Abstract Details

Title Quantitation of Hypocretin Neuropeptides (Orexin-A and -B) in Human Cerebrospinal Fluid via HPLC with MS/MS Detection

Topic Sleep

Presentation(s) P1 - Poster Session 1 (11:45 AM-12:45 PM)

Poster/Presentation
Number 4-002

Objective We have developed a highly sensitive and specific LC-MS assay for the simultaneous measurement of intact biologically active Orexin-A and -B and fully validated it for use in CSF from human subjects.

Background Orexin-A and -B are complex neuropeptides responsible for regulating physiological functions including the sleep-wake cycle, metabolism, and emotional state. The current radioimmunoassay in use for diagnosing narcolepsy and measuring orexin-A in CSF is not specific to biologically active forms, where 90% of the signal is contributed by inactive metabolites of Orexin-A or non-specific proteins, thus creating challenges in discerning orexin sleep-wake patterns in humans.

Design/Methods Six healthy male consented volunteers dosed with 5mg TAK-861, an Orexin-2 receptor agonist, QD for 10 days provided CSF sampling over 24hrs and 12 un-dosed healthy male and female consented volunteers over 12 hrs. Samples were thawed at room temperature and extracted by solid phase extraction. Chromatographic separation was achieved over 15 minutes using a Polaris C18-A HPLC column and full length Orexin-A and B were detected on a triple quadrupole API 7500 system.

Results We detected a distinct diurnal rhythm of Orexin-A and -B in human subjects dosed for 10 days QD with 5mg of TAK-861 from lumbar CSF. Measurements of Orexin-A started at 24.7 +/- 4.8 pg/mL at 8:00 a.m. and peaked 16 hours later at 12 a.m. to 38.6 +/- 4.9 pg/mL and returned to baseline by 24 hours for both Orexin-A and -B for a maximum amplitude of 56.6%, whereas the radioimmunoassay method showed a much smaller 8.8% amplitude at the same time interval.

Conclusions Our method improves upon the sensitivity of previous LC-MS methods and targets the biologically active, intact Orexin-A and Orexin-B peptides with an LLOQ of 2 pg/mL from 0.5ml of CSF, and avoids potentially measuring multiple species with signature peptide approaches which could be shared by orexin-A metabolites.

Authors/Disclosures
Anson Pierce, PhD PRESENTER	Dr. Pierce has received personal compensation for serving as an employee of Takeda Pharmaceuticals. Dr. Pierce has stock in Takeda Pharmaceuticals.
Meng Ye	No disclosure on file
Joe Palandra	Mr. Palandra has nothing to disclose.
Gus Hui, PhD	Dr. Hui has nothing to disclose.
Jian Wu, PhD	Dr. Wu has nothing to disclose.
Aaron Wheeler	Mr. Wheeler has nothing to disclose.
Walter Wiley	Mr. Wiley has nothing to disclose.
Michael S. Woolf, PhD	Dr. Woolf has nothing to disclose.
Omnia Ismaiel	No disclosure on file
Kumar Shah	Dr. Shah has nothing to disclose.
Moucun Yuan, PhD	Dr. Yuan has received personal compensation for serving as an employee of PPD, part of Thermo Fisher Scientific.
William R. Mylott, Jr., BS	Mr. Mylott has nothing to disclose.
Mike Baratta, MCAHPM	Mr. Baratta has received personal compensation for serving as an employee of Takeda Pharmaceuticals. Mr. Baratta has stock in Takeda Pharmaceuticals. Mr. Baratta has received personal compensation in the range of $500-$4,999 for serving as a Grant Reviewer with FNIH.

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