Background and Goal of Study: Remimazolam is an ultra-short acting benzodiazepine under development for procedural sedation and general anesthesia. It is a benzodiazepine ester that is rapidly hydrolyzed by carboxylesterase (CES-1A) to an inactive metabolite (CNS7054). As the performance of CES1A during long-term-exposure to remimazolam is unknown, the present study was designed to test the in-vitro metabolism of Remimazolam under conditions allowing continuous exposure to remimazolam and its principal metabolite. 

Materials and Methods: Possible changes in Remimazolam metabolism upon long-term exposure were investigated in an in vitro 3D bioreactor system, which allows for high-density perfusion culture of cells reflecting the natural situation in the organ. Bioreactors were inoculated with primary human hepatocytes (n=4) and Remimazolam was continuously infused into the bioreactors over 5 days (final concentration 3000 ng/mL =6.8 µM) in parallel with untreated control bioreactors. Static 2D cultures of hepatocytes from the same donors were investigated as a further control of cell viability and possible cytotoxicity of Remimazolam, though with non-continuous application of the drug via daily medium exchange. 

Results and Discussion: Measurements of metabolic and clinical chemistry parameters (glucose, lactate, urea, LDH, AST, ALT, GLDH) in culture supernatants indicated no noxious effect of Remimazolam or its metabolite on hepatocyte integrity in 3D bioreactors or 2D cultures. Analysis of the time-course of Remimazolam and CNS7054 in 3D bioreactors revealed values in the range of unbound concentrations observed in patients. Remimazolam levels declined within the first 8 h (initial value approx. 1,600, steady-state around 250 ng/ml). Concentrations of CNS7054 showed an inverse time-course reaching average values of 1,500 ng/ml. In 2D cultures Remimazolam was completely metabolised during 24h until the next medium exchange over the whole experiment duration of 5 days, with a corresponding increase of the metabolite. Analysis of mRNA expression levels of CES-1 indicated no changes in enzyme expression over the culture period. 

Conclusions: The results of the present study indicate a stable function of CES1A during a 5 day continuous exposure to clinically relevant concentrations of Remimazolam. Moreover, there was no indication for a harmful effect of Remimazolam exposure on integrity and metabolic activity of primary human hepatocytes.

Metabolism of remimazolam in primary human hepatocytes during continuous long-term infusion (five days) in a 3D bioreactor system