Mechanism And Impact Of A Novel Allosteric AMPA Receptor Modulator On Protection Against Respiratory Depression

Analgesics and sedative drugs are widely prescribed for clinical treatment,including for the treatment of severe or chronic pain,the induction and maintenance of anesthesia,and intravenous epidural narcosis.Different side effects arise from drug therapy.Analgesic and sedative drug-induced respiratory depression is an unresolved and significant problem in clinical treatment and it can be a leading cause of death.Specific antagonists（naloxone,flumazenil,etc.）are currently administered to counteract respiratory depression for the aim of emergency use.However,the imperfections of short-lasting and multiple side effects are unexpected with the treatment of different antagonists.More importantly,antagonists cannot be chosen appropriately when general causes of respiratory depression remains indefinite,and the scenario often delay seeking emergency treatment.In addition,respiratory stimulants（nikethamide,lobeline,etc.）are also used to alleviate respiratory depression.However,a narrow range of safety,short duration of action and other severe adverse effects go with use of respiratory stimulants.Therefore,the development of non-specific drug to support respiration could be beneficial for analgesics and sedative drug use,pain management and the discovery of medications to alleviate respiratory depression.In recent years,new pharmacological approaches to alleviate analgesic and sedative drug-induced depression of respiration have been sought.Activation of AMPA receptors by glutamate plays a vital role in promoting synaptic transmission and is critical for maintaining respiratory rhythm in the preB?tzinger complex.Ampakines are a drug class that positively regulates AMPA receptors,and specific positive allosteric AMPA modulators are considered to markedly alleviate respiratory depression triggered by different targets.The characteristics of better security and distinctive effect of abirritation,sedation,promotion of learning and memory,and neuroprotection can be conducive to the respiratory depression treatment.Therefore,AMPA receptor modulator is expected to be a promising target for emergency treatment of respiratory depression.Compared to antagonists or respiratory stimulants,AMPA modulators with wide-spectral property perform significant superiority.Based on the merits,a series of new structural AMPA receptor positive modulators have been synthesized by our Institute of Medicinal Chemistry.After the pharmacodynamics screening on protection from respiratory depression,it had been initially definite that the code-named compound LCX001 reversed respiratory depression and performed analgesic and sedation effects.To further confirm the wide-spectral property of LCX001 on protection against respiratory depression,animal models of respiratory depression induced by opioids,central inhibitors,and anesthetics were established,and the whole-body plethysmograph test was used to investigate wide-spectral property and non-specific effects of LCX001 on anti-respiratory depression.In addition,for the purpose of improving the poor water solubility and blood-brain barrier permeability of LCX001,brain-Targeted prodrugs of LCX001 were used to improve the blood-brain barrier transmission and central distribution.Screening and evaluation of prodrugs on preliminary anti-respiratory inhibition effects were conducted to obtain a more potent AMPA positive modulator.Conclusively,electrophysiological and cell imaging techniques were used to analyze the property of LCX001 on positive modulation and the mechanism of anti-respiratory depression.The function of AMPA receptors were determined by location of receptors in the membrane and state of channel opening,and both of the processes are impressed by AMPA receptor regulatory proteins.According to the theory,the effect of LCX001 on the expression of stargazing was measured firstly,and then the variation of receptor surface location and channel opening were observed.The mechanism of regulation on neuronal Ca²⁺and cell function was investigated intensively.Objective:The property and positive regulation mechanisms of protection against respiratory depression by the novel AMPA modulator LCX001 was clarified in this