Sex differences in sub-anesthetic ketamine's antidepressant effects and abuse liability

Abstract

Sub-anesthetic ketamine produces rapid antidepressant effects in patients with bipolar and unipolar major depression where conventional monoaminergic-based antidepressant drugs have been ineffective or ridden with side effects. A single ketamine infusion can produce antidepressant effects lasting up to two weeks, and multiple ketamine infusions prolong this effect. Pre-clinical studies are underway to uncover ketamine's mechanisms of action, but there are still many questions unanswered regarding the safety of its long-term use. Abuse liability is one area of concern, as recreational ketamine use is an ongoing issue in many parts of the world. Another understudied area is sex differences in responsivity to ketamine. Women are twice as likely as men to be diagnosed with depression, and they progress through stages of drug addiction more rapidly than their male counterparts. Despite this, preclinical studies in ketamine's antidepressant and addictive-like behaviors in females are limited. These intersecting factors in recent clinical and pre-clinical studies are reviewed to characterize ketamine's therapeutic potential, its limitations, and its potential mechanisms of action.

Introduction

Major depressive disorder is a multi-symptom condition that contributes significantly to the global burden of disease, and women have a twofold higher risk of depression than men (1). Treatment efforts has been hindered by monoaminergic-based antidepressants that have undesirable side effects and poor efficacy (2). Therefore, the discovery that ketamine produces rapid antidepressant effects has generated much optimism in the field of psychiatry and neuroscience. While classical antidepressants take several weeks of daily administration to produce an effect, sub-anesthetic ketamine infusions alleviates depressive symptoms hours after the first infusion, lasting approximately 7-14 days in patients with treatment-resistant depression (3). Furthermore, ketamine rapidly alleviates suicidal ideation, conferring a unique use of ketamine in the emergency room (4). These findings, however, must be tempered by a number of notable limitations in our knowledge, especially that which pertains to the safety and efficacy of chronic long-term ketamine treatment, which has not been sufficiently addressed in clinical trials (5)**. Indeed, consequences of long-term exposure to ketamine has only been assessed in recreational contexts, as ketamine is used as a popular club drug because of its euphoric effects and vivid dissociative hallucinations. Importantly, recreational ketamine use differs greatly from antidepressant ketamine in terms of dose (higher), route of administration (typically intranasal), and setting (a rave or club). Nevertheless, few studies have assessed sex differences in ketamine responsivity. The inclusion of sex as a factor improves the translatability of preclinical research, and it may uncover important sex differences with implications for the abuse liability of antidepressant ketamine. Therefore, the purpose of this review is to bridge clinical and preclinical research to summarize what is known and to identify knowledge gaps regarding sex differences in ketamine response within the context of depression and recreational ketamine use.

Sex differences in stress and ketamine's antidepressant effects

While females are included in clinical research, few studies investigating ketamine's antidepressant effects analyze sex as a variable. A recent meta-regression analysis of 6 studies (n = 103) found that sex, age, and drug use history did not contribute to ketamine efficacy (6). A larger meta-analysis analyzed 21 studies (n = 437) at 4 different time points, and they found no effect of sex at the 4h and 24h time points; only at the 7 day time point was having a greater proportion of male subjects predictive of overall higher efficacy (7). These findings are limited to a relatively short time frame and only tested one acute ketamine dose (0.5 mg/kg/40 min intravenous infusion). To date, no clinical studies have looked at dose-dependent differences in ketamine efficacy in men and women, nor has repeated ketamine infusions been analyzed by sex.

