Potential role of cerebral cytochrome P450 in clinical pharmacokinetics: modulation by endogenous compounds

Cytochrome P450 (CYP) enzymes catalyse phase I metabolic reactions of psychotropic drugs. The main isoenzymes responsible for this biotransformation are CYP1A2, CYP2D6, CYP3A and those of the subfamily CYP2C. Although these enzymes are present in the human brain, their specific role in this tissue remains unclear. However, because CYP enzymatic activities have been reported in the human brain and because brain microsomes have been shown to metabolise the same probe substrates used to assess specific hepatic CYP activities and substrates of known hepatic CYPs, local drug metabolism is believed to be likely. There are also indications that CYP2D6 is involved in the metabolism of endogenous substrates in the brain. This, along with the fact that several neurotransmitters modulate CYP enzyme activities in human liver microsomes, indicates that CYP enzymes present in brain could be under various regulatory mechanisms and that those mechanisms could influence drug pharmacokinetics and, hence, drug response.In this paper we review the presence of CYP1A2, CYP2C9, CYP2D6 and CYP3A in brain, as well as the possible existence of local brain metabolism, and discuss the putative implications of endogenous modulation of these isoenzymes by neurotransmitters.     

Update: Clinically Significant Cytochrome P-450 Drug Interactions

Recent technologies have resulted in an explosion of information concerning the cytochrome P-450 isoenzymes and increased awareness of life-threatening interactions' with such commonly prescribed drugs as cisapride and some antihistamines. Knowledge of the substrates, inhibitors, and inducers of these enzymes assists in predicting clinically significant drug interactions. In addition to inhibition and induction, microsomal drug metabolism is affected by genetic polymorphisms, age, nutrition, hepatic disease, and endogenous chemicals. Of the more than 30 human isoenzymes identified to date, the major ones responsible for drug metabolism include CYP3A4, CYP2D6, CYP1A2, and the CYP2C subfamily.     

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. II. Participation of CYP isoenzymes in the metabolism of endogenous substances and drugs

In the human organism 58 cytochrome P450 (CYP) isoenzymes belonging to 18 families have been described. These hemoproteins, with enzymatic activity characteristic for monooxygenases, show a broad affinity for chemically differentiated endo- or exogenous compounds, including drugs. CYP isoenzymes participate in metabolic pathways important for proper physiological functioning of the human organism, i.e.: cholesterol, bile acid and oxysterol biosynthesis; metabolism of fatty acids, prostaglandins, prostacyclins, leukotrienes, steroid hormones, ketone bodies, vitamines A and D. CYP isoenzymes participate in the metabolism of over 80% of drugs and other xenobiotic substances which can be present in the human organism. Differences in molecular structure and kinetics of conformational changes of particular isoenzymes of CYP superfamily monooxygenases on the one hand determine their affinity direction for chemically differentiated groups of compounds susceptible to oxidation, on the other hand determine the mechanism and position of the oxidative change of their molecules. Drugs and their metabolites and other endogenous and xenobiotic compounds may be acting not only as substrates, but also as competitive and non- competitive inhibitors, suicide inhibitors and inducers of CYP isoenzymes as well as repressors of CYP genes. These relationships are the metabolic basis of numerous multidirectional interactions between drugs, drug metabolites, food components, stimulants, environmental toxins and metabolites of these xenobiotics.     

A method for genotyping murine arylamine N-acetyltransferase type 2 (NAT2): a gene expressed in preimplantation embryonic stem cells encoding an enzyme acetylating the folate catabolite p-aminobenzoylglutamate.

Mice have three arylamine N-acetyltransferase (NAT) isoenzymes (NAT1, NAT2, and NAT3) of which NAT2 is known to be polymorphic. Humans have two polymorphic isoenzymes, NAT1 and NAT2. The isoenzymes mouse NAT1 and human NAT2 are expressed predominantly in the liver and intestine and are involved in drug and xenobiotic metabolism. Mouse NAT2 and human NAT1 have a widespread tissue distribution and the folate catabolite p-aminobenzoylglutamate (pAB-Glu) has been proposed as a candidate endogenous substrate. All mice have detectable NAT2 activity, although inbred mouse strains have either a fast or slow acetylator phenotype conferred by the presence of either NAT2*8 (fast) or NAT2*9 (slow) alleles at the NAT2 locus. In this report, we describe a simple method for distinguishing these murine alleles by polymerase chain reaction followed by restriction fragment length polymorphism analysis. We compared the tissue distribution of the acetylation activity found in both fast (C57BL/6J) and slow (A/J) acetylating strains of mice using pAB-Glu and p-aminobenzoic acid as probe substrates. It has previously been demonstrated that murine NAT2 is expressed in the neural tube prior to closure (Stanley L, Copp A, Rolls S, Smelt V, Perry VH and Sim E, Teratology 58: 174-182, 1998). We demonstrate here that murine NAT2 is expressed in preimplantation embryonic stem cells. Murine NAT2 is likely to be expressed prior to neurulation and this may be important in view of the protective role of folate in neural tube development.     

