The mystery of CYP26

Within the University of Washington School of Pharmacy, Associate Professor Nina Isoherranen’s research focuses upon the biochemical characterisation of the CYP26 and their role in retinoic acid metabolism

Firstly, what are the central aims of the The CYP26 family includes three enzymes, Have you adopted any specialist techniques project? What do you hope to discover from CYP26A1, CYP26B1 and CYP26C1 that are to complete the research? this particular piece of research? very highly conserved in vertebrates (such as xenopus, as well as zebrafish, chicken, mice We have developed a recombinant system to The central aim of the project is to establish and humans). Despite large differences in express the CYP26 enzymes. This process is the biological role of the CYP26 enzymes the sequence between the CYP26 necessary because it enables us to undertake during childhood and adult life and to enzymes, all three enzymes appear to biochemical and functional studies of these determine whether modulation of the activity metabolise retinoic acid, the active metabolite enzymes. We also use very sophisticated mass of these enzymes, via inhibition or induction of vitamin A. spectrometry approaches, in order to identify by xenobiotics, impacts human health. Our and detect atRA as well as the metabolic studies should also establish the potential What do you hypothesise is the products formed by CYP26 enzymes. In value of individual CYP26 enzymes as drug mechanism of all-trans retinoic acid addition, our research has required synthetic ASSOCIATE PROFESSOR NINA ISOHERRANEN PROFESSOR ASSOCIATE targets in specific tissues. We aim to discover (atRA) depletion? In what way have you chemistry to generate the metabolites of atRA the role that the major enzymes in the CYP26 utilised transgenic mice studies to further and selective inhibitors of CYP26 enzymes. The family play in regulating retinoid action understand this process? major gain in improving our understanding has and pharmacology, within specific tissues. been to put all of these techniques together We believe that the main mechanism of atRA with other collaborative efforts to better depletion is via clearance by the CYP26 understand the function. enzymes to hydroxylated products. However, some of these products Are there any further applications of may have actions of their own this research? that remain to be discovered. Unfortunately the knock-out Retinoids are very effective in treating skin of CYP26A1 and CYP26B1 in diseases, particularly psoriasis and acne. It is embryonic mice is lethal and, likely that the inhibition of CYP26 enzymes in as a result, we have not been the skin by novel compounds could improve able to use the knock-out mice therapy for topical treatments. Additionally, for studying the importance retinoids have been found to have potential of CYP26 enzymes after birth. utility in treatment of a multitude of diseases However, we hope that in the such as cancers and metabolic disease. Retinoids future we will be able to have also been suggested to hold promise in use conditional knock- treatment of Alzheimer’s disease and be useful in out mice for our immune system modulation. We hope that our studies. studies on the basic function of CYP26 enzymes

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and potential development of a selective CYP26 inhibitor could allow better understanding and development of therapy for these diseases.

What would you highlight as the most challenging aspects of your research?

The CYP26 enzymes are very challenging to work with. They do not express very well in recombinant systems and obtaining Retinoids: a potential good quality protein to work with is difficult. Since retinoic acid is light sensitive, all our work has to be conducted under red-yellow disease regulator lights. This can be quite difficult for lab experiments that require a lot of attention to detail and careful observations. The retinoid system overall is very complex and regulated A team from the University of Washington, USA is focusing its at multiple levels. It is very challenging to attention upon P450 enzymes and their role in the elimination of isolate the role of a single protein in the overall retinoic acid in the human body. Their groundbreaking research regulation of homeostasis as there are many protein-protein interactions and feedback has the potential to lead to improvements in the treatment of a mechanisms that can influencein vivo findings number of common diseases, from eczema to Alzheimer’s and interpretation.

