key: cord-102778-b1ul7zug authors: Serrao, Juliet.M.; Goodchild, Colin.S. title: Alfaxalone activates Human Pregnane-X Receptors with greater efficacy than Allopregnanolone: an in-vitro study with implications for neuroprotection during anesthesia date: 2020-09-06 journal: bioRxiv DOI: 10.1101/2020.09.05.284075 sha: doc_id: 102778 cord_uid: b1ul7zug Background Alfaxalone is a fast acting intravenous anesthetic with high therapeutic index. It is an analogue of the naturally-occurring neurosteroid, allopregnanolone which has been implicated in causing neuroprotection, neurogenesis and preservation of cognition, through activation of pregnane X receptors in the central nervous system. This study investigated whether alfaxalone can activate human pregnane X receptors (h-PXR) as effectively as allopregnanolone. Methods Allopregnanolone and alfaxalone were dissolved in dimethyl sulfoxide to make allopregnanolone and alfaxalone treatment solutions (serial 3-fold dilution concentration range, 50,000 – 206 nM). Activation of h-PXR by these ligand solutions compared with vehicle control was measured by an in-vitro method using human embryonic kidney cells (HEK293) expressing h-PXR hybridised and linked to the firefly luciferase gene. Ligand binding with and activation of h-PXR in those cells caused downstream changes in luciferase activity and light emission. That activity was measured as relative light units using a plate-reading luminometer, thus quantifying the changes in h-PXR activity caused by the ligand applied to the HEK293 cells. Ligand log concentration response curves were constructed to compare efficacy and potency of allopregnanolone and alfaxalone. Results Allopregnanolone and alfaxalone both activated the h-PXR to cause dose-related light emission by the linked firefly luciferase. Control solutions (0.1% dimethyl sulfoxide) produced low level light emissions. Equimolar concentrations of alfaxalone were more efficacious in activation of h-PXR: 50,000 nM, p = 0.0019; 16,700 nM, p = 0.0472; 5,600 nM, p = 0.0031 [Brown-Forsythe and Welch ANOVA]. Conclusions Alfaxalone activates human-pregnane X receptors with greater efficacy compared with the endogenous ligand allopregnanolone. These results suggest that alfaxalone sedation and anesthesia may be accompanied by beneficial effects normally caused by the physiological effects of allopregnanolone, namely neuroprotection, neurogenesis, and preservation of cognition. Pregnane X Receptor (PXR; NR112) is a nuclear receptor that binds with, and is activated by, a variety of xenobiotic and endogenous compounds, including naturally occurring steroids, pregnenolone, progesterone and allopregnanolone 1;2 . Allopregnanolone is a metabolite of progesterone synthesized in the central nervous system where it promotes neurogenesis and neuroplasticity 3;4 . PXR activation by allopregnanolone has also been linked to neuroprotection 5 . Further, these properties are due to PXR activation by allopregnanolone stimulating the production of brain-derived neurotrophic factor (BDNF) [5] [6] [7] [8] . The most important functions of BDNF include: regulation of neuro-, glio-, and synapto-genesis; neuroprotection; and control of short-and long-lasting synaptic interactions responsible for memory and cognition 9 . Alfaxalone, (3α-hydroxy-5α-pregnane-11,20-dione ) is a pregnane steroid which has potent anesthetic and sedative properties by actions at gamma aminobutyric acid type A (GABAA) receptors 10 . It is an allopregnanolone and progesterone analogue ( Figure 1 ), but it is devoid of conventional progestogen or endocrine hormonal activity 11 . An aqueous formulation of alfaxalone (Phaxan™) has been developed for use as an intravenous sedative and anesthetic 12;13 . This development occurs at a time when most commonly-used anesthetics are under investigation for neurotoxic effects [14] [15] [16] . Alfaxalone differs from those commonly used anesthetics in that it is a structural analogue of a naturally occurring neuroprotective hormone, allopregnanolone. It is unknown whether alfaxalone can activate human pregnane X receptors (h-PXR) although its structural similarity to allopregnanolone suggests that it may do so. If it is found that alfaxalone can activate mechanisms utilized by allopregnanolone to cause neuroprotection, e.g., PXR, there would be implications for the use of alfaxalone in clinical anesthetic practice. This study set out to compare the activation of h-PXR in-vitro by equimolar concentrations of allopregnanolone and alfaxalone. The test compounds alfaxalone and allopregnanolone were obtained from Sigma-Aldrich (400 Summit Drive, Burlington, MA 0180). Table 1 . The concentration of residual DMSO in all assay wells was 0.1%. Assay plates were incubated for 22-24 h in a cell culture incubator (37°C / 5% CO2 / 85% humidity). Following the incubation period, the treatment media were discarded and 100 μL of luciferase detection reagent (Indigo Biosciences) was added to each well. The resulting luminescence (relative light units; RLUs) was measured in each well using a luminometer. The recording from each well was entered into an Excel spreadsheet. Agonist concentration/activity response curves were plotted with non-linear curve fitting, using The results of the study reported here show that alfaxalone does bind with and activates