Analyses of CYP2C in porcine microsomes

The cytochrome P450 2C (CYP2C) subfamily in human beings includes four different isoenzymes that metabolize different substrates although with some cross reactivity. Some of these substrates (e.g. diclofenac and tolbutamide), have been investigated in porcine microsomes, but without identifying the specific CYP catalysing the reactions. The objective of this study was therefore to test some CYP2C substrates and identify the porcine CYP2C responsible for the reaction. Three substrates, paclitaxel, tolbutamide and omeprazole, were chosen, as they are metabolized by three different CYP2C isoenzymes in human beings. Microsomes, isolated from 20 different pigs, 12 conventional, and 8 minipigs, were incubated with these compounds, and correlations between the metabolism rates of these three substrates were found indicating that the reactions are catalysed by the same enzyme. Male minipigs tend to have higher average activity than female minipigs, which is in contrast to the gender-dependent expression seen for other CYP isoenzymes. The metabolic activities correlated with the protein level determined in Western blotting, using anti-human CYP2C9, indicating that this enzyme is responsible for the reaction. The expression of the CYP2C enzymes was analysed by real-time polymerase chain reaction, using a primer set that could amplify CYP2C8, CYP2C9 and CYP2C19. The melting curves (peaks) revealed that all three genes were present, showing very different expression levels in the various types of pigs. The area of one of the peaks, however, correlated with the CYP2C9-like enzyme concentration, suggesting that this peak represents CYP2C9. Among paclitaxel, tolbutamide and omeprazole, omeprazole is the best probe of CYP2C9-like enzyme in the pig.     

The engagement of brain cytochrome P450 in the metabolism of endogenous neuroactive substrates: a possible role in mental disorders

Abstract

The current state of knowledge indicates that the cerebral cytochrome P450 (CYP) plays an important role in the endogenous metabolism in the brain. Different CYP isoenzymes mediate metabolism of many endogenous substrates such as monoaminergic neurotransmitters, neurosteroids, cholesterol, vitamins and arachidonic acid. Therefore, these enzymes may affect brain development, susceptibility to mental and neurodegenerative diseases and may contribute to their pathophysiology. In addition, they can modify the therapeutic effects of psychoactive drugs at the place of their target action in the brain, where the drugs can act by affecting the metabolism of endogenous substrates. The article focuses on the role of cerebral CYP isoforms in the metabolism of neurotransmitters, neurosteroids, and cholesterol, and their possible involvement in animal behavior, as well as in stress, depression, schizophrenia, cognitive processes, learning, and memory. CYP-mediated alternative pathways of dopamine and serotonin synthesis may have a significant role in the local production of these neurotransmitters in the brain regions where the disturbances of these neurotransmitter systems are observed in depression and schizophrenia. The local alternative synthesis of neurotransmitters may be of great importance in the brain, since dopamine and serotonin do not pass the blood–brain barrier and cannot be supplied from the periphery. In vitro studies indicate that human CYP2D6 catalyzing dopamine and serotonin synthesis is more efficient in these reactions than the rat CYP2D isoforms. It suggests that these alternative pathways may have much greater significance in the human brain but confirmation of these assumptions requires further studies.     

Designing selective COX-2 inhibitors: molecular modeling approaches

Molecular modeling approaches have been successfully used to gain insight into the molecular mechanism of selective cyclooxygenase-2 (COX-2) inhibition. These approaches are based on X-ray structure data and results from structure-activity relationships and site-directed mutagenesis experiments. The recognition process of substrates and inhibitors by COX isoenzymes can be visualized by applying algorithms to describe local properties on the enzyme surface, thus allowing key differences in structure to be studied that may confer differential sensitivity to inhibitors. The virtual screening techniques for COX isoenzymes are rapidly improving, and the search for new selective COX-2 inhibitors has been stimulated by the tremendous success of meloxicam, celecoxib and rofecoxib in the pharmaceutical market and their potential use in new indications.     

