The Anti-Hypoxia Effect And Protective Mechanism Of Novel Compounds Targeting Histamine H3 Receptor

Hypoxia refers to inadequate supply or poor use of oxygen. It is categorized into four different kinds including hypoxic, circulatory, haemal and tissue hypoxia. High altitudes features low partial pressures of oxygen and acute exposure to it will induce various high-altitude sickness. Many strategies such as oxygen supply, gradual ascent and medicine can help to prevent or treat high-altitude illness. Pharmacological prophylaxis and treatments still remains a problem. Established drug treatments are acetazolamide and dexamethasone. And also, many drugs like free radical scavenger, erythropoietin deviates and traditional Chinese medicine are under research. However, despite the anti-hypoxia effect of acetazolamide and dexamethasone, serious side effects limit wide application in clinic. Therefore, exploring new anti-hypoxia drug targets, developing safe and effective drugs for high-altitude sickness is essential.Histamine H3 receptor is widely expressed in central nervous system. Studies have shown that histamine H3 receptor is involved in hypoxic-ischemic brain damage and plays an important role in the regulation of hypoxic respiratory response. Our previous study showed that histamine H3 receptor mRNA level changes in some brain regions of mice exposed to hypoxic hypoxia. We also found that Pitolisant, a histamine H3 receptor inverse agonist, can improve anti-hypoxic ability of mice under normobaric hypoxia condition. Further study of the anti-hypoxia effect and protective mechanism of compounds targeting histamine H3 receptor can help to discover new drugs for high-altitude illness.Objective:1. To systematically evaluate the anti-hypoxia effects and clarify the possible protective mechanism of Pitolisant.2. Screening the anti-hypoxic effects of Pitolisant derivates to obtain compounds possessing independent intellectual property right and novel chemical structure.Methods:1. Adopting normobaric and acute lethal hypobaric hypoxia animal model to determine the anti-hypoxic activities of Pitolisant. And then, the effect of Pitolisant on spontaneous locomotor activity of mice was observed;2. Simulating two kinds of continuous hypoxic conditions respectively through injection of sodium nitrite (80 mg/kg) and a walk-in a hypobaric chamber (altitude 5000 m). And then evaluating the anti-fatigue effects of Pitolisant on mice exercising with an running wheel in hypoxic environments.3. The animals were exposed to chronic hypoxia (altitude 8000 m) animal hypobaric chamber for 6h to obtain acute exposure to high altitude model. Then the cerebral protective effects of Pitolisant was evaluated by detecting lactic acid (LD) content, ATPase activity, morphology of cerebral vessel and neurons. And also hypoxia-related molecular was detected by real-time quantitative PCR;4. Agonistic and antagonistic activity of Pitolisant derivates on histamine H3 receptor was detected by protein kinase A (PKA) redistribution experiment;5. Anti-hypoxia effects of Pitolisant derivates were estimated by normobaric hypoxia tests. Effect of Pitolisant deviates on spontaneous locomotor activity of mice was also observed.Results:1. In normobaric hypoxia model, Pitolisant (1.0,3.0,10.0 mg/kg) significantly prolonged the survival time of mice versus NS. The prolongation rate was (1.1%, 11.4%,64.3%). Pitolisant (0.1,0.3,1.0 mg/kg) dose-dependently prolonged median survival time and improved survival rate of mice exposed to acute lethal hypobaric hypoxia.Pitolisant within this dose range did not show significant influence on spontaneous locomotor activity of mice;2. Pitolisant (0.1,0.3,1.0 mg/kg) dose-dependently increased running endurance of mice exposed to normobaric hypoxia (injection of sodium nitrite). Pitolisant (1.0 mg/kg) and improve running durations of mice exposed to acute and sub-acute hypoxia. Results of brain tissue HE staining showed that Pitolisant (1.0 mg/kg) can alleviate cerebral edema;3. Pitolisant (0.1,0.3,1.0 mg/kg) down regulate the compensatory increase of ATPase activity and reduce LD content by respectively 1.3%,8.8%,17.3% off. Pitolisant (1.0 mg/kg) decreased vascular endothelial growth factor (VEGF) mRNA level in cortex and hypothalamus of brain;4. PKA redistribution experiment showed that WH-1-42 (EC50=1.08±0.53 μmol), WH-3-15 (EC50=1.15±0.31μmol), WH-3-10 (EC50=9.69±1.90 nmol) behaved as antagonists of histamine H3 receptor. WH-3-10, WH-3-15 significantly prolonged the survival time of animals confined to normobaric hypoxia. Structure identification revealed that WH-3-10 and Pitolisant are the same compound (double-blind).WH-3-15 showed strong central sedative effect.Conclusion:1. Pitolisant has shown significant anti-hypoxic effects in different kinds of hypoxia including normobaric, hypobaric, static and dynamic hypoxia.2. Regulation of cerebral energy metabolism and alleviation of cerebral edema are the possible protective mechanisms of Pitolisant.