Understanding the pharmacokinetics of synthetic cathinones: Evaluation of the blood–brain barrier permeability of 13 related compounds in rats

Synthetic cathinones are the second most commonly seized new psychoactive substance family in Europe. These compounds have been related to several intoxication cases, including fatalities. Although the pharmacological effects, metabolism, and pharmacokinetics of cathinones have been studied, there is little information about the permeability of these compounds through the blood–brain barrier (BBB). This is an important parameter to understand the behavior and potency of cathinones. In this work, 13 selected cathinones have been analyzed in telencephalon tissue from Sprague–Dawley rats intraperitoneally dosed at 3 mg/kg. Our results revealed a direct relationship between compound polarity and BBB permeability, with higher permeability for the more polar cathinones. The chemical moieties present in the cathinone had an important impact on the BBB permeability, with lengthening of the α‐alkyl chain or functionalization of the aromatic ring with alkyl moieties resulting in lower concentration in telencephalon tissue. Our data suggest that transport of cathinones is a carrier‐mediated process, similar to cocaine transport across the BBB.


| INTRODUCTION
The consumption of synthetic cathinones represents an important public health problem, according to the most recent report from the United Nations Office on Drugs and Crime, 1 which illustrates that this new psychoactive substance (NPS) family is one of the most commonly seized worldwide, together with synthetic cannabinoids. 1 Most of the cathinone seizures are powder, together with pills and similar products. These compounds have also been found as adulterants in "classical" illegal drugs such as cocaine, illustrating that their prevalence of consumption could be underestimated. 2,3 In addition to the data obtained by seizure analysis, the public health problem related to cathinones is also illustrated by numerous intoxication cases related to these substances, and even some fatalities. [4][5][6] The synthetic cathinones prevalence can also be illustrated by analytical data obtained from wastewater analysis, illustrating that these compounds are being consumed worldwide. 7,8 It is almost impossible to ban all the cathinone derivatives existing nowadays due to the continuous change in structure of new compounds appearing on the market. Besides, new compounds that could replace banned ones surface in mere weeks, in a similar way to what occurs with synthetic cannabinoids. 9 To face this public health problem, the scientific community must be able to provide information about novel compounds, their chemical, pharmacological, and toxicological properties. Thus, a notable number of papers have been published, as illustrated by the reviews available in literature about the metabolism of these substances, [10][11][12] the associated pharmacological behavior, 13,14 toxicology, 5,15 and even their neurotoxicity. 16 An important pharmacological issue to highlight is how cathinones affect endogenous compounds, producing a psychoactive effect. Several studies have demonstrated that cathinones act as nonselective monoamine uptake inhibitors, increasing the levels of dopamine and serotonin, 17,18 producing effects similar to cocaine. 19,20 Thus, the potency of cathinones and other NPS may be studied using in vitro approaches, [21][22][23] in a similar way to synthetic cannabinoids. 24 Although in vitro studies provide valuable information about the intrinsic potency of a compound, the in vivo effect must be determined by the extent to which a compound reaches its site of action. One of the key barriers in this context is the blood-brain barrier (BBB), modulating the exchange of compounds between the brain and the blood. 25 The BBB is a complex system that presents different "entry routes" that can be used by drugs or hormones, 25 such as passive diffusion (usually used by nonpolar compounds such as steroids) and carrier-mediated influx 25 (used by some psychoactive substances such as cocaine 26 ), whereby a specific transporter helps the compound to cross the BBB and reach the brain. To complement the in vitro data and better understand the pharmacokinetics (and in vivo potency) of cathinones, it is therefore essential to generate accurate data on the BBB permeability of these compounds. 26 This work is the first to quantify an extensive series of cathinones in brain samples from rats intraperitoneally injected with these compounds, with the objective of relating the permeability through the BBB with their structure. To this aim, we have developed and