新型抗抑郁药的代谢与细胞色素P450介导的药物相互作用

新型抗抑郁药(选择性5-羟色胺再摄取抑制剂,SSRI)疗效肯定,耐受性好,已得到广泛应用。SSRI剂既是P 450酶的底物又是P 450酶的抑制剂,其重要特征是对P 450酶的抑制。当SSRI剂与P 450酶的其他底物合用时,可能发生明显的具有临床意义的药物相互作用。     

How and why to screen for CYP2D6 interindividual variability in patients under pharmacological treatments

Cytochromes P450 are members of a superfamily of hemoproteins that catalyze a variety of oxidative reactions in the metabolism of endogenous and exogenous hydrophobic substrates. Fifty-eight cytochrome P450 (CYP) isoenzymes belonging to 18 families have been identified in human cells; the corresponding genes are highly polymorphic, and genetic variability underlies interindividual differences in drug response. The polymorphisms of CYP2D6 significantly affect the pharmacokinetics of about 50% of the drugs in clinical use, which are CYP2D6 substrates. The number of functional CYP2D6 alleles per genome determines the existence of four different phenotypes, i.e. poor, intermediate, extensive, and ultrarapid metabolizers. CYP2D6 genetic variants include copy number variations, single nucleotide substitutions, frameshift and insertion/deletion mutations. This review reports some of the different methodological approaches used to screen for CYP2D6 variants and focuses on methods that have improved variation detection, from conventional techniques to more recent microarray technology and high throughput DNA sequencing. In addition, this review reports some results on clinical relevance of CYP2D6 polymorphisms and provides examples of variability in drug response associated with interindividual phenotypic differences.     

Structure and function of mammalian aldehyde oxidases

Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX.     

Cytochrome P450 and liver diseases

Cytochrome P-450 (CYPs) are involved in the metabolism of drugs, chemicals and endogenous substrates. The hepatic CYPs are also involved in the pathogenesis of several liver diseases. CYP-mediated activation of drugs to toxic metabolites induces hepatotoxicity. Well-known examples include acetaminophen and halothane. In some instances, covalent binding of the toxic metabolite to CYP leads to the formation of anti-CYP antibodies and immune-mediated hepatotoxicity (hydralazine, tienilic acid). Anti-CYP2D6 antibodies are also present in the serum of patients with type II autoimmune hepatitis, but the mechanism leading to their presence and their pathogenic significance remains unclear. Several studies support a role for CYP2E1 in the pathogenesis of alcoholic liver disease and non-alcoholic steatohepatitis. In these conditions, enhanced CYP2E1 activity is associated with lipid peroxidation and the production of reactive oxygen species with secondary damage to cellular membranes and mitochondria. Because of its ability to activate carcinogens, a role for CYP2E1 as a cofactor for hepatocellular carcinoma has also been postulated. On the other hand, drug metabolism is impaired in patients with liver disease, particularly that mediated by CYPs. The content and activity of CYP1A, 2C19 and 3A appear to be particularly vulnerable to the effect of liver disease while CYP2D6, 2C9 and 2E1 are less affected. The pattern of CYPs isoenzymes alterations also differs according to the etiology of liver disease. A strong relationship between the activity of CYPs and the severity of cirrhosis has been demonstrated, but the usefulness of measuring CYP activity to assess hepatic functional reserve remains uncertain.     

Features of the metabolism of various drugs involving cytochrome P-450 isoenzymes

Substrates of cytochrome P450 isoenzymes have been analyzed and systematized, and a database is created in which the substrates are classified in accordance with the chemical structure of drugs. Each substrate is characterized by the oxidation reaction and the resulting metabolites, with allowance for the Michaelis - Menten pharmacokinetic parameters. Each isoenzyme metabolises certain preferred substrates, depending on the presence of active structural groups or moieties susceptible to oxidation and on the stereoselectivity of drugs. The reaction direction also depends on the specific interactions between the active groups of a substrate and the given isoenzyme.     

Machine learning approaches for predicting compounds that interact with therapeutic and ADMET related proteins

Computational methods for predicting compounds of specific pharmacodynamic and ADMET (absorption, distribution, metabolism, excretion and toxicity) property are useful for facilitating drug discovery and evaluation. Recently, machine learning methods such as neural networks and support vector machines have been explored for predicting inhibitors, antagonists, blockers, agonists, activators and substrates of proteins related to specific therapeutic and ADMET property. These methods are particularly useful for compounds of diverse structures to complement QSAR methods, and for cases of unavailable receptor 3D structure to complement structure-based methods. A number of studies have demonstrated the potential of these methods for predicting such compounds as substrates of P-glycoprotein and cytochrome P450 CYP isoenzymes, inhibitors of protein kinases and CYP isoenzymes, and agonists of serotonin receptor and estrogen receptor. This article is intended to review the strategies, current progresses and underlying difficulties in using machine learning methods for predicting these protein binders and as potential virtual screening tools. Algorithms for proper representation of the structural and physicochemical properties of compounds are also evaluated.     

