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Dynamic hormone diagnostics

Periodic Reporting for period 3 - ULTRADIAN (Dynamic hormone diagnostics)

Reporting period: 2018-09-01 to 2020-02-29

We propose to revolutionize current endocrine management by replacing conventional time-consuming single-analyte hormone assays with ambulatory dynamic endocrine assessment. Hormonal disorders are not only accompanied by changes in absolute hormone levels, but also secretion patterns. The rapid circadian (24 hour) and ultradian (< 24 hour) cycling of hormone poses diagnostic challenges since random measurements do not always detect abnormalities. Furthermore, monitoring of treatment of endocrine diseases is often severely limited by lack of multiple measurements at different time points, which is impractical in an out-patient setting.
To overcome these hurdles, academic and commercial partners propose a novel sampling system that allows direct measurement of free hormone levels in subcutaneous tissue. Using microdialysis, samples are continuously collected for 24 hours and stored in a portable fraction collector. During this time the person continues normal daily life activities ( Minute quantities of sample are then subject to ultrasensitive assays by liquid chromatography tandem mass spectroscopy and multiplex proximity extension assays. These techniques allow simultaneously quantitation of multiples hormones at high specificity and sensitivity (currently about 30 different hormones).
Using the ULTRADIAN Diagnostic system normal ranges of different steroid hormones were defined, including cortisol, cortisone and aldosterone. Tissue-free hormone concentrations corresponded well to serum levels. Clinical trials on patients with newly diagnosed Cushing’s syndrome and primary aldosteronism enabled us to define new diagnostic criteria based on 24-hour steroid profiling in combination with patient meta-data. The technique also allowed us to define cure after treatment. Ultradian profiling also proved valuable to monitor steroid replacement therapy in adrenal insufficiency using similar algorithms.
Taken together this ULTRADIAN Diagnostic system provides a novel, simplified and cost-effective method to diagnose and follow up of patients with adrenocortical diseases. The method can easily be adapted for use in other areas of medicine, thereby increasing societal impact of the technique.
The work performed have focused on developing the microdialysate fraction sampler device, and using it to generate 24-hour hormone profiles in health and disease.

24h microfluidics sampling device
At the outset of ULTRADIAN the first University of Bristol (‘blue box’) prototype fraction collection device was available. However, it became apparent that this early prototype was not able to reliably produce 24 hour profiles. Thus, we embarked on an extensive development scheme leading to the current version, named U-RHYTHM 3.1. This prototype is the culmination of multiple improvements including significant reduction in size, installation of a rechargeable lithium polymer battery and wireless recharging dock, new manifold and pumps, volume and pressure monitoring, and pre-wound spools for sample collection. These huge efforts have led to a reliable and easy to use sampling device performing consistent samplings for up to 30 hours.

Multiplex hormone assay and detection
In order to measure hormones in minute fluid volumes, our focus has been to develop ultrasensitive multiplex assays of steroid and peptide/protein hormones. We have developed an optimized automated liquid handling system that efficiently handle the very small sample volumes of each fraction (5-15 µL) in a high-throughput setting. Hormones in the processed samples are separated by liquid chromatography with subsequent tandem mass spectrometry. A 3rd generation steroid panel has been established and is now in production mode. Protein and peptide hormone measurements have been developed using the proximity extension assay technique, which allows multiplexing and assay of multiple hormones in only 1 µl fluid. A 21-plex ULTRADIAN protein panel was established and validated. The panel performs very well in blood, but met reproducibility problems when used in microdialysis fluid. Thus, the panel was subsequently used to measure peptide hormones in plasma.

ULTRADIAN Hormone Dynamics in Healthy Controls
A prerequisite to diagnose abnormalities in hormone secretion is to have robust data on the normal variation in hormone levels over the 24-hour cycle. Thus, altogether 242 healthy subjects were sampled covering both sexes and different age groups. The sampling system is well tolerated and participants can perform daily life activities including sleep, without restriction using. Participants indicated a high level of satisfaction and minimal inconvenience through a feedback questionnaire provided at the end of each sampling session. Now in “production-mode” at least 8 out of 10 samplings are successful in giving interpretable results. No serious adverse events have been reported.

Clinical trials
Initial problems with the U-rhythm sampler delayed the trials. Furthermore, difficulties in establishing reliable assays of growth hormone and androgens made it impossible to finish trials on acromegaly, growth hormone deficiency and congenital adrenal hyperplasia. Instead the trials on Cushing’s syndrome, primary aldosteronism and adrenal insufficiency were strengthened by inclusion of more patients and post-operative samplings when relevant. This measure secured increased power to detect changes in hormone profiles.

Health economics and marketing strategies
Work is ongoing to estimate the potential value and health-economic impact of ULTRADIAN diagnostics. A systematic evaluation of the costs of has been performed showing that the “one step clinic” outpatient concept that ULTRADIAN offers have the potential to make the ULTRADIAN technique cost-effective compared to current procedures involving hospitalisation. Market evaluation has revealed a huge interest from researchers, clinicians, and stakeholders in the medical technology industry.

Dissemination and management
The consortium has had a focus on dissemination activities including lectures and broadcast material through various media including newspapers and television. A newsletter has been distributed to identified stakeholders. An video demonstrating the ULTRADIAN concept has been produced and updated with the latest version of the U-rhythm sampler. The consortium is managed through biannual meetings, phone conferences and exchange of scientific personnel between partners. Towards the end of the project period, an international conference and workshop was held in Bergen, Norway.
The Ultradian project has clearly progressed beyond the state-of-the-art demonstrating the ability to generate 24 hour free hormone profiles with unprecedented sensitivity and specificity. These profiles hold promise of faster, more reliable and cheaper diagnosis of adrenocortical diseases.
Beyond endocrine conditions, the methods could be applied to diagnosis and therapeutic monitoring of other conditions, such as epilepsy treatment, investigation of metabolism, metabolic disease, and infertility.
ULTRADIAN Diagnostics is a versatile concept and the modular ULTRADIAN concept opens for the possibility to bring kits with different components to the market. In the long term, innovative concepts of physiological hormone/drug delivery to patients should raise the interest of diagnostics and pharmaceutical industry in exploiting ULTRADIAN results.
Annual Partner Meeting in Greece, October 2016
U-Rhythm Device