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 K_d values against ketamine (86.4 ± 3.2 nM) and 6-hydroxynorketamine (74.1 ± 7.8 nM) and a 90-fold weaker K_d 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; K_d 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.

Summary

■ INTRODUCTION

Ketamine is an injectable dissociative-hypnotic that was initially approved for human and veterinary use to induce and maintain anesthesia. It has since attracted additional attention due to its utility as an adjunctive, opioid-sparing analgesic, and its rapid and sustained antidepressant effects.

Several technologies have been implemented for the detection of ketamine in biological matrices, including chromatographic, mass spectrometric, fluorometric, and immunologic approaches. The last approach, enzyme-linked immunosorbent assays (ELISA), is of unique interest in comparison to the others, as it implicitly relies on the development of antibodies that can recognize the structure of a unique small molecule.

Recent studies have used structurally specific antidrug antibodies to mechanistically dissect psychoactive chemical mixtures. However, the relationship between ketamine hapten design and resulting antibody properties remains unexplored, and the performance of antiketamine antibodies as either overdose reversal agents or as tools for mechanistic analysis in DISSECTIV depends on both affinity and selectivity.

We synthesized an exemplar hapten for norketamine, assessed the effect of varying conjugation conditions on hapten density, and determined the effect of carrier protein identity on immunologic and functional outcomes.

■ RESULTS AND DISCUSSION

We synthesized a hapten from norketamine using a five-carbon spacer, a hexanoic acid linker, and a sodium triacetoxyborohydride reaction. This hapten was then saponified using aqueous sodium hydroxide to yield a N-hexanoic acid norketamine hapten (NK-N-COOH, 11).

The hapten coupling efficiency and copy number of ketamine bioconjugates were systematically assessed using N-hydroxysuccinamide (NHS)ester chemistry. The highest copy number was achieved using sulfo-N-hydroxysuccinamide (Sulfo-NHS), an activation pH of 5.0, 20 equiv of EDC, and a reaction temperature of 4 °C.

A small-molecule conjugate vaccine is made up of three components: a hapten, an immunogenic carrier protein, and an adjuvant system. We compared the effects of two commonly used immunogenic carrier proteins, keyhole limpet hemocyanin (KLH) and a genetically modified, detoxified form of diphtheria toxin (CRM197), to deliver the vaccine candidate.

ELISA was used to assess the immunologic efficacy of these vaccine formulations. CRM-NK produced a much more robust antibody response against BSA-NK than KLH-NK, and neither mock vaccine produced antibodies that could bind BSA-NK.

In an open field task, NK-vaccinated animals demonstrated a significantly different pattern of behavior from mock-vaccinated animals when challenged with ketamine. Additionally, NK vaccination was able to mitigate the antinociceptive effects of ketamine in both reflexive and voluntary pain response assays.

We wanted to determine if we could develop vaccines to distinguish between ketamine and its active metabolites, such as the (2R,6R) hydroxynorketamine species, using the same antibody. However, the structural similarities between ketamine and 6-hydroxynorketamines make this process substantially more challenging.

Effective hapten design can lead to the generation of antibodies that can preferentially bind to one of several structurally similar compounds. We generated two additional haptens, KET-N-COOH and 6-HNK-N-COOH, and compared their performance with ketamine.

A three-injection series of CRM-KET, CRM-NK, or CRM-HNK was used to vaccinate animals. Sera from these vaccinated animals were tested against wells coated with either BSA-KET, BSA-NK, or BSA-HNK to account for potential differences in antibody reactivity against any single immobilized antigen.

CRM-NK generated the lowest mean midpoint titer, CRM-KET generated the next lowest titer, and CRM-HNK generated the highest titer to its own coating antigen.

The antibodies generated by these vaccines were tested for binding to ketamine, norketamine, or 6-hydroxynorketamine using surface plasmon resonance. The CRM-NK vaccine generated the weakest antibodies overall, with a Kd around 5 mM for norketamine and above 300 nM for ketamine and 6-hydroxynorketamine, respectively.

After vaccination with CRM-KET, CRM-NK, or CRM-HNK, the ability of these interventions to modify behavioral outcomes was assessed. The effect of vaccination with CRM-KET, CRM-NK, or CRM-HNK on reversing the effects of 15 mg/kg IP ketamine was significantly different from one another overall.

Ketamine reduced the time unvaccinated animals spent immobile in comparison to those receiving a vehicle injection. The 6-hydroxynorketamine component of ketamine is necessary for its antidepressant-like activity in rodent models.

CRM-KET vaccination did not generate high enough antibodies to reverse ketamine activity, but CRM-HNK vaccination did. This is likely because CRM-KET vaccination generated only low titers of antibodies against ketamine and 6-hydroxynorketamine.

CRM197-linked bioconjugates designed to mimic the structure of ketamine can generate robust antibody responses in vivo, and can alter the psychoactive effects of ketamine at high drug burdens. Furthermore, hapten structures containing either secondary or tertiary structures at the position of the amine can yield antibodies that bind ketamine and 6-hydroxynorketamine with approximately equal affinity.

CRM-HNK vaccination is likely to be useful in the elucidation of postmetabolic contributions to ketamine’s polypharmacologic mechanism of action, including the reduction of effects in behavioral models of antidepressant-like activity, anxiolytic-like activity, and reward-like conditioned place preference.

CRM-HNK vaccination blocks the behavioral effects of 15 mg/kg IP ketamine in the forced swim test. Although CRM-HNK-generated antibodies to recognize ketamine and norketamine have some activity, this activity is likely to be minimal.

The lack of efficacy of CRM-KET vaccination in the forced swim test demonstrates the need to optimize the concentrations of antibodies presented when considering application in the DISSECTIV approach.

In comparison to alternative methods, the DISSECTIV approach can preserve the native pharmacodynamic activity and pharmacokinetic profile of all chemical species upstream of the targeted compound. Therefore, further biochemical and behavioral studies with these haptens are planned to directly assess the pharmacokinetic consequences of vaccination with these haptens.

Funding

In this study, we used ELISA, SPR, and DISSECTIV to examine the effects of ketamine, norketamine, and 6-hydroxynorketamine on the blood-brain barrier.