Speculations on the substrate structure-activity relationship (SSAR) of cytochrome P450 enzymes.

This brief review attempts to define the SSAR of two families of cytochrome P450. With P4502D catalytic competence is achieved by tight ionic binding which gives the enzyme high regioselectivity. In contrast P4503A achieves catalytic competence by a flexible binding site relying on hydrophobic forces that allow chemically vulnerable sites to be the principal sites of metabolism. In general, the different binding mechanism should be reflected in the enzyme, such that substrates of P4502D should have lower Km values than substrates of P4503A. Thus, routes of metabolism catalysed by P4502D may be saturated at substrate concentrations lower than routes catalysed by P4503A. The apparent differences between P4502D and P4503A in terms of substrate specificity bring into question what relationships govern other families of cytochrome P450. Our analysis of data suggests that the other principal form involved, generally, in the metabolism of pharmaceuticals in humans is P4502C9 (possibly 2C8 and 2C10). The enzyme is responsible for the metabolism of phenytoin, tolbutamide, tienilic acid [4], naproxen, ibuprofen, diclofenac [38], the 7-hydroxylation of S-warfarin [39] and the 7-hydroxylation of delta 1-tetrahydrocannabinol [40]. These compounds all have areas of strong hydrogen bond [4] forming potential (Fig. 8), all distanced 5-10A from the site of metabolism. Moreover the carboxylic acid function of naproxen, ibuprofen and diclofenac (pKa 4.5) and the sulfonylurea of tolbutamide (pKa 5.4) render the compounds ionized at physiological pH. The ionised group is positioned 7-11A from the site of metabolism. It is likely, therefore, that hydrogen bonding and possibly ion-pair interactions play a major role in determining the SSAR of the P4502C isoenzymes. These interactions would suggest that the P4502C enzymes are analogous to P4502D rather than P4503A. In this regard it is noteworthy that P4502C9 is selectively and potently inhibited by sulfaphenazole (IC50 of 0.6 microM), a compound that is structurally related (Fig. 8) to the substrates in terms of potential hydrogen bonding regions [4, 41]. Simplistically we suggest that the SSAR of the various P450 enzymes ranges from the highly selective enzymes dealing with endogenous substrates, through the enzymes metabolising exogenous substrates with narrow substrate structure requirements such as P4502D to P4503A with its broad substrate structure range. It would seem logical that animals and humans would evolve such combinations of isoenzymes to deal with the vast array of exogenous xenobiotics.     

Studies of rat alkaline phosphatase

A manual system of various estimations of rat plasma alkaline phosphatase activity has been devised for small volumes of plasma which uses different inhibitors, compares the utilisation of two substrates and includes acrylamide gel electrophoresis. The different inhibitors etc. allow a degree of discrimination between alkaline phosphatase extracts of rat organs. The properties of isoenzymes, e.g. intestinal phosphatase, differ depending upon the environment in which they are studied. In conjunction, if necessary, with the methods described for the estimation of liver and intestinal alkaline phosphatase activity, it is hoped to use the system to discriminate between the isoenzymes present in the plasma alkaline phosphatase of rats in toxicological studies.     

Phosphorylation of cytochrome P450 isoenzymes in intact hepatocytes and its importance for their function in metabolic processes

Recent data show that besides the well-known long-term regulation of cytochrome P450-dependent monooxygenase activity by induction there also exists a fast regulation by phosphorylation. This phosphorylation occurs when purified cytochromes P450 are combined with purified protein kinases, and also in intact cells. This process is donor- and acceptor-selective leading to phosphorylation of defined isoenzymes by defined protein kinases. This in turn leads to fast and marked changes in metabolism which are selective for given substrates and regio- and stereo-selective for given positions. This in turn is selectively and differentially influenced by the individual control of the protein kinase in question.To facilitate it for the reader to follow the relatively complicated cytochrome P450 nomenclature, the individual isoenzymes are (as far as possible) designated by the recommended nomenclature for their (probable) genes (Nebert et al. 1987) followed in brackets by a relatively simple and relatively widely used one letter nomenclature (Bandiera et al. 1985) and — at there first occurrence in paper — very briefly characterized.     

