Aggregating clinical use and purposeful laboratory monitoring

By Nicholas Armfield

Ketamine is gaining attention as an analgesic, antidepressant and as a treatment for refractory status epilepticus. Ongoing support from the clinical laboratory is improving patient monitoring and increasing our understanding of ketamine as a clinical therapeutic. Low-dose intravenous and a relative high volume of distribution ensues a low peripheral blood concentration. Ultra-high-performance liquid chromatography–tandem mass spectrometry methods are sensitive and specific platforms to measure ketamine and metabolites for clinical monitoring.

Background

Ketamine is gathering traction for use in pain medicine. Simultaneous hypnosis, amnesia and analgesia make ketamine clinically and pharmacologically unique. Antidepressant properties and facilitation of substance abstinence have been proven too, suggesting that the applications for ketamine are as close to neuro abundant as any other pharmaceutical [1]. Ketamine has a long and textured history of more than 60 years. A derivative of phencyclidine (PCP), ketamine was developed in the 1960s at Parke-Davis Laboratories, USA (now a subsidiary of Pfizer) as an alternative to PCP. PCP had caused postoperative psychosis, and a replacement was needed imminently. Ketamine proved to be significantly less harmful with a shorter duration of action and, notably, fewer hallucinations [2]. Soon it was licensed as an anesthetic, but before long it was compartmentalized as a drug of abuse. However, clinical applications for ketamine have burrowed in the background. Low dosing with a maximum clinical effect ensures ketamine is a safe xenobiotic in intravenous (IV), intramuscular (IM) and oral routes. Nasal-sprays and sub-lingual wafers have been developed as alternative administration routes [3,4].

Ketamine is a N-methyl-D-aspartate receptor (NMDAR) antagonist. This chief characteristic is attainable as ketamine passes the blood–brain barrier freely. Theory states response to stimuli – and therefore pain – is reduced following a reduction in glutamate activity in the synaptic cleft following ketamine administration. As the dose increases, ketamine may bind to a second site on NMDAR and further depolarize the synapse [5]. Mechanistically, an increase in dose strengthens the efficacy of ketamine. Ketamine exists as an enantiomer (R-ketamine and S-ketamine). S-ketamine (branded as Esketamine) is a more potent anesthetic as it has a higher affinity for NMDAR [1].

Rationale

The Walton Centre NHS Foundation Trust (WCFT) is a neuro-specific hospital located in Liverpool, UK. A range of patients are referred to this tertiary centre with acute or chronic backgrounds requiring neurological or neurosurgical interventions. Ketamine is a near perpetual drug on the formulary at WCFT and is predominantly assigned to pain medicine. However, its use as a treatment in refractory status epilepticus is not only confined to the theory and literature but is in practice at WCFT. Chronic pain from a multitude of etiologies forms the main target cohort. Patients may be prescribed ketamine in an oral formulation or admitted for a short-stay ketamine infusion. Ketamine intravenous infusion is preferable for maximum effect where oral preparations are severely hindered by hepatic metabolism.

Ketamine is lipid and water soluble with a volume of distribution of 3L/kg at steady-state [6]. Protein binding is disputed in the literature, reportedly between 20–50% [7]. In pharmacology, Cmax is the maximum (or peak) concentration that a drug achieves in a specified compartment/test area of the body after the drug has been administered and before the administration of the second dose. Conflicts between targeted plasma Cmax levels exist with no real known therapeutic reference range to target symptoms (Table 1). Monitoring ketamine therapy has long relied on pain scores and clinical observations. At WCFT, serum levels were referred to Toxicology, City Hospital, Birmingham, UK. Although an excellent service, the referral method of high-performance liquid chromatography–UV spectroscopy (HPLC-UV) was unable to quantitate any values <300µg/L and was the only ketamine method in the UK capable of measuring ketamine in plasma/serum. Therefore, the development of an in-house method was necessary. The Neuroscience Laboratories at WCFT already provide an extensive therapeutic drug monitoring service used both locally and nationally. This repertoire includes 10 anti-epileptic analytes and midazolam for brain stem death testing. All analytes are measured using the Waters Xevo TQ-s Micro ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) platform. This analyser not only uses the specificity of UPLC-MS/MS, but incorporates StepWave™ technology to increase analytical sensitivity, lending itself well to an analyte such as ketamine that is so well distributed throughout the body and peripherally minimal. The method was published in December 2023 with a lower limit of quantitation (LLoQ) for ketamine at 36.2µg/L, lower limit of detection (LLoD) 4.5µg/L, and a linearity up to 2000µg/L. This was a 10-fold decrease in LLoQ in comparison to the referral method [9]. UPLC-MS/MS is a better approach to the target cohort of low-dose ketamine therapy monitoring.

