Generation of enantiospecific monoclonal antibodies against (2R,6R)-hydroxynorketamine.
Uriel Matthew Enriquez, Natalie M González Velázquez, Mohammad Mosharraf Hossain, Jillian L Kyzer, Ilia A. Guzei, Cody J. Wenthur
Bioorganic & Medicinal Chemistry January 17, 2026 DOI: 10.1016/j.bmc.2026.118573 (opens in new tab)
Study at a glance
AI-extracted from the abstract| Characteristics | Preclinical experimental study Peer reviewed |
|---|---|
| Interventions | (2R racemic 6-hydroxynorketamine-CRM hapten |
| Topics | Ketamine |
| Keywords | Bioconjugate chemistry Hydroxynorketamine Monoclonal antibody development |
| Key findings | Monoclonal antibody 6F11-HC1-LC2 binds (2R,6R)-hydroxynorketamine with sub-nanomolar affinity (0.4 nM) and over 150-fold selectivity over ketamine, with no cross-reactivity to the (2S,6S) enantiomer. |
Abstract
Antibodies against small psychoactive molecules have been developed for applications ranging from substance detection and overdose protection to mechanistic understanding of the actions of complex substance mixtures. In this study, we describe the design, synthesis, formulation, and animal testing of an initial immunogenic bioconjugate, as well as subsequent isolation of enantiospecific monoclonal antibodies against (2R,6R)-hydroxynorketamine, a putatively active metabolite of the dissociative-anesthetic and rapidly-acting antidepressant ketamine. Following pharmacophore synthesis, hapten generation was achieved through installation of 6-aminohexanoic acid linkers using reductive amination. Bioconjugation to the carrier protein, cross-reactive material 197 (CRM) was achieved via amide coupling. Upon administration to mice in combination with alum and CpG oligodeoxynucleotide 1826, (2R,6R)-hydroxynorketamine-CRM generated equivalent antibody titers to a comparator racemic 6-hydroxynorketamine-CRM hapten. Following creation of hybridomas arising from B-cells responsive to (2R,6R)-hydroxynorketamine-CRM exposure and subsequent screening, subcloning, sequencing, and production, we were able to identify a monoclonal antibody, 6F11-HC1-LC2, which yielded antibodies strongly responsive to (2R,6R)-hydroxynorketamine, but showed no responsiveness to (2S,6S)-hydroxynorketamine in a competitive binding enzyme-linked immunosorbent assay format. Surface plasmon resonance analysis of this monoclonal species demonstrated sub-nanomolar (0.4 nM) antibody affinity for (2R,6R)-hydroxynorketamine-BSA bioconjugates and > 150-fold selectivity in comparison to ketamine-BSA bioconjugates. Overall, these results demonstrate successful production of monoclonal antibodies capable of robustly distinguishing between hydroxynorketamine enantiomers, enabling their use in future in vivo studies to better understand their relative contributions to the rapidly-acting antidepressant properties of ketamine.
Comparable studies
Other preclinical and animal studies on ketamine, most cited first.
| Study | Year | Design | Participants |
|---|---|---|---|
| mTOR-Dependent Synapse Formation Underlies the Rapid Antidepressant Effects of NMDA Antagonists Rats | 2010 | Observational study | |
| Activation of Glutamatergic Neurotransmission by Ketamine: A Novel Step in the Pathway from NMDA Receptor Blockade to Dopaminergic and Cognitive Disruptions Associated with the Prefrontal Cortex Conscious rats | 1997 | Dose-response study with microdialysis and behavioral testing | |
| NMDAR inhibition-independent antidepressant actions of ketamine metabolites Mice | 2016 | Preclinical study | |
| The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N‐methyl‐aspartate Spinal neurons in decerebrate or pentobarbitone-anaesthetized cats and rats | 1983 | Experimental study | |
| R-ketamine: a rapid-onset and sustained antidepressant without psychotomimetic side effects Mice | 2015 | Experimental study |
Citations in the library
Cites 7
- Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function Proceedings of the National Academy of Sciences February 22, 2019
- ( 2R,6R )-hydroxynorketamine exerts mGlu 2 receptor-dependent antidepressant actions Proceedings of the National Academy of Sciences March 13, 2019
- A Phase 1 Assessment of the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of (2R,6R)-Hydroxynorketamine in Healthy Volunteers. Clinical Pharmacology & Therapeutics November 1, 2024
- Loss of the sustained antidepressant-like effect of (2R,6R)-hydroxynorketamine in NMDA receptor GluN2D subunit knockout mice. Journal of Pharmacological Sciences March 1, 2024
- The antidepressant potential of (2R,6R)-hydroxynorketamine: A detailed review of pre-clinical findings. European Journal of Pharmacology July 15, 2025
and 2 more in the library