Metabolic interactions with piperazine‐based ‘party pill’ drugs

Objectives ‘Party pills’ have found use worldwide as a substitute for amphetamine‐derived designer drugs. Whilst some information exists about the metabolism of these drugs, there is little information about their ability to inhibit the metabolism of co‐administered drugs. This study aimed to determine whether predictions can be made about global interactions between ‘party pills’ constituents and other drugs metabolised by the same cytochrome P450 (CYP) isoenzymes. Methods The inhibitory effects of seven benzyl and phenyl piperazines were measured in microsomal incubation assays of probe substrates for five major CYP isoenzymes. In addition, the metabolism of benzylpiperazine and trifluoromethylphenylpiperazine, the two most commonly used constituents of ‘party pills’, was investigated using human liver microsomes assays and known inhibitors of CYP isoenzymes. Key findings All piperazine analogues tested showed significant inhibitory activity against most, if not all, isoenzymes tested. The metabolism of benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP) involved CYP2D6, CYP1A2 and CYP3A4. Furthermore, BZP and TFMPP inhibited each other's metabolism. Conclusions Fluorophenylpiperazine, methoxyphenylpiperazine, chlorophenylpiperazine, methylbenzylpiperazine and methylenedioxybenzylpiperazine had significant inhibitory effects on CYP2D6, CYP1A2, CYP3A4, CYP2C19 and CYP2C9 isoenzymes but each piperazine had a different inhibitory profile. The metabolic interaction between BZP and TFMPP may have clinical implications, as these agents are often combined in ‘party pills’.     

Application of support vector machines to in silico prediction of cytochrome p450 enzyme substrates and inhibitors

Cytochrome P450 enzymes are responsible for phase I metabolism of the majority of drugs and xenobiotics. Identification of the substrates and inhibitors of these enzymes is important for the analysis of drug metabolism, prediction of drug-drug interactions and drug toxicity, and the design of drugs that modulate cytochrome P450 mediated metabolism. The substrates and inhibitors of these enzymes are structurally diverse. It is thus desirable to explore methods capable of predicting compounds of diverse structures without over-fitting. Support vector machine is an attractive method with these qualities, which has been employed for predicting the substrates and inhibitors of several cytochrome P450 isoenzymes as well as compounds of various other pharmacodynamic, pharmacokinetic, and toxicological properties. This article introduces the methodology, evaluates the performance, and discusses the underlying difficulties and future prospects of the application of support vector machines to in silico prediction of cytochrome P450 substrates and inhibitors.     

Cell Systems for Use in Studies on the Relationship Between Foreign Compound Metabolism and Toxicity

Abstract: Since the metabolism of most foreign compounds is predominantly controlled by hepatic in metabolism, isolated hepatocytes in most cases quite well predict the pattern of the overall metabolism of a given compound. Methods have been developed for cryopreserving isolated hepatocytes from man and other species with satisfactory maintenance of foreign compound metabolizing enzyme activities. The installation of a bank of cryopreserved hepatocytes from different species is possible and may be used for rational species extrapolation. It is necessary for some toxicological investigations to have hepatocytes which retain their differentiated status in culture for a sufficient time period. This might be achieved by co-culturing hepatocytes with diverse cell lines. However, from one cell line to the other differences in the pattern of stabilization of individual hepatocyte functions are found. In addition, questions on metabolic action of individual isoenzymes can also be addressed by the use of genetically engineered cell lines. All the in vitro systems mentioned, especially those which contain differentiated human cells or human isoenzymes are helpful in the rational species extrapolation of toxic effects from animal to man.     

CYP3A activity: towards dose adaptation to the individual

Introduction: Co-medication, gene polymorphisms and co-morbidity are main causes for high variability in expression and function of the CYP3A isoenzymes. Pharmacokinetic variability is a major source of interindividual variability of drug effect and response of CYP3A substrates. While CYP3A genotyping is of limited use, direct testing of enzyme function (‘phenotyping’) may be more promising to achieve individualized dosing of CYP3A substrates.

Areas covered: We will discuss available phenotyping strategies for CYP3A isoenzymes and causes of intra- and interindividual variability of CYP3A. The impact of phenotyping on the dose selection and pharmacokinetics of CYP3A substrates (docetaxel, irinotecan, tyrosine kinase inhibitors, ciclosporin, tacrolimus) are reviewed. Pubmed searches were conducted during March–November 2015 to retrieve articles related to CYP3A enzyme, phenotyping, drug interactions with CYP3A probe substrates, and phenotyping-guided dosing algorithms.

Expert opinion: While ample data is available on the choice appropriate phenotyping drugs (midazolam, alfentanil, aplrazolam, buspirone, triazolam), less clinical trial data is available concerning strategies to usefully guide dosing in the clinical practice. Implementation into the clinical routine necessitates further research to identify (1) an easy-to-use and cheap test for CYP3A activity that (2) adequately predicts drug exposure to (3) allow a sound decision on dose adaptation and hence (4) improve clinical outcome and/or reduce the intensity or frequency of adverse drug effects.