In the future, where do you see this field and VITAMIN A IS important for growth and from the University of Washington, led by your work progressing? development. It can be obtained through Associate Professor Nina Isoherranen, is trying foods rich in retinyl esters – the major form of to understand whether certain substances An interesting direction of our research is the vitamin A in animal-based foods – such as liver, impair normal retinoic acid homeostasis and identification of individuals (humans) with meats, milk and eggs, and non-animal based vitamin A intake, and hence cause adverse genetic mutations or deletions of the CYP26 foods rich in β-carotene – another form of effects. Their research will provide a much . We do not yet know how the CYP26 vitamin A – including carrots and other yellow- needed insight into the workings of this vital enzymes relate to disease progression. orange vegetables. Both forms of vitamin A substance in the human body. An extension of However, as we gain better understanding of are metabolised in the body to create a very their studies is to evaluate if specific increase in their function in specific tissues and during important metabolite, known as retinoic acid. retinoic acid concentrations in select tissues can different stages of life, we hope to better provide significant therapeutic benefits. understand what effect mutations in these The reason for retinoic acid’s importance, and genes have on individuals’ health. We also the scientific interest surrounding it, is its role P450 ENZYMES expect that a more in-depth understanding within the human body – it is essential for a of the CYP26 genes and their interactions variety of normal biological functions. The The overall objective of the team’s research with xenobiotics will allow better better known effects of retinoic acid relate project is to define the importance of CYP26A1 evaluation of adverse effects of drugs and to its role in supporting growth in numerous and CYP26B1, both of which are cytochrome environmental toxicology. animal species and modulating organogenesis P450 enzymes. enzymes are and differentiation during foetal development. heme proteins that generally carry out oxidative Furthermore, retinoic acid plays a role in reactions, and are involved in the elimination of neuronal differentiation, has been implicated in many drugs. P450 enzymes are also responsible mediating homeostasis and is also required for generating many of the , as well as for the maintenance of immunity. Indeed, the modulating signalling by lipid soluble vitamins. World Health Organization estimates that over The team’s specific angle is to establish the 125 million children are vitamin A deficient and role of CYP26A1 and CYP26B1 in maintaining millions of deaths could be prevented annually cellular and circulating RA homeostasis with proper vitamin A nutriture. Due to its during childhood and adult life. Furthermore, effects in immune cells, retinoic acid is also used they hope to determine how xenobiotic- in HIV vaccine development and is an approved CYP26 interactions alter retinoic acid isomer drug for the treatment of acute promyelocytic concentrations in specific target tissues and in leukaemia and Kaopsis sarcoma, as well as a serum. form of treatment for neuroblastoma and acne. To put this in context, CYP26 enzymes Despite the multitude of benefits that retinoic were discovered through genomic screening acid boasts, an excess of retinoic acid can lead, (microarrays) of retinoic acid induced in addition to teratogenicity, to bone pain, proteins in regenerating zebrafish fins. It is liver toxicity and various gastrointestinal (GI) only subsequently that genetic screening has effects. For this reason, by understanding how identified the human homologs of the same to control concentrations in target tissues, enzymes. Yet in spite of this discovery, the role therapeutic advances can be made to treat of hepatic and extrahepatic CYP26 enzymes these conditions. Therefore, a team of scientists is not well established. What the team has

