Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells

Luigia Rossi, Francesca Pierigè, Marco Agostini, Noemi Bigini, Veronica Termopoli, Yingting Cai, Fang Zheng, Chang Guo Zhan, Donald W. Landry, Mauro Magnani

Research output: Contribution to journalArticlepeer-review

Abstract

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Original languageEnglish
Article number573492
JournalFrontiers in Physiology
Volume11
DOIs
StatePublished - Sep 10 2020

Bibliographical note

Funding Information:
The authors would like to thank the staff of the Blood Transfusion Center of the Hospital “S. Maria della Misericordia” of Urbino for their collaboration in providing human blood. Funding. This work was partially supported by EryDel S.p.A. The authors declared that this study received funding from EryDel S.p.A. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Publisher Copyright:
© Copyright © 2020 Rossi, Pierigè, Agostini, Bigini, Termopoli, Cai, Zheng, Zhan, Landry and Magnani.

Keywords

  • E196-301 stability
  • RBP 8000 stability
  • cocaine degradation
  • cocaine esterase
  • cocaine use disorder
  • red blood cells as drug delivery system

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

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