A single-arm, phase 2 study of steroid-containing mouthwash for the prevention of everolimus-associated stomatitis in multiple tumor types.

BACKGROUND: Everolimus is a mammalian target of rapamycin inhibitor used in the treatment of multiple tumor types, and its most common toxicity, stomatitis, can affect patient quality of life. Recent studies in breast cancer have supported the efficacy of steroid mouthwash for the prevention of everolimus-associated stomatitis. However, a few studies have been reported to date, and none have examined this effect in other tumor types. METHODS: This single-arm phase 2 study was designed to evaluate the efficacy of steroid-containing mouthwash for the prevention of stomatitis in patients with multiple tumor types receiving everolimus. The primary outcome was incidence of grade ≥ 2 stomatitis at 8 weeks of everolimus with steroid-containing mouthwash prophylaxis. We also assessed the stability of steroid-containing mouthwash components. RESULTS: Twenty-nine patients were evaluated, of which 76% had breast cancer and 24% had neuroendocrine tumors originating in the lung, gastrointestinal tract, pancreas, or of unknown primary origin. Grade ≥ 2 stomatitis incidence at 8 weeks was 28.1% (90% CI 16.2-46.1); the higher confidence limit exceeded the prespecified threshold of 30%. No patients developed grade ≥ 3 stomatitis. Most stomatitis occurred behind the oral cavity, with no lesions observed on the lips or floor of the mouth. CONCLUSIONS: Our findings did not support a prophylactic effect of steroid-containing mouthwash on everolimus-associated stomatitis. Given the needs of prevention of everolimus-associated stomatitis in various tumor types, further studies in a larger population using a randomized controlled trial design are, therefore, required to confirm the efficacy of steroid-containing mouthwash.

Changes in hypothalamic neurotransmitter and prostanoid levels in response to NMDA, CRF, and GLP-1 stimulation.

Determination of neuroactive substances, such as neurotransmitters and prostanoids, in the extracellular space of the living brain is a very important technique in neuroscience. The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Recently, we demonstrated that thromboxane (Tx) A2 in the PVN may play an important role in adrenomedullary outflow evoked by N-methyl-D-aspartate (NMDA), corticotrophin-releasing factor (CRF), and glucagon-like peptide-1 (GLP-1) stimulation using microdialysis sampling and liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). In the present study, we investigated whether centrally administered NMDA, CRF, and GLP-1 can release five neurotransmitters, acetylcholine (ACh), histamine, glutamate (Glu), γ-aminobutyric acid (GABA), and serotonin (5-HT), along with six prostanoids, TxB2, prostaglandin (PG) E2, PGD2, 15-deoxy-∆(12,14) (15d)-PGJ2, 6-keto-PGF1α, and PGF2α in rat PVN microdialysates after optimization of LC-ITMS(n) conditions . All stimulations increased the levels of 5-HT, TxB2, PGE2, and PGF2α (except for 5-HT stimulated with GLP-1). Only NMDA increased the levels of ACh, Glu, and GABA. Treatment with dizocilpine maleate (MK-801), a noncompetitive NMDA receptor antagonist, attenuated the NMDA-induced increase in the levels of neuroactive substances except for Glu. This is the first study to use LC-ITMS(n) to analyze neurotransmitters and prostanoids in the same microdialysates from rat PVN.

Hierarchical clustering of liquid chromatography-tandem mass spectrometry data for screening of phosphodiesterase type 5 inhibitors and their analogues in adulterated dietary supplements.

Sexual enhancement dietary supplements have often been adulterated with phosphodiesterase type 5 (PDE-5) inhibitors used for treatment of erectile dysfunction, and widely distributed through online markets. As the illegal adulterants, the original PDE-5 inhibitor drugs and a numerous number of synthetized analogues, more than 80, have already been found. Therefore, analytical methods that detect various PDE-5 inhibitors and uncover newly synthesized analogues are needed. In this study, we have developed a rapid and reliable screening method for PDE-5 inhibitors and their structural analogues by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by hierarchical clustering based on similarity of MS/MS spectra. Forty reference standards of PDE-5 inhibitors/analogues were measured using a quadrupole-orbitrap mass spectrometer in data-dependent mode. The 60 most intense fragment ions were extracted from each MS/MS spectra, and the ions observed within 1.5 mDa mass tolerance were considered to be the same ion. Based on fragment ion tables representing detected ions for each compound, hierarchical clustering was performed. The resulting dendrogram showed that the reference standards were separated into seven clusters according to their characteristic structures. Subsequently, two additional standards spiked into a herbal sample were analyzed. While herbal components were clearly separated from the clusters of the reference standards, the spiked standards were clustered closely with the structurally similar standards. Furthermore, application of our method to dietary supplements allowed for detection of sildenafil and tadalafil as adulterants. These results suggest that our screening method facilitates discovery of adulterant PDE-5 inhibitors/analogues by illustrating their structural similarity.

Thromboxane A2 in the paraventricular hypothalamic nucleus mediates glucoprivation-induced adrenomedullary outflow.