study.Methods:1.Evaluation of the wide-spectral property of LCX001.Whole-body plethysmographs of breathing frequency and depth were performed on unrestrained rats to evaluate the protection effect of LCX001 against respiratory depression induced by fentanyl,TH-030418,propofol and pentobarbital（Nembutal）.2.Screening and evaluation of brain-targeted prodrugs on anti-respiratory depression.The prodrugs were tested to protect mice against acute death induced by potent opioid drug TH-030418.The effect on LD₅₀0 of the lethality of TH-030418,improvement of respiratory parameters and impact of arterial oxygen saturation,partial pressure of carbon dioxide,pH were evaluated,and candidate compounds were selected to improve the efficacy of anti-respiratory depression.3.The mechanism of LCX001 on the trafficking and location of AMPA receptors.3.1 The impact of LCX001 on the expression of AMPA receptor regulatory proteins（stargazin）was examined by Western blotting.3.2 Immunofluorescence staining and live cell imaging of GluA1 or GluA2 AMPA receptor subunits tagged with super ecliptic pHluorin（SEP）were performed to investigate the trafficking and accumulation of AMPA receptors on the surface of neurons in real-time.4.Effect of LCX001 on the kinetic properties of AMPA ion channels and cell excitability.Whole-cell patch-clamp recordings were conducted to record current amplitude and desensitization constant of AMPA ion channel on HEK293 cells that stably expressed AMPA-GluA2 receptors.The effect of LCX001 on glutamate-activated whole-cell membrane potential was investigated.The influence of LCX001 on the opening degree of AMPA ion channel,the speed of current attenuation,and other electrophysiological parameters were clarified.Stimulatory activity and positive modulation impact of LCX001 were determined as well.5.Ca²⁺imaging of neurons was conducted to detect regulation impact of intracellular calcium concentration by enhanced accumulation of AMPARs containing SEP-GluA2（R）units.Results:1.LCX001 effectively ameliorated respiratory depression induced by different anaesthetics and sedative hypnotics in rats.A fentanyl dose of 120μg/kg and a TH-030418 dose of 20μg/kg intravenously caused severe depression and a striking 70%-80%reduction in respiratory frequency and a 50%decrease in Minute Ventilation.A relatively moderate respiratory depression（respiratory rates were reduced by 30%-50%）was produced by 30 mg/kg propofol intravenously and 80 mg/kg pentobarbital intraperitoneal.With regard to varying degrees of respiration suppression by different targets,LCX001 at 10 mg/kg intravenously was simultaneously effective in rescuing and preventing depression by increasing BPM and MV.LCX001 could rapidly stabilize the respiratory function of opioid and propofol-induced respiratory depression,and restore respiratory frequency and minute ventilation to basal levels,which completely reverse the respiratory depression caused by opioid and propofol in rats.LCX001 also elevates the breathing parameters of pentobarbital-induced respiratory depression,and increased the BPM and MV values by about 20-30%gradually,which partially counteracted the respiratory depression induced by pentobarbital in rats.2.Results of screening and evaluation of brain-targeted prodrugs on anti-respiratory depression.2.1 XD-8-17C protected mice against acute death induced by TH-030418Acute toxicity experiment was used for the Screening of prodrugs.Intravenous administration of brain-targeted prodrug compound XD-8-17C（1,3,10 mg/kg）significantly increased the survival rate of mice.After the treatment of XD-8-17C,lethal dose-response curves for TH-030418 alone was right-shifted and XD-8-17C pre-treatment increased the LD₅₀ value,which was 4.7-fold higher than for TH-030418alone.2.2 The effect of XD-8-17C on enhancing arterial blood gas parametersThe test of arterial blood gas parameters showed that severe respiratory depression was induced by TH-030418（20μg/kg）in rats,and arterial oxygen partial pressure（pO₂）rapidly decreased from about 90 mmHg to 38 mmHg.Oxygen saturation（s O2）rapidly decreased from 94%to 32%.Compared with TH-030418 treatment group,intravenous administration of LCX001（10 mg/kg）or XD-8-17C（3 mg/kg）could rapidly reverse the decrease in partial pressure of oxygen and oxygen saturation,which protected the