Any new aspect of the knowledge of pharmacokinetics and metabolomics of SCs may serve for a better understanding of the clinical and toxicological findings attributable to drug poisoning and misuse cases [12]. Mass spectrometric approaches are commonly used in the study of endogenous compounds [13]. The current study used a pharmacokinetic approach and targeted metabolomics of metabolites associated with neurotransmission to assess the effects of 5F-APINAC administration in mammals. This study was performed with the aim of investigating the toxicity and possible influence of this molecule on neurotransmission systems. The pharmacokinetic profile of 5F-APINAC was dose-dependent with a rapid biotransformation, followed by a gradual elimination within 24 h. The main metabolic pathways found to be impacted were the kynurenine metabolic pathway, serotonergic system/serotonin pathway, aspartic acid innervation system and microbial tryptophan catabolism (Figure 6A–C).

It should be pointed out that, due to the low doses typical of SCs consumption, as well as their highly extensive metabolism, the pharmacokinetics assessments of SCs are complex. Only a few experiments describing the pharmacokinetic profiles of SCs in animal models have been published. The received concentration–time curve was smooth enough for the accurate assessment of the main pharmacokinetic parameters of the drug. The 5F-APINAC maximum concentrations after the 1-mg/kg and 2-mg/kg doses were higher than those after 0.1 mg/kg. The maximum concentration, as well as the AUC values, rose linearly in proportion to the administered dose, indicating linear kinetics. It should also be noted that the 5F-APINAC concentrations were substantially reduced but still detectable 24 h after the drug administration, but the concentration levels were close to the lowest limits of detection.

Since most of the SCs are full agonists of CB receptors, they provide strong neuroactive and toxic effects on the central nervous system (CNS). On the other hand, in the last decades, it has been found that tryptophan metabolism and the metabolites associated with neurotransmission are strongly associated with numerous CNS disorders. Conversely, several tryptophan derivatives have been found to display high levels of neuronal activity, possibly associated with the administration of SCs.

Tryptophan is an essential amino acid known for its influence on immunological and neurological activity, being a precursor for the synthesis of proteins and nicotinamide adenine dinucleotide (NAD), as well as nicotinic acid and serotonin [14]. Moreover, a number of its metabolites may pass the blood–brain barrier, having neuroactive properties on the brain physiology connected to behavioral changes [15,16]. Tryptophan metabolism consists mainly of two competing major metabolic pathways: the kynurenine pathway that converts approximately 95% of tryptophan and the serotonin pathway that metabolizes 5% of the amino acid. We confirmed that both the serotonergic system–serotonin pathway and, especially, the kynurenine pathway were the most-impacted pathways in our experiment. Alterations in tryptophan metabolism may appear following stress or depression. In the case of the kynurenine pathway, this is related to the control of plasma tryptophan levels through the clearance of excess of circulating tryptophan [17]. Additionally, it has been reported that the kynurenine pathway provides metabolites involved in neurotransmission, such as kynurenine, kynurenic acid and quinolinic acid [14].

Several studies have described the effects of THC on tryptophan conversion and the association with behavioral and neurological changes. It has been hypothesized that THC and cannabidiol (CBD) suppress tryptophan degradation through the activation of indoleamine-2,3-dioxygenase. This would result in the enhanced availability of tryptophan for serotonin biosynthesis, which could produce improvements of mood disturbances.

Based on our findings, it may be postulated that, after 5F-APINAC administration, the changes in tryptophan metabolism are strongly associated with the kynurenine pathway. According to the human proteome map, the expression of several enzymes related to the kynurenine pathway are much higher in the liver than in the brain. The liver plays an essential role in amino acid metabolism, being the major organ that metabolizes absorbed dietary tryptophan. The release of tryptophan metabolites into the systemic circulation has hepatic regulation. It may be postulated that precursors and side intermediates for tryptophan metabolism to other organs, including the brain, are provided by the liver, which hereby represents one of the key regulators of kynurenine metabolite levels in the bloodstream [18]. Our results were based on the identification of the biochemical features detected in the blood. We recently reported that the short- and long-term exposures of 5F-APINAC in a zebrafish model were mainly characterized by alterations in the kynurenine pathway but, also, in other neurotransmitter systems such as the gamma-aminobutyric acid/glutamic acid, dopaminergic/adrenergic and cholinergic neurotransmitter systems not found to be altered in rabbit plasma [8]. This may be explained by the fact that these alterations were measured in the entire organism of the zebrafish, reflecting the systemic biochemical alterations from the organs and not just from the blood, as in the present study.