Ketamine is extensively metabolized with an initial demethylation step to norketamine by cytochrome P450 3A4 (CYP3A4), CYP2B6 and CYP2C9. Norketamine has 80% of the pharmaceutical strength of ketamine as an active metabolite [10,11]. Norketamine is a prime candidate as an additional metabolite for monitoring ketamine therapeutics and is a welcome addition to the WCFT repertoire. Curiously, S-ketamine is metabolized faster than R-ketamine [12]. Although Esketamine has been used on the formulary at WCFT and is preferred as it maintains efficacy at lower doses, the availability of Esketamine in the UK is reduced. The assay developed therefore targeted ketamine as a racemate. Also, all oral ketamine formulations are a racemic mixture.

Table 1. Plasma Cmax (µg/L) reports in targeted symptoms or adversities following ketamine therapy
Sourced: Zanos P, Moaddel R, Morris P et al. Correction to “KetamineMetabolite Pharmacology: Insights into Therapeutic Mechanisms”. Pharmacol Rev 2018;70(4):879–879 [1], Driesen N, McCarthy G, Bhagwagar Z et al. The impact of NMDA receptor blockade on human working memory-related prefrontal function and connectivity. Neuropsychopharmacology 2013;38(13):2613–2622 [2], and Krystal JH, Karper LP, Seibyl JP et al. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Arch Gen Psychiatr 1994;51(3):199–214 [8].

Patient results

The assay has been in use for WCFT patients since May 2023. In this period, the Neuroscience Laboratories has received 67 requests for ketamine and norketamine analysis on patient sera. This included 38 patients receiving IV and 29 receiving oral administration. Timing of the patient sample for oral administrations is critical. The dose is taken and blood is drawn 30 minutes later. Timing for IV samples is throughout the 11-step infusion, usually 2–3 samples spaced evenly or when clinically indicated. Figure 1 displays all data for ketamine and norketamine for May 2023 – April 2024. Significant differences are noted between ratios of ketamine and norketamine in IV and oral formulations (P<0.001). This is in part due to excess circulating ketamine from the continuous infusion in IV, but also due to heavy hepatic metabolism a single bolus will encounter. Figure 1 provides clarity in using norketamine as the analyte of choice when monitoring oral ketamine formulations where all serum ketamine measurements are <LLoQ.

In reference to Table 1, there are conflicting reports for therapeutic peripheral blood levels of ketamine. This is a dose- and administration-method-dependent phenomenon, akin to other xenobiotics of a similar chemistry. Figure 2 displays inter-subject variability (CVi) in several patients receiving IV ketamine infusions at 300µg/kg/hr. The time into the infusion is not normalized (4.15–50.15hrs). Nonetheless, CVi is 24.3% in ketamine serum concentrations and 45.6% in norketamine serum concentrations. Pharmacogenomics will account for the wider difference noticed in conversion to norketamine, but samples taken earlier into the infusion largely gave lower norketamine concentrations. Generally, the CVi for IV ketamine is narrow for normalized doses, albeit a small sample size. Oral ketamine CVi for 50mg doses QDS (taken four times daily) gave similar results for serum norketamine concentrations at 30.5% (n=10). However, Figure 2 again displays

Figure 1. Box-and-whisker plots intravenous (IV) ketamine (n=38) vs oral ketamine (n=29)
(a) Serum ketamine and norketamine concentrations in IV and oral formulations (T=30mins post-bolus). IV serum ketamine vs oral ketamine (P<0.001). Oral serum ketamine vs oral ketamine (P<0.04). (b) Ketamine:norketamine in IV and oral formulations (P<0.001). Samples measured by UPLC-MS/MS. Heteroscedastic paired t-test.

the limited use for measuring serum ketamine in monitoring oral users. All values were <LLoQ, but >LLoD. This displays the heavy first pass hepatic metabolism and distribution of ketamine.

Considerations are made to protein binding of a drug in therapeutic monitoring. The free-protein fraction of a drug may have a narrower window in which the lines between sub-therapeutic, therapeutic and toxic ranges are fine. Phenytoin is a prime example in which 90% is protein bound [source: The Neurosciences Labora-tories reference range (https://www.thewaltoncentre.nhs.uk/neurobiochemistry-diagnostic-tests.htm)]. Free ketamine and norketamine has been explored at WCFT labs following the disparity in the literature reporting the protein-bound fraction of ketamine. Centrifree® Centrifugal filter units by Millipore® were used prior to the method described in Armfield et al. 2023 [9]. Analysis from patients receiving either oral or IV ketamine yielded results seen in Figure 3 (n=18). There is a significant difference between the mean free ketamine (38.4%) and free norketamine (49.5%), (P<0.001). This suggests that norketamine has a higher affinity for protein than ketamine, which could account for the 20% lower efficacy of norketamine described in the literature. Furthermore, increased serum albumin or total protein, increases the percentage bound to protein for both ketamine and norketamine. For percentage bound ketamine to serum albumin R2 = 0.55 and to serum total protein 0.63. For percentage bound norketamine to serum albumin R2 = 0.58 and to serum total protein 0.55. These analyses did not yield any significant differences nor have a clear linear relationship. This data is suggestive that ketamine and norketamine do not need to be measured in their free-protein fractions and the mean percentage free is large enough to ensure measuring a total ketamine/norketamine is appropriate for patient monitoring.

