This mouse study investigated the development of immunopharmacotherapies to generate antibodies against ketamine and its metabolites. It finds that specific hapten designs successfully elicited antibody responses with high affinity for ketamine or 6-hydroxynorketamine, offering a potential pathway for treating overdose or restricting metabolite access to the brain.
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.
Papers cited by this study that are also in Blossom
Zanos, P., Moaddel, P. J., Morris, P. J. et al. · Nature (2016)
Zanos, P., Thompson, S. M., Duman, R. S. et al. · CNS Drugs (2018)
Ketamine is a dissociative-hypnotic with growing therapeutic uses—anesthetic, adjunctive analgesic and a rapid-acting antidepressant in certain formulations—yet it is also widely misused and can cause overdose for which no specific antidote exists. Earlier work has established that conjugate vaccines can elicit high-affinity antibodies against small-molecule drugs (e.g. opioids, nicotine) that sequester drug in the periphery and prevent blood–brain barrier penetration; however, antiketamine antibodies have been developed primarily for detection (ELISA) and their potential as immunopharmacotherapeutics or mechanistic tools has not been systematically explored. A particular mechanistic question motivating this study is whether ketamine's rapid antidepressant effects arise from the parent compound, from metabolites such as 6-hydroxynorketamine (6-HNK), or from a combination of both. Zheng and colleagues set out to examine how rational hapten design and carrier/conjugation conditions affect the affinity and selectivity of antibodies raised against ketamine and its metabolites. The study synthesised three related haptens—NK-N-COOH (norketamine-targeting), KET-N-COOH (ketamine-targeting), and 6-HNK-N-COOH (6-hydroxynorketamine-targeting)—optimised conjugation to carrier proteins, assessed resulting antibody titres and affinities using ELISA and competitive surface plasmon resonance (SPR), and tested functional consequences on ketamine-induced behaviours (locomotion, nociception, and an antidepressant-like forced swim test). The broader aim was both to evaluate immunopharmacotherapy for ketamine intoxication and to provide selective immunologic tools for dissecting metabolite contributions to ketamine's behavioural effects.
Male Swiss Webster mice (6–8 weeks old) were used for all in vivo experiments; the authors note known sex differences in ketamine pharmacology and plan a parallel study in females. All animal procedures complied with institutional and NIH guidelines. Haptens were prepared by reductive amination to append a six-carbon (hexanoic acid) linker to either norketamine, ketamine, or 6-hydroxynorketamine, yielding NK-N-COOH, KET-N-COOH, and 6-HNK-N-COOH, respectively; full organic synthesis and characterisation details are reported in the Supporting Information. Hapten–protein conjugates were formed by NHS-ester chemistry and quantified by MALDI-ToF mass spectrometry to determine hapten copy number per carrier (copy number = (MW conjugate − MW carrier)/(MW hapten − MW water)). Vaccine formulations combined 100 μg hapten–protein conjugate per mouse with alum (500 μg) and CpG ODN 1826 (50 μg). Animals were randomly assigned and immunised subcutaneously on days 0, 14 and 28; serum was collected on days 21 and 35 for midpoint antibody titre determination by ELISA. ELISAs used BSA-hapten coating antigens and HRP-conjugated anti-mouse IgG for detection; titres were defined as the dilution yielding 50% of maximum absorbance. For affinity and selectivity, purified IgG (Protein G eluate) underwent competitive binding SPR on BSA-hapten-coated chips; increasing concentrations of free ketamine, norketamine or 6-HNK (1 nM–1 μM) were preincubated with antibody and injected to derive IC50/Kd estimates from nonlinear competitive binding fits. Behavioural assays probed functional effects of vaccination. Open-field locomotor activity was measured for 1 h after IP ketamine at 12.5, 25 or 50 mg/kg, with repeated testing and ≥48 h washout between sessions. Antinociception was assessed at 50 mg/kg ketamine using tail-flick and hot-plate tests with standard cutoffs and % maximum possible effect (%MPE) calculation. Antidepressant-like activity was examined with the forced swim test (FST): mice received 15 mg/kg IP ketamine or saline and were tested 1 h later for immobility over a 6 min session (analysis on final 4 min). Statistical analyses included two-way ANOVA for dose × vaccine interactions, one-way ANOVA for group comparisons in the FST, and post hoc tests (Sidak’s or Tukey’s); significance was set at α = 0.05.