The tryptophan concentrations immediately decreased at all doses within the first two hours after 5F-APINAC administration. It is generally observed that the circulating levels of tryptophan are mainly dependent on its exogenous supply of the amino acid. However, cytokine-induced tryptophan degradation may constitute an additional important mechanism potentially justifying, through the serotonergic system, the anti-depressive effects of cannabinoids [19]. It should be taken into consideration that, as mentioned above, approximately 95% of tryptophan metabolism passes through the kynurenine pathway. In this case, the first step of the pathway is generally affected by the enzyme tryptophan pyrrolase. Its release is activated in stress conditions, resulting in a decrease of blood tryptophan concentrations. Additionally, it has been found in the active conversion of tryptophan into kynurenine in depressed individuals [20].

L-kynurenine represents a central metabolite of the kynurenine pathway. The experimental data showed a dose-dependent increase of plasma concentrations in rabbits receiving 1-mg/kg and 2-mg/kg 5F-APINAC. However, the group of rabbits receiving the lowest dose (0.1 mg/kg) did not present a significant difference compared to the vehicle control group. Due to the ability of transportation through the blood–brain barrier by the neutral amino acid carriers, L-kynurenine may easily reach the central nervous system and be captured and metabolized by the glial cells [21,22]. Notably, Stone et al. hypothesized that kynurenine does not directly affect the neuronal electrical activity in brains or in isolated neurons and has no direct depressant influence on cardiac and smooth muscle tissues [23]. At the same time, kynurenine has been shown to increase the expression and synthesis of the nerve growth factor that plays an importance role in the tropism of different neurons [24]. Kynurenine may be further metabolized along three neurotoxic branches, with the consequent formation of 3-hydroxy-L-kynurenine, kynurenic acid and anthranilic acid. In general, it has been reported that metabolites in the kynurenine pathway take part in different pathophysiological processes, including neurodegenerative and neurological diseases and even psychiatric disorders such as schizophrenia or depression [16].

Kynurenic acid, found to be elevated in the tested groups at all doses (with a maximum increase in a medium concentration) in our study, is generated by the action of a specific enzyme kynurenine aminotransferase and represents one of the most widely studied neuroactive tryptophan metabolites. It is mainly present in the brain, being an antagonist for the receptors of excitatory amino acids [25]. Elevated levels of kynurenic acid have shown neuroprotective and neuroinhibitory properties associated with its action as a competitive antagonist at the glycine site of NMDA receptors [26]. Kynurenic acid inhibits noncompetitive α7-nicotinic acetylcholine receptors, which are the primary endogenous targets of kynurenic acid, and binds α-bungarotoxin, which blocks the action of acetylcholine at the postsynaptic membrane, inhibiting the ion flow [27,28]. Considering the interaction of kynurenic acid with different receptors, as well as its effect on the extracellular levels of glutamate, dopamine, acetylcholine and γ-aminobutyric acid, L. A. Ramos-Chávez et al. considered this molecule a neuromodulator. It is also worth considering that these neurotransmitters take part in neuronal development and plasticity, thus affecting cognition, behavior and memory processes [29]. The regulation of the effects of these neurotransmitters by kynurenic acid may contribute to the knowledge of the mechanisms of the neurological effects of cannabinoids.