| Reagents and chemicals
Research chemicals containing cathinones were provided by Energy Control (ABD Foundation, Barcelona, Spain). All the compounds were characterized and purity tested by UHPLC-HRMS and nuclear magnetic resonance, following the same procedures already reported in literature. 42,43 Cathinone stock solutions were prepared at approximately 1 mg/ml in methanol (0.01-mg accuracy). HPLC-grade water was obtained by purifying demineralized water using a Milli-Q system  Two rats were dosed per compound, and the brains were pooled in order to avoid possible animal differences. In total, 26 rats received intraperitoneal injections of the 13 different cathinones at a dose of 3 mg/kg in 300 μl of physiological saline solution containing 5% ethanol-the latter for increasing the solubility of the synthetic cathinones. Four additional animals were injected with the same volume of the vehicle and used to obtain blank brain tissue samples, to be used to prepare quality control samples and matrix-matched calibration curves. After 20 min, the rats were anesthetized with CO 2 and decapitated immediately. The brain was dissected (avoiding blood that could contaminate it), and the telencephalon (both cerebral hemispheres) was isolated, quickly frozen in liquid nitrogen and stored at −23 C until analysis.

| Sample treatment
Brain tissue samples were homogenized and crushed with dry ice (Praxair, Valencia, Spain) using an electric grinder, followed by a The sample treatment procedure was adapted from literature, 19 with the only difference being the homogenization procedure. In the present study, homogenization was performed using an electric grinder and dry ice, followed by extraction with acetonitrile and 1% formic acid, a freezing step as clean-up and dilution of the supernatant with HPLC-grade water. Sample weight, extraction volumes, and dilutions were designed according to information available in literature, 40 with some modifications to improve method sensitivity. For a detailed description on analytical methodology validation and the results obtained, see the Supporting Information.

| RESULTS
Cathinone concentrations found in the brain displayed wide differences, from 762 ng/g brain tissue for N,N-dimethylpentylone to 10 596 ng/g for N-ethyl-pentylone, the concentrations for most of the remaining compounds ranging between 1000 and 4000 ng/g. In all cases, the concentrations were well above the analytical performance, in terms of sensitivity and limits of quantification of our methodology. In addition, reliability of the analytical methodology was supported by analysis of quality control samples in duplicate, spiked at 1 and 10 ng/g, included in the sample batch. Recoveries between 70% and 120% were obtained, confirming the correct quantification of the cathinones in telencephalon tissue.
The concentrations found in telencephalon samples for all the compounds are shown in Tables 1-3. The differences observed in the cathinone levels suggest that the BBB permeability of these compounds is structure dependent, being associated with their polarity, as discussed further.

