Diurnal Fluctuations of Orexin‑A and -B in Cynomolgus Monkey Cerebrospinal Fluid Determined by a Novel Analytical Method Using Antiadsorptive Additive Treatment Followed by Nanoflow Liquid Chromatography–High-Resolution Mass Spectrometry

Orexin-A (OXA) and -B (OXB) are involved in the regulation of multiple physiological functions including the sleep–wake states; therefore, it is critical to monitor their levels under various conditions. Unfortunately, the widely used radioimmunoassay has insufficient specificity for OXA. Although liquid chromatography–tandem mass spectrometry (LC–MS/MS) has higher specificity for OXA, previously reported OXA levels in human cerebrospinal fluid (CSF) measured using this technique are still inconsistent. Moreover, to the best of our knowledge, OXB has not been detected in the CSF. In this study, we established a novel method for OXA and OXB measurement. We noticed that OXA and OXB in the CSF was sticky; thus, citric acid and Tween 80 were used to prevent their nonspecific binding. Then, highly specific and sensitive nanoflow liquid chromatography–high-resolution mass spectrometry (nanoLC-HRMS) was used to measure OXA and OXB levels. Evaluation of the diurnal fluctuations of OXA and OXB in cisternal and lumbar CSF samples from cynomolgus monkeys revealed a sharp increase in the early light period, followed by a gradual increase to the maximum levels at the end of the light period, and then a sharp drop to the minimum levels during the early dark period. OXB levels were lower than OXA levels in cisternal CSF. Although basal OXA levels in individual monkeys showed substantial variations, the ratios between the maximum and minimum OXA levels of each monkey were similar. Our method for accurate OXA and OXB measurement should help improve our knowledge of orexin biology

BTZO-15 activates the GST Ya ARE by interacting with MIF.

<p>(A) Chemical structures of BTZO-15. (B) Sensorgram showing binding of BTZO-15 to immobilized hMIF on a CM5 sensor chip using an SPR biosensor. (C) Displacement studies with BTZO-15 by the SPA showing inhibition of BTZO-1 binding to captured rMIF on SPA beads. Results shown are the mean ± SD, n = 3. (D) Nucleotide sequence of the rat GST Ya ARE. The core ARE sequence is indicated by the nucleotides in boldface type. (E) Reporter gene assays showing the effect of BTZO-15 on ARE activity. H9c2 cells transiently transfected with either pGL3-ARE-Luc or pGL3-mARE-Luc reporter plasmids were treated with the indicated concentrations of BTZO-15 for 24 h and luciferase activities were measured. Results shown are the mean ± SD, n = 3. (F) BTZO-15 induced ARE-regulated cytoprotective proteins in H9c2 cells. The cells were treated with the indicated concentrations of BTZO-15 for 6 h (HO-1) or 21 h (GST Ya). HO-1 mRNA levels were measured by real-time PCR and normalized by GAPDH mRNA. Results shown are the mean ± SD, n = 3. (G) Effects of reducing the cellular MIF protein level on BTZO-15-induced GST Ya and HO-1 gene expression in H9c2 cells. H9c2 cells transfected with control siRNA (control siRNA) or siRNAs directed against rMIF (MIF siRNA) were treated with 3 µM BTZO-15 with or without 0.8 µM rMIF for 24 h. Messenger RNA levels of MIF, GST Ya, and HO-1 were measured by real-time PCR. MIF protein level in cell lysates was measured by ELISA.</p

<i>In vitro</i> autoradiography (ARG) using [³H]TAK-063 in sagittal rat brain sections.

<p>The chemical structure of [³H]TAK-063 (A). Sections adjacent to those used for <i>in vitro</i> ARG of [³H]TAK-063, were stained with hematoxylin and eosin (B). The autoradiogram shows the high accumulation of [³H]TAK-063 in the caudate putamen (CPu; white arrow), nucleus accumbens (NAc; black arrow), globus pallidus (GP; white arrow head), substantia nigra (SN; black arrow head), and striatonigral projection (gray arrow; C). <i>In vitro</i> ARGs in the presence of an excess amount of MP-10 (D) or TAK-063 (E) were performed with adjacent sections. Radioactivity levels in several brain regions were represented as photostimulated luminescence (PSL) values in the presence or absence of an excess amount of MP-10 or TAK-063 (F). Statistical analyses were performed using Dunnett's test (*<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01 vs total binding, n = 3). Fcx, frontal cortex; Thal, thalamus; Bs, brainstem; Hipp, hippocampus; Cb, cerebellum.</p

<i>In vitro</i> autoradiography (ARG) using [³H]TAK-063 in mouse brain sections.

