Alfaxalone activates Human Pregnane-X Receptors with greater efficacy than Allopregnanolone: an in-vitro study with implications for neuroprotection during anesthesia

Abstract

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 (hPXR) 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 to 206 nM). Activation of hPXR by these ligand solutions compared with vehicle control was measured by an in vitro method using human embryonic kidney cells (HEK293) expressing hPXR hybridised and linked to the firefly luciferase gene. Ligand binding with and activation of hPXR 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 hPXR 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 hPXR 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 hPXR: 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.

Synthetic neuroactive steroids as new sedatives and anaesthetics: Back to the future

“…Coincubation with allopregnanolone reversed these effects. Another possible mechanism comes from an unpublished study by Serrao and Goodchild 169 …”

General Anesthesia and the Young Brain: The Importance of Novel Strategies with Alternate Mechanisms of Action

“…Another possible explanation could be that their pharmacological effects are not only mediated via the cellular membrane receptors, but also via the nuclear receptors. For example, the work by Serrao et al (2020) suggests that both allopregnanolone and alphaxalone activate pregnane-x-receptor (PXR) [82], a ligand-activated nuclear receptor that controls inducible expression of biotransformation enzymes and drug transporters [83,84]. Importantly, PXR is known to cross-talk with other nuclear receptors, thus controlling coactivation and activity of many transcription factors [83,84].…”

Alfaxalone Alleviates Neurotoxicity and Cognitive Impairment Induced by Isoflurane Anesthesia in Offspring Rats

“…12 ALF is a steroid anesthetic and an analog of tetrahydro-progesterone. 13 A study demonstrated that ALF was not associated with abnormal neuronal apoptosis or persistent impairment of spatial memory in young animals. 14…”

“…12 ALF is a steroid anesthetic and an analog of tetrahydro-progesterone. 13 A study demonstrated that ALF was not associated with abnormal neuronal apoptosis or persistent impairment of spatial memory in young animals. 14 This study aimed to determine the protective effects of ALF on gestationally exposed fetuses and postnatal offspring rats against isoflurane-induced neurotoxicity and cognitive impairment.…”