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ROLE OF CYP26A1 AND CYP26B1 IN MAINTAINING CELLULAR AND CIRCULATING RA HOMEOSTASIS demonstrated so far, is that CYP26A1 is the As well as finding the answers to their specific main liver CYP26 enzyme and is responsible for questions, the result of the studies could also OBJECTIVES retinoic acid clearance in the liver. Additionally, provide important information of CYP26 The overall objective of the studies is the project members think it likely that both enzymes in terms of drug targets and regulators to determine the role of CYP26A1 and CYP26A1 and CYP26B1 play important roles of disease progression. Such information CYP26B1 in regulating all-trans-retinoic in regulating retinoic acid concentrations could prove groundbreaking in treatment of acid levels in vivo, with the goal of outside the liver. In this vein, they have any diseases that are believed to be related to understanding how modification of enzyme detected the expression of both enzymes in altered AtRA concentrations and homeostasis function (induction, inhibition or genetic many extrahepatic tissues and some clinical and can be treated with retinoids. These could polymorphisms) might affect retinoic acid studies have shown that inhibition of CYP26 include psoriasis, acne, eczema and certain homeostasis and an individual’s health. enzymes results in increased retinoic acid types of cancer as well as Alzheimer’s disease, Research in the lab includes experiments concentrations in serum. dementia and obesity. As part of their research the group, in collaboration with medicinal that characterise the basic biochemistry chemistry group from University of Montana of the CYP26 enzymes as well as the TESTING HYPOTHESES led by Dr Philippe Diaz, is also investigating the regulation of their expression and their All-trans retinoic acid (atRA), the biologically therapeutic potential of novel compounds that pharmacogenetics. active form of vitamin A, is essential for diverse specifically inhibit CYP26 enzymes and may KEY COLLABORATORS biological functions and while the positive increase retinoic acid concentrations in specific effects of atRA are well documented, the fact tissues such as the brain during conditions that Dr John Amory, School of Medicine, that they are concentration dependent is are related to retinoid deficiency. University of Washington • Dr Philippe slightly less well understood. It is known that Diaz, University of Montana • Dr Wendel compounds which inhibit atRA metabolism MEDICAL PROGRESS Nelson, Department of Medicinal can provide great therapeutic benefits in Chemistry, University of Washington • specific diseases by increasing cellular and Retinoids have already shown promise in DermaXon circulating atRA concentrations, but if AtRA treating some cancers and are approved therapy concentrations are increased too high the for neuroblastoma, acute promyelocytic FUNDING inhibition may cause multiple detrimental leukaemia (APL) and Kaposi’s sarcoma. National Institutes of Health Medicine effects on individual’s health. However the Resistance to retinoid therapy in APL patients Institute of General Medicine grant no. reasons surrounding this are not well founded. is generally believed to be due to CYP26A1 R01GM081569 and T32GM07750 induction. Resistance to retinoids in other While substantial progress has been made cancers has also been suggested to be a result CONTACT in our understanding of retinoid metabolism of increased metabolism of retinoic acid in Nina Isoherranen and function, the mechanisms that control cancer cells and/or in the liver. Therefore, better Principal Investigator circulating and tissue atRA concentrations in understanding of the structure-function of humans are still uncertain. This is precisely the CYP26 enzymes is vital, as it could allow University of Washington the knowledge-gap that the Washington development of compounds that can be given School of Pharmacy team seeks to fill. In particular, there is very together with retinoic acid to improve patient PO Box 357631 limited knowledge on how xenobiotics affect outcomes and combat resistance. Seattle, WA 98195, USA AtRA homeostasis and whether xenobiotic- AtRA interactions cause toxicity or can be There also seems to be a link between late T +1 206 543 2517 used for drug therapy. With a desire to learn onset Alzheimer’s disease and defective retinoid E [email protected] more about these phenomena, the team has signalling. To date, clinical studies have found sop.washington.edu/pharmaceutics been working to test their hypotheses that that treatment with retinoic acid improves hepatic and extrahepatic CYP26 enzymes memory and cognitive function. Retinoic sop.washington.edu/pharmaceutics/ are responsible for AtRA clearance and that acid is also known to stimulate neuronal isoherranen-lab/isoherranen-lab-home.html the selective inhibition of CYP26A1 and/or differentiation and regulate immune responses CYP26B1 will increase circulating and tissue that potentially play a role in Alzheimer’s. NINA ISOHERRANEN, PhD, is Associate AtRA concentrations resulting in fine-tuning Hence, increasing retinoic acid levels in the Professor of Pharmaceutics within of atRA concentrations in tissue specific brain may be beneficial in its treatment. This the School of Pharmacy, University of manner. The specific aims of the research are could be obtained either via treatment with a Washington. She earned her BS and MS all designed to establish a comprehensive synthetic retinoid or with inhibiting retinoic from the University of Helsinki before understanding of tissue specific clearance of acid clearance that would increase endogenous pursuing PhD studies at the Hebrew atRA, its contribution to atRA homeostasis concentrations of retinoic acid. The team in University of Jerusalem, Israel. Isoherranen and its susceptibility to inhibition by drugs and Washington hopes to better establish the has published nearly 60 research papers other xenobiotics. In order to draw conclusions role of CYP26 enzymes in regulating retinoic in peer-reviewed journals and four book on this, the researchers will characterise the acid concentrations in the brain, so to further chapters. She is also a member of several importance of the CYP26 enzymes in clearance elucidate upon the potential for medical professional societies and journal editorial of RA isomers and metabolites. improvements. boards and has two US patents regarding new antiepileptic drugs.

Clinical studies have found that treatment with retinoic acid improves memory and cognitive function

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