways that are protective or toxic (directly or through sensitisation). Subsequently, pro- and anti-inflammatory cascades of the innate immune system are simultaneously activated, the balance of which plays a major role in determining the progression and severity of APAP-induced hepatotoxicity. The threshold and susceptibility to APAP hepatotoxicity is determined by the interplay of injury promoting and inhibiting events downstream of the initial production of toxic metabolite. The environmental and genetic control of these intracellular and intercellular responses to toxic metabolites may be of critical importance in determining susceptibility to APAP hepatotoxicity and presumably idiosyncratic drug hepatotoxicity.

Clinical implications of CYP3A polymorphisms.

Abstract: Due to their enormous substrate spectrum CYP3A4, -3A5 and -3A7 constitute the most important drug-metabolising enzyme subfamily in humans. CYP3As are expressed predominantly, but not exclusively, in the liver and intestine, where they participate in the metabolism of 45 - 60% of currently used drugs and many other compounds such as steroids and carcinogens. CYP3A expression and activity vary interindividually due to a combination of genetic and nongenetic factors such as hormone and health status, and the impact of environmental stimuli. Over the past several years, genetic determinants have been identified for much of the variable expression of CYP3A5 and -3A7, but not for CYP3A4. Using these markers, an effect of CYP3A5 expression status has been demonstrated beyond doubt for therapies with the immunosuppressive drug tacrolimus. Further associations are likely to emerge for drugs metabolised predominantly by CYP3A5 or -3A7, especially for individuals or tissues with concomitant low expression of CYP3A4. However, as exemplified by the controversial association between CYP3A4*1B and prostate cancer, the detection of clinical effects of CYP3A gene variants will be difficult. The most important underlying problems are the continuing absence of activity markers specific for CYP3A4 and the strong contribution of nongenetic factors to CYP3A variability.

Involvement of enzymes other than CYPs in the oxidative metabolism of xenobiotics.

Abstract: Although the majority of oxidative metabolic reactions are mediated by the CYP superfamily of enzymes, non-CYP-mediated oxidative reactions can play an important role in the metabolism of xenobiotics. The (major) oxidative enzymes, other than CYPs, involved in the metabolism of drugs and other xenobiotics are: the flavin-containing monooxygenases, the molybdenum hydroxylases (aldehyde oxidase and xanthine oxidase), the prostaglandin H synthase, the lipoxygenases, the amine oxidases (monoamine, polyamine, diamine and semicarbazide-sensitive amine oxidases) and the alcohol and aldehyde dehydrogenases. In a similar manner to CYPs, these oxidative enzymes can also produce therapeutically active metabolites and reactive/toxic metabolites, modulate the efficacy of therapeutically active drugs or contribute to detoxification. Many of them have been shown to be important in endobiotic metabolism, and, consequently, interactions between drugs and endogenous compounds might occur when they are involved in drug metabolism. In general, most non-CYP oxidative enzymes appear to be noninducible or much less inducible than the CYP system, although some of them may be as inducible as some CYPs. Some of these oxidative enzymes exhibit polymorphic expression, as do some CYPs. It is possible that the contribution of non-CYP oxidative enzymes to the overall metabolism of xenobiotics is underestimated, as most investigations of drug metabolism in discovery and lead optimisation are performed using in vitro test systems optimised for CYP activity.

Active transport across the human placenta: impact on drug efficacy and toxicity

Abstract: The human placenta expresses a large number of transport proteins. The ATP-binding cassette (ABC) family of active efflux pumps, predominantly localised to the maternal-facing syncytial membrane of placental microvilli, comprise the major placental drug efflux transporters. A variety of other transporters are also expressed in the placenta that can facilitate xenobiotic transfer in both the maternal and fetal directions. Many drugs administered in pregnancy are ABC transporter substrates, and many are either teratogenic or fetotoxic. The in vitro, in vivo and clinical evidence reviewed in this article argues that active efflux of drugs by placental transporters helps to maintain its barrier function, reducing the incidence of adverse fetal effects. ABC transporter polymorphisms may explain the wide variability observed in fetal drug concentrations, incidence of teratogenesis or drug failure in pregnancies exposed to therapeutic agents. Although our understanding of the molecular mechanics and dynamics of placental drug transfer is advancing, much work is needed to fully appreciate the significance of placental drug transporters in the face of increasing drug administration in pregnancy.