<p>[³H]TAK-063 selectively accumulated in the caudate putamen (CPu; black arrow) and nucleus accumbens (NAc; white arrow) of wild-type (WT) mouse brain sections (A). The selective accumulation of [³H]TAK-063 in these areas did not occur in <i>Pde10a</i>-KO mouse brain sections (B). Radioactivity levels in the CPu of brain sections in the presence and absence of an excess amount of MP-10 are represented as a percent of total binding of WT mice (C). Data are represented as mean ± SEM.</p

BTZO-15 induces HO-1 and GST Ya protein expression in the rectum of rats with DSS-induced colitis.

<p>Colitis was induced in rats by daily treatment with 2.3% DSS solution in drinking water for 7 days. During this period, the rats were orally administered BTZO-15 (3 mg/kg, b.i.d.) or vehicle (0.5% MC). Rectal HO-1 (A) and GST-Ya (B) protein expression was assessed after excision and homogenization in PBS supplemented with 0.1% NP-40. The amount of GST Ya and HO-1 protein was quantified using ELISA. Results shown are the mean ± S.E.M, n = 8. * P≤0.05: compared the DSS (−) vehicle control group with DSS (+) vehicle control group using Aspin-Welch test. # P≤0.05: compared the DSS (+) vehicle control group with DSS (+) + BTZO-15 treated groups with Student's t test.</p

Concentration of T-773 in the rat brain and displacement by TAK-063.

<p>The data (ng/g tissue) are represented as mean (n = 2 at 0.3 mg/kg) or mean ± SEM (n = 3).</p><p>Concentration of T-773 in the rat brain and displacement by TAK-063.</p

BTZO-15 ameliorates TNBS-induced colitis in rats.

<p>Colitis was induced in male F344/Du rats by topical intracolonic application of a TNBS solution. BTZO-15 was orally administered to rats at the indicated doses of 10 mg/kg (b.i.d.) for 4 days from experimental day 1 to 4 (A). The efficacy of BTZO-15 was evaluated using the size of the rectal ulcer (B), MPO activity (C), and TNF-α level in rectum. Results shown are the mean ± S.E.M. TNBS-treated group, n = 6; TNBS untreated group, n = 2. ** P≤0.01: compared the DSS (−) vehicle control group with DSS (+) vehicle control group by Aspin-Welch test, and # P≤0.05 compared the DSS (+) vehicle control group with DSS (+) + BTZO-15 treated groups with Student's t test (B). * P≤0.05: compared the DSS (−) vehicle control group with DSS (+) vehicle control group using Aspin-Welch test, and # P≤0.05 compared the DSS (+) vehicle control group with DSS (+) + BTZO-15 treated groups by Aspin-Welch test (C and D).</p

Percent inhibition of enzymes by TAK-063 at 10 μM.

<p>EGF, epidermal growth factor; HMG CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A. Negative value of percent inhibition indicates activation of enzyme activity.</p><p>Percent inhibition of enzymes by TAK-063 at 10 μM.</p

<i>In vivo</i> ARG of [¹⁴C]TAK-063 in rats.

<p>The chemical structure of [¹⁴C]TAK-063 (A). The asterisk denotes the labeled position. Autoradiograms of head sections were obtained from male rats 6 h after single oral administration of [¹⁴C]TAK-063. The autoradiograms of 40 μm sagittal sections between 2.1 to 2.4 mm lateral to midline were taken (B). The locations for each coronal section relative to the bregma were 1.7 to 1.2 mm (C), 0.48 to −0.26 mm (D), −0.4 to −0.8 mm (E), −2.8 to −3.1 mm (F), −6.0 to −6.3 mm (G), and −12.7 to −12.8 mm (H). Acc, nucleus accumbens; Cb, cerebellum; Cpu, caudate putamen; Ctx, cortex; Fcx, frontal cortex; GP, globus pallidus; Hipp, hippocampus; MO, medulla oblongata; OT, olfactory tubercle; SN, substantia nigra; Thal, thalamus; VP, ventral pallidum.</p

Percent inhibition of receptors by TAK-063 at 10 μM.

<p>AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartic acid. Negative value of percent inhibition indicates stimulation of receptor activity.</p><p>Percent inhibition of receptors by TAK-063 at 10 μM.</p