The hydroxylation of kynurenine by kynurenine 3-monooxygenase results in the formation of 3-hydroxykynurenine, which is further metabolized into xanthurenic acid and quinolinic acid. Actually, the levels of xanthurenic acid in the test group of rabbits were elevated at all levels of the administered drug, although without any dependence on the 5F-APINAC doses. Despite the fact that it is structurally similar to kynurenic acid, its biological role remains unclear. Several authors have suggested that xanthurenic acid might play a role in neurotransmission, neuromodulation and synaptic signaling, in relation with an active intake by synaptic vesicles in the brain [30]. In the present study, the levels of quinolinic acid found in rabbits administered with 5F-APINAC were also elevated in a dose-dependent manner. Quinolinic acid is a potent excitotoxin, as well as a strong NMDA receptor agonist [14]. By its interaction with NMDA-type glutamate receptors, quinolinic acid has been reported to cause convulsions and other excitatory effects [31]. On the other hand, quinolinic acid causes axon-sparing lesions in the brain, associated with mitochondrial damages and free radical generation [28]. Simultaneously, other neurotoxicity effects of quinolinic acid are associated with reactive oxygen species formation and destabilization of the cytoskeleton [32,33]. Moreover, quinolinic acid toxicity affects oligodendrocytes, neurons and motor neurons [34,35,36]. Thus, elevated levels of quinolinic acid in our study may be connected to the neurotoxic effects of 5F-APINAC.

We found that the concentration levels of aspartic acid were significantly increased in the treated group of animals. This can be explained by the fact that an increase of the concentration of quinolinic acid may cause the release of glutamate and aspartic acid from the cerebral cortex in vivo, which predisposes excitotoxic brain damage. To protect itself from such damage, the brain rapidly uptakes extracellular glutamate and aspartic acids from nerves and glial cells [37,38]. Quinolinic acid acts selectively on the methyl-d,l-aspartate type of amino acid receptor and consequently produces excitation of the central neurons [23,39]. Quinolinic acid requires putative glutamate pathways to produce neurotoxic effects [40]. Connick et al. demonstrated the release of aspartate and glutamate by quinolinate in vivo. This mechanism may explain the induction of neurotoxic effects accelerated by quinolinate [30]. Therefore, elevated levels of aspartic acid in the treated groups may indicate neuroprotection of the organism as a response to 5F-APINAC administration.

Additionally, several metabolites from the different tryptophan catabolism pathways were affected after 5F-APINAC administration. Interestingly, we did not observe any significant differences in the levels of serotonin, the major neuromodulator usually affected by cannabinoids [19]. Blood serotonin is produced by specific intestine cells and released into the circulation, but, generally, the levels of serotonin in the blood are not directly associated with the production of serotonin in the brain. However, both brain and blood serotonin have been reported to be similarly regulated by endogenous processes, including key enzymes, as well as the uptake, transport and storage mechanisms of serotonin [41]. Besides the aforementioned main metabolic pathways of tryptophan, it has been recently shown that tryptophan metabolism occurs also in the gut, being supported by bacterial tryptophanases. Tryptophan is also identified as a key intermediate of host gut microbiome signaling, and its bioavailability is associated with the microbial balance within the gut [42]. Thus, gut microbiota-derived tryptophan metabolites are suggested to mediate gut–brain communications, acting as major mediators possessing neuroprotective properties [43]. In the present study, elevated levels of indole-3-propionic acid and indole-3-carboxaldehyde were found in the rabbits treated with 5F-APINAC. It was suggested that indole-3-propionic acid, acting as a free-radical scavenger, may reduce the oxidative stress in Alzheimer’s disease associated with β-amyloid formation, as well as the neuronal damage [44,45]. On the contrary, the levels of indole-3-lactic acid were found to decrease with a pattern similar to tryptophan, characterized by an extreme fall in the first two hours and a subsequent increase of the metabolite concentrations to reach the basal levels as a U shape.

Due to ethical reasons, there are several restrictions concerning controlled SC administration experiments in humans. Thus, controlled human studies for pharmacokinetic and toxicity evaluations of SCs are not conducted, and only several studies performed in experimental animals have been published. In terms of extrapolation, the pharmacokinetic properties of 5F-APINAC characterized after intravenous administration in the rabbit model may serve as a tool for the prediction of 5F-APINAC pharmacokinetics near to what we expect would happen in humans. We found important changes in plasma concentrations of metabolites related to the kynurenine pathway, i.e., kynurenine, kynurenic acid and quinolinic acid, which probably are at the level of being neurotoxic. Overall, the kynurenine pathway takes an essential role in several fundamental physiological processes, being a mediator of the interactions between the immunological and neuronal functions of the body that are part of the pathophysiology of many diseases. Future experiments considering behavioral and neurotoxic functional outcomes and, also, other biological tissues, such as the brain and cerebrospinal fluid, are warranted to be performed to confirm the degree of neurotoxicity.