Using pharmacokinetics to predict the effects of pregnancy and maternal–infant transfer of drugs during lactation

Abstract: Knowledge of pharmacokinetics and the use of a mechanistic-based approach can improve our ability to predict the effects of pregnancy for medications when data are limited. Despite the many physiological changes that occur during pregnancy that could theoretically affect absorption, bioavailability does not appear to be altered. Decreased albumin and α1-acid glycoprotein concentrations during pregnancy will result in decreased protein binding for highly bound drugs. For drugs metabolised by the liver, this can result in misinterpretation of total plasma concentrations of low extraction ratio drugs and overdosing of high extraction ratio drugs administered by non-oral routes. Renal clearance and the activity of the CYP isozymes, CYP3A4, 2D6 and 2C9, and uridine 5′-diphosphate glucuronosyltransferase are increased during pregnancy. In contrast, CYP1A2 and 2C19 activity is decreased. The dose of a drug an infant receives during breastfeeding is dependent on the amount excreted into the breast milk, the daily...

High-throughput pKa screening and prediction amenable for ADME profiling

Abstract: Recent technological advances have made it possible for several new pK(a) assays to be used in drug screening. In this review, a critical overview is provided of current new methodologies for high-throughput screening and prediction of pK(a). Typical applications of using pK(a )constants and charge state for absorption, distribution, metabolism and excretion (ADME) profiling and quantitative structure-activity relationship modelling complements the methodological comparisons and discussions. The experimental methods discussed include high-throughput screening of pK(a) by multiplexed capillary with ultraviolet absorbance detection on a 96-capillary format instrument, capillary electrophoresis and mass spectrometry (CEMS) based on sample pooling, determination of pK(a) by pH gradient high-performance liquid chromatography, and measurement of pK(a) by a mixed-buffer liner pH gradient system. Comparisons of the different experimental assays are made with emphasis on the newly developed CEMS method. The current status and recent progress in computational approaches to pK(a) prediction are also discussed. In particular, the accuracy limits of simple fragment-based approaches as well as quantum mechanical methods are addressed. Examples of pK(a) prediction from in-house drug candidates as well as commercially available drug molecules are shown and an outline is provided for how drug discovery companies can integrate experiments with computational approaches for increased applications for ADME profiling.

Drug-drug interactions involving membrane transporters in the human kidney

Abstract: The kidneys play a critical role in the elimination of xenobiotics. Factors affecting the ability of the kidney to eliminate drugs may result in marked changes in the pharmacokinetics of a given compound. Drug-drug interactions due to competitive inhibition of renal organic anion or cation secretion systems have been noticed clinically for a long time. However, our understanding of the physical sites of interactions, that is, the specific transport proteins that the interacting drugs act on, has just begun very recently. This review summarises the latest progress in molecular identification and functional characterisation of major drug transporters in the human kidney. In particular, the review focuses on relating cloned renal drug transporters to clinically observed drug-drug interactions. The authors' opinion on the current status and future directions of research in these areas is also offered.

Enhancement of drug absorption by noncharged detergents through membrane and P-glycoprotein binding.

Abstract: Noncharged detergents are used as excipients in drug formulations. Until recently, they were considered as inert compounds, enhancing drug absorption essentially by improving drug solubility. However, many detergents insert into lipid membranes, although to different extents, and change the lateral packing density of membranes at high concentrations. Moreover, they bind to the efflux transporter P-glycoprotein (P-gp) and most likely to related transporters and metabolising enzymes with overlapping substrate specificities. If their affinity to P-gp is higher than that of the coadministered drug they act as modulators or inhibitors of P-gp and enhance drug absorption. Inhibition of P-gp and related proteins can, however, cause severe side effects. This paper first reviews the membrane binding propensity of different noncharged detergents (including poloxamers) and discusses their ability to bind to P-gp. Second, literature data on drug uptake enhancement by noncharged detergents, obtained in vivo and in vitro, are analysed at the molecular level. The present analysis provides the tools for an approximate and simple prior estimate of the membrane and P-gp binding ability of noncharged detergents based on a modular binding approach.

The use of isotopes in the determination of absolute bioavailability of drugs in humans

Abstract: Absolute bioavailability studies in humans are not routinely performed as part of the drug registration process. They tend to be reasonably demanding, not least because toxicology data are required...

Pregnane X receptor and natural products: beyond drug–drug interactions

Abstract: The pregnane X receptor (PXR, NR1I2) is a member of the nuclear receptor superfamily that is activated by a myriad of compounds and natural products in clinical use. Activation of PXR represents the basis for several clinically important drug–drug interactions. Although PXR activation has undesirable effects in patients on combination therapy, it also mediates the hepatoprotective effects exhibited by some herbal remedies. This review focuses on PXR activation by natural products and the potential therapeutic opportunities presented. In particular, the biological effects of St. John’s Wort, gugulipid, kava kava, Coleus forskolii, Hypoxis, Sutherlandia, qing hao, wu wei zi, gan cao and other natural products are discussed. The impact of these natural products on drug metabolism and hepatoprotection is highlighted in the context of activation and antagonism of PXR.

