The Calcitriol Study A Randomised Placebo Controlled Clinical Trial to Test the Effects of Calcitriol in Steroid Resistant Asthma A Thesis presented by Alexandra M Nanzer registered at Centre for Primary Care and Public Health, Blizard Institute Barts and The London School of Medicine and Dentistry London E1 2AT and MRC & Asthma UK Centre in Allergic Mechanisms of Asthma King’s College London SE1 9RT for the degree of Doctor of Philosophy 2015 I, Alexandra M Nanzer, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. For Philip 3 Abstract Background Over five million people in the UK are receiving treatment for asthma. Corticosteroids (steroids) are the mainstay of asthma therapy but some patients do not respond fully to steroid treatment, they are characterised as being steroid resistant (SR) and are at high risk of morbidity and mortality. Earlier data from our laboratory has shown evidence that in vitro treatment with the active form of vitamin D, - 1!,25-dihydroxyvitamin D3 or calcitriol - enhanced responsiveness to steroids for induction of the anti inflammatory cytokine IL-10. This thesis discusses the results of a proof of concept clinic trial - ‘The Calcitriol Study’ - which hypothesized that concomitant in vivo treatment with oral calcitriol improves the clinical responsiveness to systemic steroid (prednisolone) therapy. The study further allowed investigation of the in vitro cytokine profile of patients with SR and steroid sensitive (SS) asthma. Th17 cells and their hallmark cytokine IL-17A are proposed to play a role in the pathology of severe asthma, including SR asthma, and this work tested the susceptibility of IL-17A and other pro inflammatory cytokines important in asthma to inhibition by steroids and calcitriol in vivo and in culture. Methods Adult patients with moderate/severe asthma (FEV1 <80% predicted) with demonstrable reversibility of airways obstruction underwent a two-week, pharmacodynamically standardised course of oral prednisolone (screening phase) to delineate steroid resistance a <10% improvement in FEV1. Patients were then randomly assigned to receive calcitriol (n=12) or indistinguishable placebo (n=11) for four weeks, with a repeat course of prednisolone during the final two weeks (treatment phase). Changes in lung function (!FEV1) in response to prednisolone were compared between the placebo and calcitriol groups in the treatment phase, and within groups between the screening and treatment phases. Asthma Control Questionnaires and fractional exhaled nitric oxide (FeNo) were scored and analysed as secondary endpoints. All participants had serum 25(OH)D levels measured at baseline. CD8-depleted PBMCs were isolated from SS and SR asthmatics and healthy controls and cultured with or without dexamethasone and / or calcitriol. Cytometric bead array, ELISA, qPCR and intracellular cytokine staining were used to assess cytokine production. Results Treatment with calcitriol improved the clinical response to steroids in patients classified as clinically steroid resistant (SR) in a within group comparison of changes in FEV1. However, there was no significant difference seen between the two groups from screening to the end of the trial. A striking dichotomy was observed between SR and SS asthma patients in terms of their cytokine profiles; SS patients, who showed the biggest improvement in lung function after a course of prednisolone had the highest levels of IL-10 in culture in response to dexamethasone, whereas SR patients, whose lung function failed to improve, had significantly greater levels of IL-17A. Treatment with steroids appeared to aggravate production of this pro-inflammatory cytokine but in vitro and in vivo calcitriol not only resulted in a significant reduction of IL-17A levels but also restored the impaired, steroid-induced anti-inflammatory IL-10 response in SR patients. Serum 25(OH)D levels at baseline correlated positively with IL-13 in culture, a Th2 cytokine known to be associated with steroid responsive asthma. Conclusion Calcitriol may have the potential to improve the clinical responsiveness of asthma patients to systemic steroid therapy in SR asthma. These data identify immunological pathways that likely underpin the beneficial clinical effects of calcitriol in SR asthma, by directing the SR cytokine profile towards a more SS type, suggesting strategies to characterise vitamin D responder immune phenotypes. 5 Acknowledgements I would like to thank my supervisors Professor Chris Griffiths and Professor Catherine Hawrylowicz for an exciting opportunity to conduct this clinical trial and to let me read for this thesis. I would like to thank Professor Hawrylowicz in particular for her mentoring and unswerving encouragement and Professor Chris Griffiths for his support throughout this time. I would also like to thank Professor Chris Corrigan, who provided a wealth of advice and support durig the conduct of this trial. A special thank you to Dr Emma Chambers, whith whom I shared the scientific work that resulted from this trial, she is a great friend and source of inspiration and education. I would like to thank Grainne Colligan for her help with recruitment and care of the patients enrolled in the study and Dr Anna Freeman for her invaluable help during her three months placements at the laboratory during two consecutive years. Dr Kimuli Ryanna has always offered much appreciated support during the conduct of the trial. I would like to thank Drs Dave Richards, Zoe Urry, Emanuel Xystrakis and Sarah Dimeloe, members of Professor Hawrylowicz’s laboratory, everyone was most helpful and provided much appreciated advice. Ich danke meinen Eltern herzlichst fuer all ihre Unterstuetzung waehrend vielen Jahren. Most of all I would like to thank Philip, Henry and Isla for their wonderful love, patience and encouragement. Sadly my dear mother in law, Dr. Joan Kelly, passed away shortly before this thesis was completed. She has always shown a great interest in my work. As a doctor and mother herself, she has been incredibly inspirational and always supportive. We often talked about medicine and motherhood and I shall miss our conversations dearly. 6 Table of Contents !"#$%&'$ (((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((()! !'*+,-./01/2/+$# ((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((3! 45#$6,768&"./#(((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((( 99! !""%/:5&$5,+# (((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((( 9;! <=&>$/%69((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((( 93! ?+$%,0@'$5,+((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((93! 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