Preclinical studies indicate a heightened sensitivity to ketamine in females. Stress-naïve female rodents consistently respond to a lower dose of ketamine than males on behavioral assays related to depression: specifically using measures of antidepressant efficacy (forced-swim test) and anxiety-induced neophagia (novelty-suppressed feeding) (8-11), summarized in Table 1. The assessment of ketamine's effects on anhedonia, a signature feature of depression that is measured in rodents using the sucrose preference test, have produced conflicted findings. This is in part due to sex differences in baseline sucrose intake and ceiling effects (8). Discussed in a review by Kokras and Dalla (12), methodological factors such as duration of access to the bottles differentially affect the sucrose intake of males and females, thereby complicating interpretations of sex differences in depression-like behaviors. Non-consummatory measures of anhedonia such as intracranial self-stimulation of the lateral hypothalamus has shown that ketamine does not have an anti-anhedonic effect on socially defeated male mice while it rescues other depressionlike behaviors induced by the social defeat stress (13). Other models that utilize chronic stressors such as social isolation have found that female rats appear to be more resilient to social isolation stress, as they do not display a decrease in sucrose preference after 8 weeks of social isolation, which is sufficient to induce anhedonia in males (14). When chronic ketamine is tested (10 mg/kg daily, for 21 days), males displayed an antidepressant-like phenotype but females showed pro-depressive and anxiogenic behavioral traits (15). This study highlights the importance of including females in these studies, and furthermore it illustrates that sex differences can emerge following exposure to various drug dosages and treatment regimens, or exposure to different types of stressful stimuli. Another recent example of this comes from Hodes et al. (16), where subchronic social defeat stress (which produces a depression-like behavioral profile in female mice but not males, lending this model good face validity) results in a distinct pattern of gene expression in the nucleus accumbens of females compared to males, specifically differential expression of genes that control the DNA methylation machinery. The epigenetic mechanisms underlying sex-specific stress and ketamine responsiveness may be a compelling target of future research.

Table 1.

Summary of recent findings investigating behavioral sex differences in ketamine response.

Measure	Mode of delivery	Behavioral outcome	Citations
Forced-swim test	acute, IP	♀ respond to lower dose than ♂; estrus cycle-dependent	8, 9, 10, 11, 14
Forced-swim test	repeated, IP	♀ prodepressive; ♂ antidepressant	15
Novelty-suppressed feeding	acute, IP	♀ respond to lower dose than ♂	8
Sucrose preference test	acute + repeated, IP	mixed/conflicting results; hormone-dependent	8, 9, 14, 19
Locomotor sensitization	repeated, IP	♀ respond to lower dose than ♂	26, 27
Conditioned place preference/avoidance	repeated, IP	mixed/conflicting results, dose-dependent	26, 27, 28
Self-administration	repeated, IV	♀ in proestrus similar to ♂, but diestrus lower than either.	30

IP, intraperitoneal; IV, intravenous;

Ketamine's potential mechanisms of action

Antidepressant ketamine's putative mechanism of action is due to a rapid alteration of corticolimbic signaling and structural remodeling; however, the omission of females in these studies raises questions as to the translatability of these findings, as there has since been evidence that ketamine's mechanism may diverge in a sex-specific manner. In males, ketamine results in disinhibition of excitatory neurons in the medial prefrontal cortex (mPFC) via blockade of the n-methyl-d-aspartate (NMDA) receptors on inhibitory interneurons; this increased excitation leads to activation of downstream signaling cascades like mammalian target of rapamycin (mTOR) via Akt phosphorlylation, and the rapid synthesis of proteins that ultimately promotes synaptogenesis and increased spine density, thereby reversing effects of chronic stress (17). In line with this, males that underwent chronic social isolation showed decreased sucrose preference, mPFC spine density, and expression of synaptic proteins postsynaptic density protein 95 (PSD-95), synapsin, and GluA1 (14). All effects were reversed with acute ketamine 3 h later. Females, on the other hand, did not show a stressed-induced decrease in sucrose preference; they did show stressed-induced decreases in mPFC spine density and synaptic proteins, but ketamine did not rescue these alterations (14). Sex differences in glutamate neurotransmission have also been reported, where ketamine-treated males show increased hippocampal glutamate and females had no differences; while in the mPFC females had increased aspartate levels and males had no differences (9). Additionally, increased hippocampal serotonin turnover was observed in females but not males (9).

Circulating gonadal hormones may underlie the female ketamine response in rodents. Indeed, female rats that undergo gonadectomy, thereby depleting levels of circulating estradiol and progesterone, displayed increased anxiety-like and depression-like behaviors which were alleviated by a dose of ketamine shown to work in both males and females (10 mg/kg) (18). At a lower dose that is only effective in females (2.5 mg/kg), replacing estradiol and progesterone levels in gonadectomized females was necessary to produce ketamine's antidepressant-like effects (8). Another study from our lab using gonadectomized rats demonstrated that activational effects of estradiol and progesterone (but not testosterone) are necessary in females, but not males, for 2.5 mg/kg ketamine's pro-hedonic effects on the sucrose preference test (19). Additionally, pharmacological agonism of the two nuclear estrogen receptors (but not a progesterone receptor agonist) promoted ketamine behavioral response on the forced-swim test in intact diestrus 1 female mice (11). Together these findings suggest that estrogen may have both protective effects on mood and a facilitative effect on ketamine responsiveness, warranting consideration for clinical studies of estrogen as an adjuvant with ketamine.