Mechanistic biomarkers for cytotoxic acute kidney injury.

Abstract: Acute kidney injury is a common condition and is associated with a high mortality rate It has been recognised that routinely used measures of renal function, such as levels of blood urea nitrogen and serum creatinine, increase significantly only after substantial kidney injury occurs and then with a time delay Insensitivity of such tests delays the diagnosis in humans, making it particularly challenging to administer putative therapeutic agents in a timely fashion Furthermore, this insensitivity affects the evaluation of toxicity in preclinical studies by allowing drug candidates, which have low, but nevertheless important, nephrotoxic side effects in animals, to pass the preclinical safety criteria only to be found to be clinically nephrotoxic with great human costs This review presents the current status of sensitive and specific biomarkers to detect preclinical and clinical renal injury and summarises the techniques used to quantitate these biomarkers in biological fluids

Methods of evaluating immunotoxicity

Abstract: Immunotoxicology is an important aspect of the safety evaluation of drugs and chemicals. Immunosuppression, (unspecific) immunostimulation, hypersensitivity and autoimmunity are the four types of immune-mediated adverse effects. However, the nonclinical assessment of immunotoxicity is at present often restricted to animal models and assays to predict unexpected immunosuppression. There is, however, no general consensus that a variety of assays can be considered depending on the compound to be tested. A major issue is whether histological examination of the thymus, spleen, lymphoid organs and Peyers patches is a reliable predictor of immunosuppression or whether immune function should also be assessed. A T-dependent antibody response assay, either the plaque-forming cell assay or anti-keyhole limpet haemocyanin enzyme-linked immunosorbant assay, is recommended as a first-line assay. A variety of assays, including lymphocyte subset analysis, natural killer-cell activity, lymphocyte proliferation, delayed-ty...

Chemical toxicology: reactive intermediates and their role in pharmacology and toxicology

Abstract: Reactive intermediates formed during the metabolism of drugs have been investigated extensively over the past decades. Today, interest in reactive intermediates in drug discovery is focused on minimising bioactivation in hopes of reducing the risk of causing so-called idiosyncratic toxicity. These efforts are justified based on the 'hapten hypothesis', namely, that on binding to protein, reactive intermediates may elicit an immune response to the modified protein, leading to a cascade of events that ultimately manifests as a toxic outcome. However, the pharmacological action of certain drugs depends on reactive intermediates that modify critical amino acid residues of proteins, typically enzymes, thereby altering their activity. Thus, the notion that reactive intermediates are inherently dangerous is unjustified. When a reactive intermediate is necessary for the desired pharmacological effect of a drug, the selectivity it displays towards the target protein is crucial, as off-target binding may produce unwanted toxicities. On the other hand, reactive intermediates may play no role in toxicity. This review provides a balanced perspective, primarily focusing on the proposed role of reactive intermediates in drug toxicity, while also highlighting examples in which they are involved in causing the desired pharmacology. It is hoped that this knowledge can help scientists involved in drug discovery and development in their challenging task of producing safe and effective drugs.

Pharmacokinetics, toxicology and safety of lamotrigine in epilepsy.

Abstract: Optimisation of pharmacotherapy for epilepsy requires consideration of the impact of drug metabolism and toxicology on the therapeutic profiles and clinical use of antiepileptic drugs (AEDs). This review discusses the pharmacokinetics and toxicology of the AED lamotrigine, and considers the implications of these data for optimising its use in the management of epilepsy. Lamotrigine has good absorption, minimal plasma protein binding and linear pharmacokinetics. Partly because of these properties, frequent dosing adjustments are generally unnecessary, and therapeutic monitoring is not required under most circumstances. Lamotrigine is not associated with clinically significant neurological, cognitive, metabolic, hepatic or reproductive endocrine toxicity. Like other AEDs, including carbamazepine and phenytoin, lamotrigine has been associated with serious rash. With some exceptions, lamotrigine has relatively few clinically relevant drug interactions, a characteristic important in reducing safety risks, especially among patients who require polytherapy. The clinical impact of pharmacokinetic interactions between lamotrigine and enzyme-inducing AEDs or valproate can be minimised by adhering to recommended dose-escalation schedules with demonstrated reliability in clinical trials and clinical practice. Likewise, adhering to recommended dosing guidelines can minimise the risk of lamotrigine-associated rash. The pharmacokinetic, toxicology and safety profiles of lamotrigine make the drug suitable for use across a spectrum of patients with epilepsy.