| Cathinone penetration through the BBB
The main objective of our study was to evaluate the relationship between the structure of cathinones and their BBB permeability, in order to get better acquainted with the pharmacological behavior of these substances.
Pronounced concentration differences were observed in the telencephalon for different cathinones, ranging from 762 ng/g (N,N-dimethylpentylone) to 10 596 ng/g (N-ethyl-pentylone). This difference is surprising given the very high structural similarity of these two compounds, which only differ in the amine functionalization (dimethyl vs. ethyl). Table 1 shows the concentrations found in telencephalon tissue for cathinones that differ by the functionalization of the amine moiety (N). In the two groups (those without aromatic ring substitution and those with a 3,4-methylenedioxy substituent), cathinones with an Nmethyl (pentedrone and pentylone) moiety were found at a higher concentration than those with a pyrrolidine ring (α-PVP and MDPV).
Regarding the compounds with an N-ethyl group, N-ethyl-pentedrone had lower permeability than the N-methyl analog whereas N-ethyl-pentylone had a higher permeability than the N-methyl analog. It is also remarkable that the cathinone with a N,N-dimethyl group (N,Ndimethylpentylone) seemed to have the lowest permeability of the BBB.
N-ethyl-pentylone was, by far, the cathinone with the highest concentration in telencephalon tissue. Also known as ephylone or bk-EBDP, this substance is a recently reported cathinone that has been involved in numerous recent intoxication cases 45,46 including 151 deaths between 2014 and 2018, 47 which raises high concerns regarding the toxicity of this compound. The high N-ethyl-pentylone concentration found in telencephalon tissue is in line with a recent study about the pharmacokinetic behavior of this cathinone, which also suggested a high BBB permeability, 39 which could explain its elevated toxicity.
Another common modification seen in cathinone analogs is altering the length of the alkyl chain. In this study, we evaluated three pairs of cathinones that only differed from each other in the length of the alkyl chain. (Table 2): buphedrone and pentedrone, N-ethylpentedrone and N-ethyl-hexedrone, and butylone and pentylone. In all three cases, lengthening the alkyl chain led to a reduction of the BBB permeability, as shown in Table 2. These results are in concordance with data reported in a similar study, 41 where the permeability of methylone, butylone, and pentylone through the BBB was evaluated. The reported concentrations in cerebrospinal fluid were around 13 mg/L for butylone and 7 mg/L for pentylone after dosing Sprague-Dawley rats at 20 mg/kg. These results are coherent with those of the present study, where around 6000 and 3700 ng/g butylone and pentylone, respectively, were found in telencephalon for rats dosed at 3 mg/kg. Based on these data, the increment of the nonpolarity of the cathinones due to the increase of the alkyl chains produces a reduction of the BBB permeability. Strangely, the most potent cathinone analogs in terms of dose reported by consumers are those that have a three-carbon alkyl chain: MDPV, pentylone, α-PVP, pentedrone, and so forth, with the dose being higher if the length is shortened or increased further in most cases. 48 This could indicate that the mechanisms of toxicity of these compounds are not directly linked to their BBB permeability. As can be seen in the case studies for bk-EBDP intoxications, users frequently report a long duration of action for this compound, which is not so common for other cathinones. Perhaps the duration of effects indicates that bk-EBDP lingers in the body for an unusually long amount of time, and some of the toxicity may stem from this phenomenon.
The last typical change in the cathinone structure is functionalization of the aromatic ring. As can be observed in Table 3, the functionalizations studied were the addition of a 3,4-methylenedioxy moiety, a methyl group, a 3,4-dimethoxy group, and the addition of an halogen atom (in this case, a fluorine). Three of the four cathinone couples with/without a 3,4-methylenedioxy moiety (buphedrone and butylone, pentedrone and pentylone, and α-PVP and MDPV) presented a reduction of the permeability through the BBB when this moiety was added to the molecule (Table 3), and it could also be related to the increment of the nonpolarity of the compound by this modification. The remarkably low brain tissue concentration (860 ng/g) for MDPV is in line with previously reported concentrations for MDPV in rat brain, quantified around 260 ng/g at 30 min when dosing a rat at 1 mg/kg. 38 Only for the couple N-ethyl-pentedrone and N-ethyl-pentylone, the cathinone with the 3,4-methylenedioxy moiety presented a higher concentration in telencephalon tissue. Similar to the results obtained when analyzing the N-functionalization (Table 1) It is possible that the presence of these terminal methyl groups allows for easier passing through the BBB. In order to confirm this, more cathinones with these aromatic ring changes should be evaluated.
The concentration differences discussed above, when changing the N-functionalization, alkyl chain length, and aromatic ring substitution, point at a positive correlation between polarity and BBB permeability, as also suggested by others. 41 However, based on an in vitro model using TY09 conditionally immortalized human brain capillary endothelial cells, Simmler and colleagues suggested the opposite: these authors reported that a decrease in polarity of cathinones produces an increment of the permeability of the BBB, with nonpolar cathinones presenting a particularly high transendothelial permeability. 17 Although these in vitro data apparently contradict our findings and those of the previous literature, 38,41 the use of live animals instead of a cell culture is a closer representation of the real pharmacokinetic behavior of these compounds in a process as complex as BBB permeability.
In fact, the BBB is not only composed of endothelial cells but also includes associated cell elements such as astrocyte endfeet, pericytes, and microglia. There are several important routes of transport across the BBB, 25  whereas the presence of a fluorine atom increased BBB transport.
All these data, together with information available in literature from similar studies 41 and the BBB transport 26 suggest that cathinones cross the BBB through a carrier-mediated process. Additionally, this study shows that studying the pharmacology and pharmacokinetics of cathinones, and NPS as a whole, is crucial for a better understanding of the in vivo potency of these compounds, complementing other studies such as dopamine and serotonin uptake inhibition. Our future work will be focused on the study of additional cathinones that will appear on the continuously evolving NPS market, in order to support the role of carrier-mediated processes in the BBB passage of cathinones.