Glucoprivation stimulates a rapid sympathetic response to release and/or secrete catecholamines into the bloodstream. However, the central regulatory mechanisms involving adrenoceptors and prostanoids production in the paraventricular hypothalamic nucleus (PVN) that are responsible for the glucoprivation-induced elevation of plasma catecholamines are still unresolved. In this study, we aimed to clarify whether glucoprivation-induced activation of noradrenergic neurons projecting to the PVN can induce α- and/or ß-adrenergic receptor activation and prostanoids production in the PVN to elevate plasma catecholamine levels. We examined the effects of α- and ß-adrenergic receptor antagonists, a cyclooxygenase inhibitor, a thromboxane A synthase inhibitor, and a PGE2 subtype EP3 receptor antagonist on intravenously administered 2-deoxy-D-glucose (2-DG)-induced elevation of noradrenaline in the PVN and plasma levels of catecholamine in freely moving rats. In addition, we examined whether intravenously administered 2-DG can increase prostanoids levels in the PVN microdialysates. Intracerebroventricular (i.c.v.) pretreatment with phentolamine (a non-selective α-adrenergic receptor antagonist) suppressed the 2-DG-induced increase in the plasma level of adrenaline, whereas i.c.v. pretreatment with propranolol (a non-selective ß-adrenergic receptor antagonist) suppressed the 2-DG-induced elevation of the plasma level of noradrenaline. I.c.v. pretreatment with indomethacin (a cyclooxygenase inhibitor) and furegrelate (a thromboxane synthase inhibitor) attenuated the 2-DG-induced elevations of both noradrenaline and adrenaline levels. Furthermore, 2-DG administration elevated the thromboxane B2 level, a metabolite of thromboxane A2 in PVN microdialysates. Our results suggest that glucoprivation-induced activation of α- and ß-adrenergic receptor in the brain including the PVN and then thromboxane A2 production in the PVN, which are essential for the 2-DG-induced elevations of both plasma adrenaline and noradrenaline levels.

Centrally administered isoproterenol induces sympathetic outflow via brain prostaglandin E2-mediated mechanisms in rats.

Brain ß-adrenoceptor stimulation can induce elevations of plasma levels of noradrenaline. However, there have been no detailed studies related to signaling pathways downstream of ß-adrenoceptors responsible for central sympathetic outflow. In the present study, we pharmacologically examined the possibility that centrally administered isoproterenol can induce elevations of plasma noradrenaline levels in a brain prostaglandin-dependent manner. In addition, we also examined whether or not intracerebroventricular administration of isoproterenol could release endogenously synthesized prostaglandin (PG) E2 in the hypothalamic paraventricular nucleus (PVN) by using the brain microdialysis technique combined with liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). Under urethane anesthesia, a femoral venous line was inserted for infusion of saline and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed by high-performance liquid chromatography with electrochemical detection. Quantification of PGE2 in rat PVN microdialysates was performed by the LC-ITMS(n) method. We demonstrated that centrally administered isoproterenol-induced elevations of plasma noradrenaline could be mediated via activation of ß-adrenoceptors and the downstream phospholipase A2-cyclooxygenase pathway. Furthermore, PGE2 in the PVN and the PGE2 receptor EP3 subtype appear to play an important role in the process. Our results suggest that central isoproterenol-induced sympathetic outflow is mediated via brain PGE2 in a PGE2 receptor EP3 subtype-dependent manner.

Mass spectrometric determination of prostanoids in rat hypothalamic paraventricular nucleus microdialysates.

The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Several kinds of prostanoids, such as prostaglandin (PG) E2, are considered to act in the PVN as mediators of autonomic responses. In the present study, we used liquid chromatography ion trap tandem mass spectrometry (LC-ITMS(n)) to simultaneously quantify four prostanoids, thromboxane (Tx) B2, PGE2, PGD2 and 15-deoxy-∆(12,14) (15d)-PGJ2 in PVN microdialysates from urethane-anesthetized rats. The quantification limits were estimated to be 0.05ng/mL for TxB2, 0.025ng/mL for PGE2, 0.1ng/mL for PGD2, and 0.5ng/mL for 15d-PGJ2. The RSD% obtained from all prostanoids was <15%, indicating an acceptable level of reproducibility. LC-ITMS(n) analysis of rat PVN microdialysates revealed that TxA2 may play an important role in adrenomedullary outflow evoked by centrally administered N-methyl-d-aspartate, corticotrophin-releasing factor and glucagon-like peptide-1. This is the first study to use LC-ITMS(n) to analyze prostanoid levels in rat PVN microdialysates. This LC-ITMS(n) method will be useful for investigating the potential involvement of prostanoids in brain function.

Phosphoprotein analysis for investigation of in vivo relationship between protein phosphatase inhibitory activities and acute hepatotoxicity of microcystin-LR.

Microcystin-LR (MCLR) produced by freshwater cyanobacteria is a potent hepatotoxin and inhibits protein serine/threonine phosphatases 1 and 2A (PP1 and PP2A). Okadaic acid (OA) is a similar phosphatase inhibitor, which has less affinity to PP1 than PP2A. MCLR and OA behave similarly with primary culture hepatocytes with the induction of phosphorylation of the cytokeratins, morphological changes, and apoptosis. The purpose of this study was to investigate the in vivo relationship between the protein phosphatase inhibitory activities and the acute hepatotoxicity of MCLR compared to OA. MCLR and OA were intraperitoneally administrated to mice at approximately 220 microg/kg. After 30 min, the liver of only the MCLR-treated mouse was dark-colored and heavier than that of the control mouse. Subsequently, the phosphoproteins of the mouse liver were chemically modified with reversible biotinylation reagent and selectively analyzed by LC/MS/MS. Consequently, the phosphorylated Ser 354 of formyltetrahydrofolate dehydrogenase, which is an abundant enzyme in the liver cytoplasm, was observed in the MCLR- and the OA-treated mice 9.5 and 5.3 times more intensely than in the control mouse respectively, suggesting that MCLR and OA inhibited PP2A and induced the resulting phosphorylation. These results supported the hypothesis that the acute hepatotoxicity is possibly caused by the PP1 inhibition, and not by the PP2A inhibition.