There are also intriguing sex differences in the metabolism of ketamine, where female rodents have higher brain levels of ketamine metabolites norketamine and hydroxynorketamine (HNK) than males (Saland et al., unpublished; (10), suggesting that differential pharmacokinetics may underlie the behavioral and molecular effects observed. Additionally, administration of the metabolite (2S,6S; 2R,6R)-HNK produces NMDA receptor-independent antidepressant-like effects without ketamine's adverse side effects (10), however, others have recently challenged this view (20, 21). Furthermore, the antidepressant-like effects of this metabolite were only tested in males, thereby limiting the implications of these findings (10, 21). In humans, higher plasma levels of (2S,6S; 2R,6R)-HNK was observed in females than males, however plasma levels of this specific metabolite were not correlated with treatment nonresponse as it was for other ketamine metabolites such as (2S,5S;2R,5R)-HNK in bipolar patients (22, 23). The pharmacologic effects of ketamine's active metabolites may underlie the prolonged antidepressant effects that extend beyond ketamine's half-life, and sex-specific differences in these metabolites may contribute to the differences in responsiveness observed in pre-clinical findings. More research is needed to fully characterize the metabolic pathways of ketamine and the different pharmacologic effects of each metabolite.

Ketamine abuse/addiction and implications for mood disorders

Ketamine is a popular club drug worldwide, and a number of detrimental health consequences are associated with its long-term use, including the development of addiction, increased tolerance over time, uncontrollable cravings, and symptoms of withdrawal upon cessation in many cases (24). As individuals who exclusively use ketamine are rare, most observational ketamine studies involve poly-drug users. Studies investigating recreational ketamine use have uncovered some notable trends with regards to sex differences and comorbidity with mood disorders. Female ketamine users self-report greater levels of cognitive impairment and greater withdrawal effects compared to males (25), suggesting a potential sex-specific sensitivity to ketamine's effects. Additionally, women who use ketamine along with other amphetamine-type drugs are more likely to suffer from mood disorders compared to women who only use amphetamine-type drugs, and more than males (26). Ignoring sex, club drug users (including ketamine) are more prone to bouts of depression than “hard” drug users (the authors defined as cocaine or heroin) (27). This association between mood disorders and ketamine use could be explained by individuals self-medicating in an attempt to alleviate their depressive symptoms, or that long-term ketamine use exacerbates the symptoms. A recent study found decreased resting state thalamocortical functional connectivity in chronic ketamine users from a sample that was 80% male (28). This decreased functional connectivity is thought to underlie the transition from drug use to uncontrollable addiction. Additionally, female ketamine users have higher depression scores than males and differential resting state functional connectivity of subregions of the prefrontal cortex, an area greatly implicated in the pathophysiology of depression. Specifically, subgenual anterior cingulate cortex (sgACC) connectivity to the superior temporal gyrus in males and dorsomedial prefrontal cortex in females was correlated with a higher depression score (29)*. More research is needed to understand the structural and functional brain changes in male and female ketamine users, especially as it pertains to risk of depression.

Preclinical studies used to characterize ketamine's addiction-like properties involve behavioral assays such as locomotor sensitization, conditioned place preference (CPP), and intravenous self-administration, and sex difference studies have recently started to emerge. In line with females' increased sensitivity to antidepressant effects of ketamine discussed above, female rats also show increased locomotor sensitization to intermittent (weekly and every other day, respectively) repeated ketamine at depression-relevant doses (2.5 – 10.0 mg/kg intraperitoneally, i.p.) (30, 31). Locomotor sensitization after repeated exposure to drugs of abuse is indicative of plasticity in the reward circuitry that may underlie the transition to addiction. Interestingly in these studies, the same rats that displayed sensitization did not form a CPP to ketamine at any dose tested (30, 31); in fact, females displayed a conditioned place aversion to 5.0 mg/kg. Together, these findings suggest that divergent mechanisms may underlie the locomotor-activating effects and the associative rewarding effects of ketamine. However another group testing higher ketamine doses (6-14 mg/kg, daily) found females displayed a greater CPP than males (32). This study also found distinct urine metabolic profiles in males and females, warranting further research into sex-specific pharmacokinetics of ketamine.