Hapten synthesis and optimisation established an N-hexanoic-acid linker strategy to present ketamine-related scaffolds while enabling NHS-mediated conjugation to carrier proteins. Systematic variation of NHS chemistry parameters for NK-N-COOH conjugation to BSA showed that lower activation pH, use of sulfo-NHS, increased EDC equivalents and lower temperature improved hapten loading; the highest mean copy number observed for BSA-NK was 11.3 under optimal conditions. CRM197 (detoxified diphtheria toxin) conjugates prepared using the same conditions yielded comparable copy numbers (CRM-NK ≈ 11.5); KLH conjugate copy numbers could not be assessed by MALDI due to size. Immunogenicity differed by carrier. When NK-N-COOH was conjugated to CRM197 (CRM-NK) versus KLH (KLH-NK) and administered with alum and CpG, CRM-NK produced substantially higher midpoint ELISA titres—approximately 40-fold greater at day 35—while carrier-only controls produced no anti-hapten signal. Purified IgG analysed by SPR showed that CRM-NK elicited relatively weak antibodies overall, with an approximate Kd ≈ 5 μM for norketamine and >300 nM for ketamine and 6-HNK, although the authors note the BSA-NK probe performed poorly in some assays. Behavioural testing indicated functional blockade by NK vaccination at high ketamine doses. In the open-field test, a significant dose × vaccine interaction was found (two-way ANOVA Finteraction(3,48) = 6.258, p = 0.001); both mock and NK-vaccinated mice showed hyperlocomotion at 12.5 and 25 mg/kg, but at 50 mg/kg mock-vaccinated mice displayed net sedation whereas NK-vaccinated mice exhibited a significantly different hyperlocomotive profile (Sidak’s p = 0.015 vs mock; p = 0.022 vs 0 mg/kg). NK vaccination also blocked ketamine-induced antinociception at 50 mg/kg in both tail-flick (t-test p = 0.002) and hot-plate assays (t-test p = 0.033). To probe selectivity, the authors synthesised KET-N-COOH and 6-HNK-N-COOH and conjugated them to CRM197 (CRM-KET and CRM-HNK). CRM-KET and CRM-HNK achieved higher hapten densities (CRM-KET ≈ 15.4 copies; CRM-HNK ≈ 21.8 copies). ELISA midpoint titres across groups showed CRM-NK generated the lowest titres, CRM-KET intermediate, and CRM-HNK the highest mean midpoint titre; CRM-HNK was the only vaccine whose strongest ELISA response was against its own coating antigen (BSA-HNK). SPR competitive binding revealed distinct affinity/selectivity profiles. CRM-KET antibodies bound ketamine and 6-HNK with Kd values around 80 nM and had much weaker interactions with norketamine. CRM-HNK antibodies showed the strongest and most selective binding to 6-HNK, with a Kd around 33–40 nM for 6-HNK and much weaker binding to ketamine (≈1.29 μM) and norketamine (≈3.97 μM). CRM-NK produced the weakest overall antibodies by SPR. These results align with the abstract-reported figures that KET-N-COOH and 6-HNK-N-COOH haptens yielded 5–10-fold affinity improvements over NK-N-COOH and that CRM-HNK offered 33–105-fold selectivity for 6-HNK versus ketamine or norketamine. Functional consequences in the forced swim test (15 mg/kg ketamine) supported a role for 6-HNK in ketamine’s antidepressant-like effect. Groups differed overall (ANOVA F = 7.68, p = 0.0001). Unvaccinated mice showed reduced immobility after 15 mg/kg ketamine (Tukey’s p = 0.0015 vs vehicle). CRM-NK vaccination did not reverse ketamine’s effect (Tukey’s p = 0.9897 vs 15 mg/kg), whereas CRM-HNK fully reversed ketamine-induced reduction in immobility (Tukey’s p = 0.0095 vs 15 mg/kg; not different from vehicle). CRM-KET showed an intermediate, non-significant phenotype. A post hoc analysis showed a significant linear trend when groups were ordered by overall ELISA titre (test for linear trend p = 0.0230), indicating that antibody titer contributed to functional reversal.
Zheng and colleagues interpret these data to show that rational hapten design can tune both affinity and selectivity of antibodies raised against ketamine and closely related metabolites. They highlight that CRM197 is an effective carrier for generating high titres relative to KLH in their system and that hapten copy number is sensitive to conjugation chemistry parameters such as activation pH, EDC equivalents and NHS reagent. The authors note that haptens presenting tertiary or secondary amine motifs at the nitrogen position can elicit antibodies that bind both ketamine and 6-HNK with similar affinities, whereas incorporation of a 6-hydroxyl moiety into a secondary-amine-bearing hapten (6-HNK-N-COOH) produced antibodies with markedly higher affinity and selectivity for 6-HNK. Relating their findings to the DISSECTIV strategy for dissecting metabolite contributions, the authors argue that CRM-HNK vaccination—which produced roughly 33-to 105-fold selectivity for 6-HNK—provides a practical tool for selectively sequestering 6-HNK in vivo without substantially perturbing upstream pharmacokinetics of parent compounds. Behavioural data from the forced swim test are interpreted as evidence that 6-HNK activity is a necessary component of ketamine’s antidepressant-like activity in this rodent assay, because only CRM-HNK (selective for 6-HNK) fully reversed the antidepressant-like effect of 15 mg/kg ketamine, whereas CRM-KET and CRM-NK did not. The authors acknowledge several constraints and future needs. They emphasise that antibody concentration (titer) and affinity jointly determine functional reversal and that CRM-KET’s intermediate phenotype likely reflects relatively low titres despite reasonable affinity. Consequently, isolation and production of monoclonal antibodies derived from these vaccines is proposed as a next step to control dose and timing of antibody administration and to remove titer variability. Additional limitations flagged include the potential influence of linker positioning and chemistry on selectivity, absence of hapten designs presenting a primary amine at the critical position, and the need to explore enantiomerically pure haptens to distinguish (2R,6R)- versus (2S,6S)-hydroxynorketamine effects. Finally, the authors recommend follow-up pharmacokinetic, biochemical and behavioural studies to clarify how vaccination alters drug distribution and downstream molecular or circuit-level endpoints (e.g. BDNF changes, hippocampal oscillations).
The study demonstrates that CRM197-linked bioconjugates based on rational hapten design can elicit circulating antibodies that alter ketamine’s psychoactive effects at high doses and that hapten structure controls antibody affinity and selectivity. In particular, a 6-hydroxynorketamine-directed hapten (6-HNK-N-COOH) produced antibodies with the highest affinity and strong selectivity for 6-HNK and was able to block ketamine’s antidepressant-like effect in the forced swim test at 15 mg/kg. The authors conclude that such selective antibodies both offer a potential avenue for mitigating ketamine overdose symptoms and provide a promising immunologic tool (within the DISSECTIV framework) for parsing the contributions of parent drug and metabolites to ketamine’s polypharmacologic actions. They propose further development of monoclonal antibodies, exploration of linker and stereochemical variants, and additional pharmacokinetic and behavioural studies as future directions.