Focus on success: using a probabilistic approach to achieve an optimal balance of compound properties in drug discovery.

Abstract: The success of any drug will depend on how closely it achieves an ideal combination of potency, selectivity, pharmacokinetics and safety. The key to achieving this success efficiently is to consider the overall balance of molecular properties of compounds against the ideal profile for the therapeutic indication from the earliest stages of a drug discovery project. The use of insilico predictive models of absorption, distribution, metabolism and elimination (ADME) and physicochemical properties is a major aid in this exercise, as it enables virtual molecules to be assessed across a broad range of properties from initial library generation, through to candidate selection. Of course, no measurement, whether insilico, invitro or invivo, is perfect and the uncertainties in any data should be explicitly taken into account when basing conclusions on test results. In addition, in the early stages of drug discovery, when designing a library that is lead seeking or building compound structureactivity relationships,...

Genetic polymorphisms of drug transporters: pharmacokinetic and pharmacodynamic consequences in pharmacotherapy

Abstract: There has been increasing appreciation of the role of drug transporters in pharmacokinetic and pharmacodynamic consequences in pharmacotherapy. The clinical relevance of drug transporters depends on the localisation in human tissues (i.e., vectorial movement), the therapeutic index of the substrates and inherent interindividual variability. With regard to variability, polymorphisms of drug transporter genes have recently been reported to be associated with alterations in the pharmacokinetics and pharmacodynamics of clinically useful drugs. A growing number of preclinical and clinical studies have demonstrated that the application of genetic information may be useful in individualised pharmacotherapy for numerous diseases. However, the reported effects of variants in certain drug transporter genes have been inconsistent and, in some cases, conflicting among studies. Furthermore, the incidence of almost all known variants in transporter genes tends to be racially dependent. These observations suggest the necessity of considering interethnic variability before extrapolating pharmacokinetic data obtained in one ethic group to another, especially in the early phase of drug development. This review focuses on the impact of genetic variations in the function of drug transporters (ABC, organic anion and cation transporters) and the implications of these variations for pharmacotherapy from pharmacokinetic and pharmacodynamic viewpoints.

Sex differences in pharmacokinetics and toxicity of antiretroviral therapy

Abstract: Worldwide, the rate of new HIV infections continues to increase among women. Over the past 5 years there has been a growing focus on the clinical aspects of HIV infection among women. Historically, the majority of subjects enrolled in HIV clinical trials were male patients. Consequently, most knowledge about antiretroviral efficacy and toxicity has been derived from studies of predominately male subjects. More recently, results from clinical trials, especially those that have focused on HIV-infected female subjects, suggest that there may be clinically important gender-related differences in several aspects of HIV disease. These include, but are not limited to, differences in natural history, efficacy and safety of drug treatment. This chapter reviews current data on gender differences in antiretroviral pharmacokinetics and toxicity.

Functional evolution of the pregnane X receptor

Abstract: The pregnane X receptor (PXR; NR1I2) is a nuclear hormone receptor (NR) that transcriptionally regulates genes encoding transporters and drug-metabolising enzymes in the liver and intestine. PXR activation leads to enhanced metabolism and elimination of xenobiotics and endogenous compounds such as hormones and bile salts. Relative to other vertebrate NRs, PXR has the broadest specificity for ligand activators by virtue of a large, flexible ligand-binding cavity. In addition, PXR has the most extensive sequence diversity across vertebrate species in the ligand-binding domain of any NR, with significant pharmacological differences between human and rodent PXRs, and especially marked divergence between mammalian and nonmammalian PXRs. The unusual properties of PXR complicate the use of in silico and animal models to predict in vivo human PXR pharmacology. Research into the evolutionary history of the PXR gene has also provided insight into the function of PXR in humans and other animals.

Recent progress in cytochrome P450 enzyme electrochemistry.

Abstract: Cytochrome P450 (CYP) enzymes perform crucial functions in humans, including the metabolism of drugs and hormone synthesis. The catalytic reactions performed by these enzymes (typically monoxygenation) require the transfer of electrons. Thermodynamic and mechanistic detail of the electron transfer component of these catalytic processes has been obtained traditionally from potentiometric titrations. More recently, voltammetric approaches (that are inherently simpler and require less sample) have been used. This has been made possible by the creation of biocompatible electrode surfaces at which the P450 enzyme is confined and able to undergo physiologically relevant electron transfer processes. The continuing challenge has been to obtain an in vivo-like enzyme response, and to provide the basis for the creation of an artificial bioprocess in vitro. A powerful instrumental electrochemical method, employing Fourier-transformed large-amplitude ac voltammetry, offers the potential for greater insight and new opportunities to understand the nuances of the electron transfer process. This review highlights several recent advances in the electrochemistry of P450 enzymes rather than providing a comprehensive review of P450 electrochemistry.