suppressed respiration.At the detection on time point of 60 min,XD-8-17C performed better effect on recovering pO2 and sO2 than LCX001.2.3 The effect of XD-8-17C on enhancing respiratory parametersThe pulmonary test suggested that severe respiratory depression was induced by TH-030418（20μg/kg）in rats,and the respiratory rate was decreased to about 30%of the baseline,and the minute ventilation was reduced to about 50%of the baseline.Compared with TH-030418 treatment group,intravenous administration of XD-8-17C（3 mg/kg）rapidly and effectively reversed opioid-induced respiratory parameters,and gradually restored and stabilized the respiration to the normal state.The effect of XD-8-17C（3 mg/kg）was basically consistent with LCX001（10 mg/kg）on the promotion of respiratory parameters.3.LCX001 facilitated the expression of stargazin and the surface expression of GluA2（R）-containing AMPA receptors in neurons.LCX001 treatment increase the expression of stargazin（AMPA receptor regulatory proteins）.Immunofluorescence staining and live cell imaging demonstrated that a significant rapid rise of fluorescent intensity was produced after LCX001 application and the rise was stable for at least 7 min.This finding indicated that LCX001 caused a rapid and steady increase in the number of surface GluA2 subunits,which was closely bound up with positively modulation on the behavior of AMPA-GluA2 receptors.4.Effect of LCX001 on the kinetic properties of AMPA ion channels and cell excitability.LCX001 significantly enhanced the amplitude of AMPA receptor inward current,facilitated channel opening,and slow down the process of currents attenuation.When cells were recorded in the presence of modulators,co-application of LCX001 caused a significant leftward shift in the dose-response curve for glutamate,and the EC₅₀ value was decreased from 3.43 mM to 2.76 mM after LCX001 treatment.This evidence indicates that LCX001 facilitated the binding ability and sensibility of endogenous neurotransmitter glutamate on recombinant AMPA receptors.100μM LCX001potentiated the glutamate（3.5 mM）response by 711±20（pA）as compared with that of432±27（pA）in the control group.At 10 mM glutamate evoked amplitudes,LCX001 at100μM increased the potency of glutamate induced currents by 1120±60（pA）,compared with that of 752±35（pA）in the control group.LCX001 also prominently promoted steady state/peak amplitude ratio.The results indicated that LCX001significantly slowed down the desensitization rates of the AMPA ion channel,and inhibited current decay.5.LCX001 regulated intracellular calcium concentration and decreased abnormal intracellular Ca²⁺load by fentanyl or TH-030418In the physiological state,predominant numbers of AMPARs in the central nervous system contain heteromers with GluA2（R）,which was impermeable to extracellular calcium influx.The results of Ca²⁺imaging indicated that fentanyl or TH-030418 could prominently enhance and up-regulate intracellular Ca²⁺.However,enhanced surface expression of GluA2（R）triggered by LCX001 regulated intracellular calcium concentration and decreased abnormal intracellular Ca²⁺load by fentanyl or TH-030418.The extraordinary property of LCX001 played a neuroprotective effect,and indirectly participated in respiratory protection.Conclusion:1.A novel ampakine compound,LCX001,which can alleviate suppressed respiration by different analgesics and sedative hypnotics.2.As a LCX001 brain-targeted prodrug of LCX001,XD-8-17C achieved an identical or better therapeutic effect than LCX001 on respiratory protection at a lower dose,the results indicated that XD-8-17C was more potent compound on alleviation of respiratory depression.3.By improving the expression of stargazing,LCX001 facilitated the binding ability and sensibility of glutamate on recombinant AMPA receptors,slowing down the desensitization rates of the AMPA ion channel,and increase the surface expression of GluA2（R）as well,which increased the number of receptors in cell surface.These all resulted in a typical positive modulatory impact on AMPAR-mediated function and cell excitability.Importantly,LCX001 application generates the increase in GluA2（R）surface expression,and potently restrains the abnormal intracellular Ca²⁺load,which is possibly a new action of ampakines in modulating breathing and neuroprotection.