Composition for competitive inhibition of orexin receptors
A technology of orexin receptors and compositions, which is applied in the field of inhibiting the binding of orexin to its receptors, can solve problems that are not yet known, and achieve excellent antagonism and inhibition of orexin receptors and the effect of inhibiting the binding of orexin to its receptors
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Embodiment 1
[0056] The orexin receptor antagonist activity of the test substance was measured using an accessory of the Orexin 2 Receptor Reporter Assay Kit (Cayman Chemical Company).
[0057] In this evaluation system, cells expressing orexin receptors on the cell surface are used. When an agonist (orexin) binds to this orexin receptor, secreted alkaline phosphatase (SEAP) is secreted. The secreted SEAP was reacted with an alkaline phosphatase substrate, and the enzymatic activity of SEAP was measured using a photometer. Substances having orexin receptor antagonistic action (orexin receptor antagonist action) reduce the enzyme activity.
[0058] (1) Use Dulbecco's Modified Eagle's Medium-high glucose (With 4500mg / Lglucose, L-glutamine , sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture) (Sigma-Aldrich Company), using HEK293T cells to reach 2.5×10 5 cells / mL were prepared.
[0059] (2) Sow 200 μL of the culture medium containing HEK293T cells...
Embodiment 2
[0070] For the pyrroloquinoline quinone disodium salt-added group of Example 1, cell proliferation after the addition of pyrroloquinoline quinone disodium salt was measured using WST-8 Cell Proliferation Assay Kit (Cayman Chemical Company). The determination method is based on the attached experimental guide. It is known that dehydrogenase activity is directly proportional to the number of viable cells. This kit is a kit for measuring the dehydrogenase activity released from cells into the culture medium using this principle. For the negative control group and the positive control group in Example 1, the cell proliferation after the addition of DMSO or Suvorexan was also measured in the same way.
[0071] figure 2It is a graph showing dehydrogenase activity in a negative control group (N.C.), a positive control group (P.C.), and a pyrroloquinoline quinone disodium salt-added group (PQQ). The results (mean ± standard error) are expressed as relative values with the dehydr...
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