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.