Pharmacogenetics of ketamine metabolism and immunopharmacology of ketamine

Abstract

Ketamine is an anaesthetic agent that is being increasingly used at sub-anaesthetic doses as an analgesic or co-analgesic in the management of postoperative pain and chronic pain. In most countries, ketamine is administered as a racemic compound consisting of two enantiomers: (S)- and (R)-ketamine at a ratio of 1:1. Ketamine analgesia is frequently restricted by the low efficacy and large interindividual variability in drug response, which may be associated with the differences in the plasma pharmacokinetics. Previous in vitro studies suggested that ketamine is primarily cleared to its active metabolite, norketamine, by two hepatic CYP enzymes: CYP2B6 and CYP3A4, whose expression and catalytic activities show vary large variability in humans due to genetic and environmental influences. Therefore, it is logical that the variability in these enzymes contributes to the variability in ketamine pharmacokinetics. Additional variability in analgesic response may arise from the heterogeneous nature of pain, as ketamine is expected to be more effective against hyperalgesia and allodynia (neuropathic pain). Although these anti-hyperalgesic and anti-allodynic mechanisms have been primarily associated with the non-competitive antagonism of neuronal NMDA receptors, it has been speculated that the attenuation of proinflammatory response may also contribute to ketamine analgesia, since there is evidence to suggest important roles of proinflammatory cytokines in the pathogenesis of neuropathic pain. Thus, the major aims of this thesis were to examine the influence of variability in enzyme activity, especially that due to CYP2B6 genetic polymorphisms, on ketamine pharmacokinetics in vitro and ex vivo in chronic pain patients. The secondary aims of this thesis were to examine the effects of ketamine and norketamine enantiomers on proinflammatory cytokine production in vitro, using interleukin-6 (IL-6) as a marker of cytokine production; and to explore the mechanistic characterisation of the drug actions using both in silico docking simulations and in vitro experiments. The in vitro experiments showed that, at clinically relevant concentrations, CYP2B6 but not CYP3A4 is the major isoform responsible for ketamine metabolism to norketamine in human liver microsomes (HLM). Moreover, the presence of the CYP2B6*6 allele, the most common allelic variant of the CYP2B6 gene, reduced the intrinsic clearance of both ketamine enantiomers in HLMs and cDNA-expressed proteins by at least 62%. This substantial CYP2B6*6 allele-induced decrease in ketamine intrinsic clearance in vitro was also observed ex vivo in chronic pain patients who received 24 h continuous subcutaneous infusions of 100 mg to 500 mg ketamine. The impact of the CYP2B6*6 allele, by itself and in combined with the age of the patient, explained approximately 40% and 60% of interindividual variation in plasma ketamine concentrations at steady-state, respectively, whereas sex, disease and other medications had no significant influences. The decrease of ketamine clearance may be associated with the adverse effects of ketamine, as patients who experienced adverse effects showed approximately 15% lower steady-state plasma clearance of ketamine than those who did not. However, no evidence linking the plasma pharmacokinetics of ketamine and norketamine and the analgesic efficacy was found. One possible explanation for this lack of concentration-response relationship is the overwhelming effect of heterogeneous nature of pain on analgesic response, since ketamine analgesic efficacy was higher in patients suffering from neuropathic pain than other pain types. This finding may reflect the fact that the analgesic activity of ketamine is more likely due to the attenuation of pain hypersensitivity rather than the direct suppression of nociceptive transmission. The in vitro experiments on the inhibition of IL-6 by ketamine and norketamine enantiomers showed that pre-incubation with these drugs, at biologically relevant concentrations (1 to 100 μM), stereoselectively attenuated stimulated IL-6 production in recombinant cells in a concentration- and time-dependent manner. (R)-ketamine inhibited stimulated IL-6 production by approximately 60% at all exposure duration, an inhibitory effects that were up to 2-fold greater than (S)-ketamine after short term exposure (less than 2 h). However (S)-ketamine was as potent as (R)-ketamine after long-term exposure (4 to 8 h), as its inhibitory effects were significantly enhanced with exposure duration. In addition, (S)-norketamine also attenuated IL-6 response in a time-dependent manner with approximately half the potency of (S)- ketamine. Further in vitro experiments and in silico docking simulation suggested that this time-dependent effects of (S)-ketamine and (S)-norketamine may indicates a mechanisticallybased difference between acute and chronic effects of (S)-enantiomers on IL-6 production. This findings extend the current knowledge of the innate immune pharmacology of ketamine that may lead to a new direction for future research into ketamine analgesia. In summary, this thesis demonstrates a substantial impact of the CYP2B6*6 allelic variant on the clearance of ketamine, which may contribute to the interindividual variability in drug concentration. However, other factors such as the heterogeneity in the nature of pain and the inflammatory state should be taken into consideration to provide a more accurate prediction on ketamine analgesic response.