As discussed above, ovarian hormones mediate responsivity to ketamine's pro-hedonic effects (19). There is also evidence that the reinforcing effects of other drugs of abuse, like cocaine, is augmented by estrogen via its interactions with the dopaminergic reward circuitry (33). Freely-cycling female rats that have access to ketamine self-administration only on days when they are in proestrus (when estradiol and progesterone levels peak) maintain their ketamine intake at levels comparable to males, but females with ketamine access only during diestrus 1 (when estradiol and progesterone are low) fail to maintain their intake (34). This suggests that the peak of gonadal hormones that coincides with proestrus supports the reinforcing effects of ketamine. Additionally, adolescent female rats display a stronger locomotor response to ketamine than males, and this effect was not seen in preadolescent rats whose circulating gonadal hormones have not yet peaked (35). It is important, however, to note that several factors likely contribute to this behavioral effect including developmental changes in brain structures as well as potential pharmacokinetic differences related to different developmental stages. While these findings add to the growing body of evidence suggesting a contribution of ovarian hormones in responsivity to ketamine, a more systematic approach (for example, (19)) must be taken to fully elucidate the activational and organizational effects of gonadal hormones on the reinforcing properties of ketamine.

Rodent models have been used to study the effects of stress (the single greatest predictor of depression) on addiction-like behavior for a variety of drugs (36), and ketamine is beginning to be studied in this context. A recent study utilized the bulbectomy model in male rats, which is a model of depression that potentiates cocaine addiction-like behavior. Interestingly, while bulbectomy increased ketamine self-administration, it did not potentiate relapse (37), suggesting a different mechanism at play. This bulbectomy paradigm has yet to be tested in females, but female rats are more sensitive to stress-induced reinstatement of alcohol (38), and females are more responsive to corticotropin releasing factor (CRF) signaling in the context of cocaine withdrawal (39). Sex differences in CRF signaling as it relates to ketamine's effects have yet to be studied, but it may be an attractive mechanism by which to explain the intersection of stress and addiction.

Different molecular adaptations between acute ketamine and chronic self-administration of ketamine has shown that a single ketamine infusion increases hippocampal brain-derived neurotropic factor (BDNF), while a history of chronic ketamine self-administration decreased hippocampal BDNF (40). This group also found Akt was oppositely regulated by single vs repeated ketamine. Akt signaling and subsequent phosphorylation of mTOR may be the critical mechanism by which ketamine exerts its effects in the prefrontal cortex and hippocampus, but this may be a sex- and region- specific effect, as ketamine's protracted effects on synaptic plasticity in the nucleus accumbens of males, a critical center of reward processing, occurs independently of mTOR activation (41). Additionally, differential nucleus accumbens phosphorylation of the glutamate receptor GluA1 has been observed with a single injection (increased (41)) vs chronic self-administration (decreased (42)). As the aforementioned studies only include males, it will be critical to characterize these effects in females as well.

Conclusions and future directions

The discovery of ketamine's rapid antidepressant effects has invigorated the field of psychiatry and given hope to millions of patients and caretakers. However, little is known about the long-term effects of repeated exposure to ketamine, and the addiction field has demonstrated that the neurobiological effects of acute vs chronic ketamine result in very different molecular profiles. A question clinicians may have is: where is the threshold at which the benefits of therapeutic, antidepressant ketamine becomes overshadowed by its risk of addiction? As women are twice as likely to develop depression and progress through the stages of addiction faster than men, are they at an increased risk? Preclinical literature reviewed therein suggests that female rats are more sensitive to ketamine's effects, but more research is needed in preclinical and clinical studies to fully understand sub-anesthetic ketamine.

Highlights.

• - Clinical studies rarely analyze sex as a factor in ketamine's effects.

• - Preclinical rodent studies indicate that females are more sensitive to ketamine's antidepressant-like effects.

• - While a number of recent reports demonstrate behavioral sex differences, few studies have assayed sex-specific molecular or structural effects on the brain following ketamine.

• - The putative mechanism of action for ketamine's antidepressant effects may be different in females.