Figure 2. Inter-subject variability (CVi) for normalized doses ketamine IV at 300µg/kg/hr (n=9) and oral ketamine bolus 50mg QDS (n=10)
Oral ketamine blood sampled 30 minutes after dose.
CV, coefficient of variation; SD standard deviation.

Future considerations

Evidently, more measurements need to be made and correlated to clinical observations to develop a therapeutic reference range. Some patients experience low-grade hepatocellular injury on dose titration. This does not correlate with current serum measurements alone. It is hypothesized that this adverse reaction is related to chirality of ketamine and subsequent metabolites, speed of titration and pharmacogenomics. Further work will be undertaken at WCFT to detail kinetic profiles of patients receiving low-dose IV racemic ketamine. This will help to define reference ranges and understand this patient cohort.

Ketamine is a well-defined drug in the laboratory and a fundamental compound measured in toxicology. It is not defined in clinical monitoring at low doses. UPLC-MS/MS using the Waters™ Xevo TQ-s Micro has enabled better understanding of ketamine in clinical laboratory medicine. WCFT looks forward to providing future contributions to this subject.

Acknowledgements

Dr Bernhard Frank, Associate Clinical Professor and Consultant in Pain Medicine, spearheaded use of ketamine at WCFT and began the project for measuring serum ketamine. Carrie Chadwick, WCFT Laboratory Director and Consultant Clinical Scientist, facilitated the project and procured laboratory equipment. All staff at WCFT Neurosciences Laboratories are involved in analysis and handling of ketamine requests.

Figure 3. Box-and-whisker plots showing percentage unbound free ketamine and norketamine (n=18)
Heteroscedastic paired t-test.

The author

Nicholas Armfield MSc, MIBMS
The Neuroscience Laboratories, The Walton Centre NHS
Foundation Trust, Liverpool, L9 7LJ, UK

Email: Nicholas.Armfield@nhs.net

 References
1. Zanos P, Moaddel R, Morris P et al. Correction to “KetamineMetabolite Pharmacology: Insights into Therapeutic Mechanisms”. Pharmacol Rev 2018;70(4):879–879.
2. Li L, Vlisides PE. Ketamine: 50 years of modulating the mind. Front Hum Neurosci 2016;10:612 (https://www.frontiersin.org/articles/10.3389/fnhum.2016.00612/full).
3. Reif A, Godinov Y, Bitter I. Esketamine nasal spray versus quetiapine for resistant depression. N Engl J Med 2024;390(1):93–95 (https://www.nejm.org/doi/full/10.1056/NEJMc2313230).
4. Tohira H, Brink D, Davids L et al. Use of ketamine wafer for pain management by Volunteer Emergency Medical Technicians in rural Western Australia. Emerg Med Australas 2023;35(5):786–791 (https://onlinelibrary.wiley.com/doi/10.1111/1742-6723.14226).
5. Driesen N, McCarthy G, Bhagwagar Z et al. The impact of NMDA receptor blockade on human working memory-related prefrontal function and connectivity. Neuropsychopharmacology 2013;38 (13):2613–2622 (https://www.nature.com/articles/npp2013170).
6. Hijazi Y, Boulieu R. Protein binding of ketamine and its active metabolites to human serum. Eur J Clin Pharmacol 2002;58(1):37–40 (https://link.springer.com/article/10.1007/s00228-002-0439-4).
7. Pai A, Heining M. Ketamine. Continuing Education in Anaesthesia, Critical Care & Pain 2007;7(2):59–63 (https://e-safe-anaesthesia.org/e_library/03/Ketamine.pdf).
8. Krystal JH, Karper LP, Seibyl JP et al. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Arch Gen Psychiatr 1994;51(3):199–214 (https://jamanetwork.com/journals/jamapsychiatry/article-abstract/496531).
9. Armfield N, Frank B, Chadwick C. A rapid, sensitive method for clinical monitoring of ketamine and norketamine by ultra-high-performance reverse-phase liquid chromatography tandem mass spectrometry. Ann Clin Biochem 2023;45632231224215 (https://journals.sagepub.com/doi/10.1177/00045632231224215)
10. Mion G, Villevieille T. Ketamine pharmacology: an update (pharmacodynamics and molecular aspects, recent findings). CNS Neurosci Ther 2013;19(6):370–380 (https://onlinelibrary.wiley.com/doi/10.1111/cns.12099).
11. Hijazi Y, Boulieu R. Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes. Drug Metabol Dispos 2002;30(7):853–858 (https://dmd.aspetjournals.org/content/30/7/853).
12. Desta Z, Moaddel R, Ogburn E et al. Stereoselective and regiospecific hydroxylation of ketamine and norketamine. Xenobiotica 2012;42(11):1076–1087 (https://www.tandfonline.com/doi/full/10.3109/00498254.2012.685777).