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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ULTRADIAN (Dynamic hormone diagnostics)

Teaser

Summary

We propose to revolutionize current endocrine diagnostics by replacing conventional, time-consuming single-analyte hormone assays with ambulatory dynamic endocrine diagnostics. 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 to develop a novel sampling system where fluid from the adipose tissue is continuously collected for 24 hours from a microdialysis catheter into a fraction collector device while the person tends to daily life activities (https://vimeo.com/212217246). The minute quantities of fluid are then subject to novel ultrasensitive assays by liquid chromatography tandem mass spectroscopy (LCMS/MS) and multiplex proximity extension assays (Proseek). These techniques allow simultaneously quantitation of multiples hormones (currently about 30 different hormones).

During the project the Ultradian Diagnostic system will be used to improve diagnostic procedures of hormonal diseases and to monitor their treatment. We believe the technique will improve the quality of diagnostics and at the same time simply them by reducing the need for hospitalisation. Moreover, the technique can easily be used in other areas of medicine, thereby increasing societal impact of the technique.

Work performed

The work performed in the first reporting period has focused on developing the microdialysate fraction sampler device and testing it in healthy subjects. Furthermore, we have developed and optimized the analysis panels.

1. 24h microfluidics sampling device (WP1)
At the outset of Ultradian the first University of Bristol (blue box) prototype fraction collection device was available. After manufacturing additional 15 devices for use in the clinical trials, feedback from partners indicated that did not perform reliably for 24 hours. We therefore included spool shifts midway in sampling to improve success rates in the initial testing.To overcome these problems Designworks made a huge effort to redesign and develop the U-rhythm prototype that now perform consistent sampling for up to 30 hours in the patient setting.

2. Multiplex hormone assay and detection (WP2)
In order to measure hormones in minute fluid volumes, one of the main has been to develop ultrasensitive multiplex assays of steroid and peptide/protein hormones.

a. A main challenge has been to handle the very small sample volume (5-15 ÂµL) in a high volume/high-throughput setting. We achieved this by developing an optimized code to control an automated liquid handler system. This code implements intelligent handling of variations in sample volume, which is crucial for reliable measurements. Validation procedures on the sample preparation step have documented good precision. Hormones in the processed samples are separated by liquid chromatography (LC) with subsequent tandem mass spectrometry (MS/MS) using. Using these techniques the 1st generation steroid panel has been established and validated with various microdialysis probes (20 and 100 kDa) to assess recovery and reproducibility. The panel contains the main hormones needed for clinical trials.

b. Protein and peptide hormone measurements have been developed using the proximity ligation technique which allows multiplexing and assay of multiple hormones in only 1 Âµl fluid. Partners compiled a wish list of proteins that would be relevant to assay including growth hormone and adrenocorticotropic hormone. A total of 55 protein assays were evaluated, targeting 45 different proteins. These experiments formed the basis for selection of assays constituting the 1st Ultradian multiplex hormone panel now successfully developed.

3. ULTRADIAN Hormone Dynamics In Healthy Controls (WP3)
The work has concentrated on delivering a practical demonstration of the Ultradian system in healthy volunteers. The sampling system is well tolerated and patients are able to perform daily life activities, although with some difficulties regarding dressing and toileting. A Device satisfaction questionnaire is used. With the newest prototype, the U-rhythm sampler, we anticipate increased user satisfaction.

4. Clinical trials (WP4)
All ethic committee confirmations for the clinical studies have been obtained. The start of clinical trials has been delayed about 6, but is now on track. The delay was used to test various catheters and coating conditions to optimize sample recovery and reproducibility of samplings.

5. Health economics and marketing strategies (WP6)
Work is ongoing to estimate the potential value and health-economic impact of Ultradian diagnostics in 4 European health care systems. For a systematic review Cushingâ€™s syndrome, primary hyperaldosteronism and growth hormone deficiency will be focused on. User satisfaction will be evaluate by the generic EQ-5D questionnaire and a specific Ultradian questionnaire.

6. Dissemination and management (WP7 and 8)
The consortium has had a great focus on dissemination activities including lectures and broadcast through various media including newspapers and television. A newsletter has been distributed to identified stakeholders and a Ultradian video has been produced. The consortium is managed through biannual meetings, phone conferences and exchange of scientific personnel between partners

Final results

The Ultradian project has clearly progressed beyond the state of the art in hormone diagnostics as we are able to reliably assay 24 hour profiles of free hormones in subcutaneous tissue microdialysis fluid. The development of a robust fraction collector device and novel ultrasensitive assay methods has made this possible.

We believe we can reach anticipated impacts including improved diagnosis of and monitoring tools for pituitary and adrenal diseases. Novel hormone reference ranges integrating variability between sufficient numbers of individuals will have the potential to refine current cut-off values with consequences for diagnosis and therapy, and pave way for personalized treatment.

The methods we are developing should be applicable to diagnosis and therapy monitoring of other diseases, naming epilepsy treatment, metabolic diseases, and infertility as a few examples.

ULTRADIAN diagnostics is a versatile concept and the modular ULTRADIAN concept opens for the possibility to bring kits with modulated 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.