Family of Structurally Related Bioconjugates Yields Antibodies with Differential Selectivity against Ketamine and 6-Hydroxynorketamine

As ketamine is being increasingly used in therapeutic settings, this in vivo study explored the possibility of developing an antidote for ketamine intoxication/overdose. To do so, three hapten molecules (small molecules that elicit an immune response only when attached to a large carrier such as a protein) similar in structure to ketamine were synthesized. All three haptens elicited immune responses in mice, leading to the production of antibodies with varying affinities for ketamine. These findings indicate a potential approach toward mitigating ketamine overdose and further studying the antidepressant effects of ketamine and its metabolites.

Abstract

The dissociative-hypnotic compound ketamine is being used in an increasingly wide range of therapeutic contexts, including anesthesia, adjunctive analgesia, treatment-resistant depression, but it also continues to be a notable substance of abuse. No specific antidotes exist for ketamine intoxication or overdose. Immunopharmacotherapy has demonstrated the ability to offer overdose protection through production of highly specific antibodies that prevent psychoactive drug penetration across the blood–brain barrier, although antiketamine antibodies have not yet been assessed or optimized for use in this approach. Moreover, generation of specific antibodies also provides an opportunity to address the role of 6-hydroxynorketamine metabolites in ketamine’s rapid-acting antidepressant effect through selective restriction of metabolite access to the central nervous system. Hapten design is a critical element for tuning immune recognition of small molecules, as it affects the presentation of the target antigen and thus the quality and selectivity of the response. Here, we report the synthesis and optimization of carrier protein and conjugation conditions for an initial hapten, norketamine-N-COOH (NK-N-COOH), to optimize vaccination conditions and assess the functional consequences of such vaccination on ketamine-induced behavioral alterations occurring at dissociative-like (50 mg/kg) doses. Iterating from this initial approach, two additional haptens, ketamine-N-COOH (KET-N-COOH) and 6-hydroxynorketamine-N-COOH (HNK-N-COOH), were synthesized to target either ketamine or 6-hydroxynorketamine with greater selectivity. The ability of these haptens to generate antiketamine, antinorketamine, and anti-6-hydroxynorketamine immune responses in mice was then assessed using enzyme-linked immunosorbent assay (ELISA) and competitive surface plasmon resonance (SPR) methods. All three haptens provoked immune responses in vivo, although the KET-N-COOH and 6-HNK-N-COOH haptens yielded antibodies with 5- to 10-fold improvements in affinity for ketamine and/or 6-hydroxynorketamine, as compared to NK-N-COOH. Regarding selectivity, vaccines bearing a KET-N-COOH hapten yielded an antibody response with approximately equivalent Kd values against ketamine (86.4 ± 3.2 nM) and 6-hydroxynorketamine (74.1 ± 7.8 nM) and a 90-fold weaker Kd against norketamine. Contrastingly, 6-HNK-N-COOH generated the highest affinity and most selective antibody profile, with a 38.3 ± 4.7 nM IC50 against 6-hydroxynorketamine; Kd values for ketamine and norketamine were 33- to 105-fold weaker, at 1290 ± 281.5 and 3971 ± 2175 nM, respectively. Overall, these findings support the use of rational hapten design to generate antibodies capable of distinguishing between structurally related, yet mechanistically distinct, compounds arising from the same precursor molecule. As applied to the production of the first-reported anti-6-hydroxynorketamine antibodies to date, this approach demonstrates a promising path forward for identifying the individual and combinatorial roles of ketamine and its metabolites in supporting rewarding effects and/or rapid-acting antidepressant activity.”

Authors: Zhen Zheng, Jillian L. Kyzer, Adam Worob & Cody J. Wenthur

Notes

Ketamine is increasingly being used in therapeutic settings to treat mental disorders such as anxiety and depression. However, ketamine continues to be a notable drug of abuse and despite its relatively safe pharmacological profile, ketamine intoxication/overdose can occur at high doses. At present, there is no antidote available to reverse the effects of ketamine overdose.

The present study explored the possibility of generating highly specific antibodies which would prevent ketamine from crossing the blood-brain barrier once bound to the drug. To do so, researchers developed carrier protein and three hapten molecules similar in structure to ketamine and its metabolites. These hapten molecules are small molecules that elicit an immune response only when attached to a large carrier such as a protein. The ability of these haptens to generate anti-ketamine immune responses was assessed in mice, with the evoked immune response being assessed using an ELISA assay and SRP methods.

The main findings:

  • All three haptens provoked an immune response in vivo although KET-N-COOH and 6-HNK-N-COOH haptens yielded antibodies with 5- to 10-fold improvements in affinity for ketamine and/or 6-hydroxynorketamine
  • 6-hydroxynorketamine-N-COOH (HNK-N-COOH) generated the highest affinity and most selective antibody profile
  • The circulating antibodies generated from the immune response demonstrated the ability to alter the psychoactive effects of ketamine

The present study provides preliminary evidence for the use of vaccination against ketamine as a potential approach toward mitigating ketamine overdose symptoms. In theory, the antibodies should bind to freely circulating ketamine which creates a complex too large to cross the blood-brain barrier and therefore, prevent ketamine from bringing about both its rewarding and adverse effects.

Additionally, the methodology used here can be used to further explore metabolite-driven polypharmacologic mechanisms in the antidepressant effects of ketamine i.e how ketamine and its metabolites bind to different targets/receptors in the body to exert therapeutic or adverse effects.

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