Francesca Toselli

Echinacea metabolism and drug interactions: The case for standardization of a complementary medicine

The herbal medicine, Echinacea, is used for treatment and prevention of upper respiratory tract infections. Among the phytochemicals found in Echinacea, the bioavailable alkylamides are thought to be the compounds responsible for its effects on the human immune system. Cytochrome P450 enzymes (P450s) appear to be the principal system responsible for the metabolism of Echinacea components and most of the main hepatic and some extrahepatic isoforms appear to be involved. Epoxide formation, N-dealkylation and hydroxylation are the main metabolic pathways mediated by P450s. Interactions with P450s determine the circulating concentrations and duration of action of these phytochemicals as well as any potential interactions with other chemicals. Most research to date has focused on the potential of Echinacea to interact with other drugs. Literature reports are equivocal and comparisons between studies are difficult as the phytochemical composition of the preparations examined is rarely assessed. Certain alkylamides containing a terminal acetylene appear to exert a time- and NADPH-dependent inhibition on the metabolism of other compounds. However as there are no industry standardization requirements, differences in the relative concentrations of individual alkylamides between preparations could alter the potential for interactions. A thorough phytochemical analysis of samples investigated is necessary in further studies so that sound conclusions can be drawn regarding the potential for inter-individual variation in pharmacokinetics and therapeutic effects and interactions with other chemicals. Moreover standardization of alkylamide content may allow the exploitation of beneficial interactions between alkylamide components to enhance the therapeutic effect of this widely used complementary medicine.

Differential Expression of Cytochrome P450 Enzymes from the CYP2C Subfamily in the Human Brain

Cytochrome P450 enzymes from the CYP2C subfamily play a prominent role in the metabolic clearance of many drugs. CYP2C enzymes have also been implicated in the metabolism of arachidonic acid to vasoactive epoxyeicosatrienoic acids. CYP2C8, CYP2C9, and CYP2C19 are expressed in the adult liver at significant levels; however, the expression of CYP2C enzymes in extrahepatic tissues such as the brain is less well characterized. Form-specific antibodies to CYP2C9 and CYP2C19 were prepared by affinity purification of antibodies raised to unique peptides. CYP2C9 and CYP2C19 were located in microsomal fractions of all five human brain regions examined, namely the frontal cortex, hippocampus, basal ganglia, amygdala, and cerebellum. Both CYP2C9 and CYP2C19 were detected predominantly within the neuronal soma but with expression extending down axons and dendrites in certain regions. Finally, a comparison of cortex samples from alcoholics and age-matched controls suggested that CYP2C9 expression was increased in alcoholics.

Differential Expression of Human Cytochrome P450 Enzymes from the CYP3A Subfamily in the Brains of Alcoholic Subjects and Drug-Free Controls

Cytochrome P450 enzymes are responsible for the metabolism of most commonly used drugs. Among these enzymes, CYP3A forms mediate the clearance of around 40–50% of drugs and may also play roles in the biotransformation of endogenous compounds. CYP3A forms are expressed both in the liver and extrahepatically. However, little is known about the expression of CYP3A proteins in specific regions of the human brain. In this study, form-selective antibodies raised to CYP3A4 and CYP3A5 were used to characterize the expression of these forms in the human brain. Both CYP3A4 and CYP3A5 immunoreactivity were found to varying extents in the microsomal fractions of cortex, hippocampus, basal ganglia, amygdala, and cerebellum. However, only CYP3A4 expression was observed in the mitochondrial fractions of these brain regions. N-terminal sequencing confirmed the principal antigen detected by the anti-CYP3A4 antibody in cortical microsomes to be CYP3A4. Immunohistochemical analysis revealed that CYP3A4 and CYP3A5 expression was primarily localized in the soma and axonal hillock of neurons and varied according to cell type and cell layer within brain regions. Finally, analysis of the frontal cortex of chronic alcohol abusers revealed elevated expression of CYP3A4 in microsomal but not mitochondrial fractions; CYP3A5 expression was unchanged. The site-specific expression of CYP3A4 and CYP3A5 in the human brain may have implications for the role of these enzymes in both normal brain physiology and the response to drugs.

Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N‐isobutyldodeca‐2E,4E,8Z,10Z‐tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N‐dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N‐dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic‐metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified. Copyright

Emerging roles for brain drug-metabolizing cytochrome P450 enzymes in neuropsychiatric conditions and responses to drugs

Abstract P450s in the human brain were originally considered unlikely to contribute significantly to the clearance of drugs and other xenobiotic chemicals, since their overall expression was a small fraction of that found in the liver. However, it is now recognized that P450s play substantial roles in the metabolism of both exogenous and endogenous chemicals in the brain, but in a highly cell type- and region-specific manner, in line with the greater functional heterogeneity of the brain compared to the liver. Studies of brain P450 expression and the characterization of the catalytic activity of specific forms expressed as recombinant enzymes have suggested possible roles for xenobiotic-metabolizing P450s in the brain. It is now possible to confirm these roles through the use of intracerebroventricular administration of selective P450 inhibitors in animal models, coupled with brain sampling techniques to measure drug concentrations in vivo, and modern neuroimaging techniques. The purpose of this review is to discuss the evidence behind the functional importance of P450s from the “xenobiotic-metabolizing” families, CYP1, CYP2 and CYP3 in the brain. Approaches used to define the quantitative and qualitative significance of these P450s in determining tissue-specific levels of xenobiotics in brain will be considered. Finally, the possible roles of these enzymes in brain biochemistry will be examined in light of the demonstrated activity of these enzymes in vitro and the association of particular P450 forms with disease states.

Gene expression profiling of cytochromes P450, ABC transporters and their principal transcription factors in the amygdala and prefrontal cortex of alcoholics, smokers and drug-free controls by qRT-PCR

Abstract 1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.

Oxetane Substrates of Human Microsomal Epoxide Hydrolase

Oxetanyl building blocks are increasingly used in drug discovery because of the improved drug-like properties they confer on drug candidates, yet little is currently known about their biotransformation. A series of oxetane-containing analogs was studied and we provide the first direct evidence of oxetane hydrolysis by human recombinant microsomal epoxide hydrolase (mEH). Incubations with human liver fractions and hepatocytes were performed with and without inhibitors of cytochrome P450 (P450), mEH and soluble epoxide hydrolase (sEH). Reaction dependence on NADPH was investigated in subcellular fractions. A full kinetic characterization of oxetane hydrolysis is presented, in both human liver microsomes and human recombinant mEH. In human liver fractions and hepatocytes, hydrolysis by mEH was the only oxetane ring-opening metabolic route, with no contribution from sEH or from cytochrome P450-catalyzed oxidation. Minimally altering the structural elements in the immediate vicinity of the oxetane can greatly modulate the efficiency of hydrolytic ring cleavage. In particular, higher pKa in the vicinity of the oxetane and an increased distance between the oxetane ring and the benzylic nitrogen improve reaction rate, which is further enhanced by the presence of methyl groups near or on the oxetane. This work defines oxetanes as the first nonepoxide class of substrates for human mEH, which was previously known to catalyze the hydrolytic ring opening of electrophilic and potentially toxic epoxide-containing drugs, drug metabolites, and exogenous organochemicals. These findings will be of value for the development of biologically active oxetanes and may be exploited for the biocatalytic generation of enantiomerically pure oxetanes and diols.