h-PXR and further that it is a more efficacious ligand than allopregnanolone in activating h-PXR in this model. Conclusions about the relative potency of the two pregnane ligands were not possible because the plateau portion of the alfaxalone log10 concentration response curve was not demonstrated. This was due to the inability to dissolve alfaxalone in water using 0.1% DMSO at concentrations higher than 50,000 nM. This result suggests that sedation and anesthesia achieved using alfaxalone may be accompanied by the effects caused by interaction of allopregnanolone with brain PXR such as stimulating the production of BDNF [5] [6] [7] [8] , which has been shown to have important roles in regulation of neurogenesis, gliogenesis, and synaptogenesis, as well as in neuroprotection, and control of short-and long-lasting synaptic interactions that determine memory and cognition 9;17 . See Figure 3 for a schematic representation of the relationships between neurosteroids, PXR and BDNF and their effects on brain and neuronal function under conditions of anesthesia, stress, trauma, and inflammation. PXR activation by alfaxalone may also inhibit microglial hyperinflammatory responses in the central nervous system via neuroimmune regulatory proteins, such as CD55 18;19 . Glial-mediated inflammation in the CNS is caused by many factors such as trauma, hypoxia, stress, and also by β-amyloid in Alzheimer's disease, all of which are common co-morbidities that complicate surgery and lead to poorer postoperative cognitive function [19] [20] [21] . Glial-mediated inflammation is also a cause of deficits in cognition after bacterial and viral infections, such as COVID-19 22 . Alfaxalone sedation and anesthesia with simultaneous activation of PXR as described above clearly has the potential for improved CNS recovery. Arrows denote a causal relationship, red for deleterious and blue for beneficial effects on brain function. A "+" or "-" next to an arrow indicates a positive effect or negative effect on the target of the arrow. Small numbers adjacent to arrows refer to papers in the reference list, red background denoting deleterious effects and blue denoting beneficial effects Alfaxalone is being developed for sedation and anesthesia in humans 12;13 . This occurs at a time of controversy in anesthesia and critical care. Many preclinical studies published in the last two decades have reported neuronal damage and long-lasting cognitive impairment after administration of anesthetics, particularly to neonates and juveniles 23 have been linked with acute delirium and long-term deficits in cognition in older persons 24 . These have been linked to low levels of BDNF 25 . There are reports citing precise mechanisms for neurotoxicity of anesthetic drugs per se. Liu et al showed that propofol causes dose-dependent neuronal cell death at clinically relevant concentrations through a mitochondrial pathway; Akt (protein kinase B)/glycogen synthase kinase-3 (GSK3) 26 . See Figure 3 . This mechanism for neurotoxicity is also activated by ketamine and inhalational agents such as isoflurane [27] [28] [29] . Further, the apoptosis caused by persistent neuroinflammation has been shown to involve the same Akt pathway as shown in inflammation is well established as a mechanism 34 . Preclinical studies have shown that progesterone and allopregnanolone suppress that inflammatory response and the apoptotic enzyme caspase-3, and also decrease cerebral infarct volume after experimental brain injury 35 . Cervantes et al showed that a single anesthetic dose of alfaxalone administered immediately after restoration of normal brain perfusion prevented severe neurological damage caused by 8 minutes cardiorespiratory arrest in cats 36 . More recently it has been reported that alfaxalone is not neurotoxic to the developing brain 14 . This report is reassuring but the results of the study reported herein suggest that alfaxalone anesthesia may also be accompanied by positive effects on neuronal function mediated by PXR activation, protecting neurons from the adverse effects of surgical stress and other comorbidities commonly found in patients presenting for surgery [37] [38] [39] . The limitation of this study lies in question of relevance of the in vitro system to the in vivo situation. The in vitro cell culture approach provides benefits of investigating h-PXR activation under controlled conditions without interference of confounding physiological or pathological factors seen in the intact animal model. However, the in vitro system is artificial and interactions between different cell types in intact animals is missing. It is therefore important to confirm in vitro findings using an intact animal model. To that end, it is important to note data already published by Yawno and colleagues that showed normal anesthetic doses of alfaxalone can replace allopregnanolone in the control of apoptosis in fetal lambs, an effect of allopregnanolone-PXR referred to above 33 . Although the study reported herein used in vitro methods, the naturally occurring and active hormone, allopregnanolone, was used as an active control. The results show that allopregnanolone was active in this model and further that alfaxalone was more efficacious than allopregnanolone in h-PXR activation. 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