Molecular mechanisms for drug interactions with hERG that cause long QT syndrome.

Abstract: The human ether-a-go-go-related gene (hERG) encodes the pore-forming alpha-subunit of a voltage-gated potassium (K(+)) channel A variety of unrelated compounds reduce K(+ )current in the heart by blocking the pore or disrupting trafficking of the hERG channel to the membrane surface This induces a syndrome known as long QT, which arises from abnormalities in action potential repolarisation and can degenerate into lethal cardiac arrhythmias As a result, this undesirable side effect has severely hindered safe drug development This review describes progress in understanding the molecular basis for drug binding to hERG, outlines the characteristics of hERG ligands and discusses experimental and in silico approaches for identifying compounds with QT liabilities Recent developments should enable recognition of hERG-positive compounds at the early stages of their development

The role of amine oxidases in xenobiotic metabolism.

Abstract: The amine oxidases of mammalian tissues are a heterogeneous family of enzymes that metabolise various monoamines, diamines and polyamines produced endogenously, or being absorbed as dietary or xenobiotic substances. The heterogeneous class of amine oxidases can be divided on an arbitrary basis of the chemical nature of their cofactors into two types. Monoamine oxidase (MAO) and an intracellular form of polyamine oxidase (PAO) contain flavin adenine dinucleotide (FAD) as their cofactor, whereas a second group of amine oxidases without FAD contain a cofactor possessing one or more carbonyl groups, making them sensitive to inhibition by carbonyl reagents such as semicarbazide; this group includes semicarbazide-sensitive amine oxidase (SSAO) and the connective tissue enzyme, lysyl oxidase. This article focuses on the general aspects of MAO's contribution to the metabolism of foreign toxic substances including toxins and illegal drugs. Another main objective of this review is to discuss the properties of PAO and SSAO and their involvement in the metabolism of xenobiotics.

Developmental and tissue-specific expression of human flavin-containing monooxygenases 1 and 3.

Abstract: Substantial changes occur in drug and toxicant disposition during early life stages that can impact therapeutic efficacy and adverse reactions to drugs and toxicants. Of the many parameters involved, alterations in drug metabolism are of major importance. Although the cytochrome P450-dependent monooxygenases are accepted as playing a substantial role in drug and toxicant metabolism, the flavin-containing monooxygenases (FMOs) also have an important role. Apparently unique to the human, FMO3 is the most abundant FMO family member in the adult human liver, whereas FMO1 dominates in most animal models. However, early studies documented that FMO1 is the most abundant FMO enzyme in the human fetal liver, whereas FMO3 is essentially absent. This review focuses on recent studies characterising human FMO ontogeny and, in particular, the 'switch' in hepatic FMO enzyme expression. Because it is so closely related, tissue-specific expression patterns also are examined. Finally, a summary of what is known in animal models is presented as a point of comparison.

Exploiting Streptomyces coelicolor A3(2) P450s as a model for application in drug discovery

Abstract: One of the surprising discoveries about the genomics of the cytochrome P450 (CYP) superfamily is the large number of CYPs in the bacterial class of actinomycetes. It had previously been imagined that bacteria have small numbers of CYPs or none at all. Particularly intriguing is that the bacterial genus Streptomyces, which produce a large number of secondary metabolites with important medical application, has a large CYP complement reflecting the ecological niche that the organism finds itself in. In 2001 the first complete Streptomyces species genome (Streptomyces coelicolor A3[2]) was published, revealing the presence of 18 CYP genes. Subsequently, genomes for Streptomyces avermitilis, with 33 CYPs, and Streptomyces peucetius, with 15 CYPs, have been reported. Although a certain number of these CYPs have known functions in secondary metabolism, as identified biochemically or through gene locus organisation, in the vast majority of Streptomyces species, CYP functions are unknown. The first detailed analysis of the CYP complement from a Streptomyces species genome has begun in the laboratories of Waterman et al. The long-term goal of this effort is to identify orphan CYP function, to establish their high resolution structure and to establish a strategy for producing novel secondary metabolites that have new biomedical function. This chapter provides an overview of CYP systems in Streptomyces species and provides a plan of how new drugs might be generated from streptomycetes by modifying the structure of specific CYPs.