pharmaceuticals Article Disposition of Phytocannabinoids, Their Acidic Precursors and Their Metabolites in Biological Matrices of Healthy Individuals Treated with Vaporized Medical Cannabis Francesco Paolo Busardò 1,† , Ana Pilar Pérez-Acevedo 2,3,† , Roberta Pacifici 4 , Giulio Mannocchi 5 , Massimo Gottardi 6 , Esther Papaseit 2,3,† , Clara Pérez-Mañá 2,3 , Soraya Martin 2 , Lourdes Poyatos 2,3 , Simona Pichini 4, * and Magí Farré 2,3 1 Department of Excellence-Biomedical Sciences and Public Health, Università Politecnica delle Marche, 60121 Ancona, Italy; fra.busardo@libero.it 2 Clinical Pharmacology Unit, Hospital Universitari Germans Trias i Pujol and Institut de Recerca Germans Trias i Pujol (HUGTiP-IGTP), 08916 Badalona, Spain; appereza.germanstrias@gencat.cat (A.P.P.-A.); epapaseit.germanstrias@gencat.cat (E.P.); cperezm.mn.ics@gencat.cat (C.P.-M.); smartins.mn.ics@gencat.cat (S.M.); lpoyatos@igtp.cat (L.P.); mfarre.germanstrias@gencat.cat (M.F.) 3 Department of Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, Spain 4 National Centre on Addiction and Doping, Istituto Superiore di Sanità, 00161 Rome, Italy; roberta.pacifici@iss.it 5 School of Law, University of Camerino, 62032 Camerino, Italy; giulio.mannocchi@unicam.it 6 Comedical S.Rl, 38123 Trento, Italy; massimo.gottardi@comedical.biz  * Correspondence: simona.pichini@iss.it; Tel.: +39-0649906545  † These authors share first authorship. Citation: Busardò, F.P.; Pérez-Acevedo, A.P.; Pacifici, R.; Abstract: Inhalation by vaporization is a useful application mode for medical cannabis. In this study, Mannocchi, G.; Gottardi, M.; Papaseit, we present the disposition of ∆9-tetrahydrocannabinol (THC), cannabidiol (CBD), their acidic precur- E.; Pérez-Mañá, C.; Martin, S.; sors, and their metabolites in serum, oral fluid, and urine together with the acute pharmacological Poyatos, L.; Pichini, S.; et al. effects in 14 healthy individuals treated with vaporized medical cannabis. THC and CBD peaked Disposition of Phytocannabinoids, firstly in serum and then in oral fluid, with higher concentrations in the first biological matrices Their Acidic Precursors and Their and consequent higher area under the curve AUCs. Acidic precursors ∆-9-tetrahydrocannabinolic Metabolites in Biological Matrices of acid A (THCA) and cannabidiolic acid (CBDA) showed a similar time course profile but lower Healthy Individuals Treated with concentrations due to the fact that vaporization partly decarboxylated these compounds. All THC Vaporized Medical Cannabis. and CBD metabolites showed a later onset with respect to the parent compounds in the absorption Pharmaceuticals 2021, 14, 59. https://doi.org/10.3390/ph14010059 phase and a slower decrease to baseline. In agreement with serum kinetics, THC-COOH-GLUC and 7-COOH-CBD were the significantly most excreted THC and CBD metabolites. The administra- Received: 1 January 2021 tion of vaporized medical cannabis induced prototypical effects associated with the administration Accepted: 12 January 2021 of cannabis or THC in humans, with a kinetic trend overlapping that of parent compounds and Published: 13 January 2021 metabolites in serum. The pharmacokinetics of cannabinoids, their precursors, and their metabolites in biological fluids of individuals treated with vaporized medical cannabis preparations showed a Publisher’s Note: MDPI stays neu- high interindividual variability as in the case of oral medical cannabis decoction and oil. Inhaled tral with regard to jurisdictional clai- medical cannabis was absorbed into the organism earlier than decoction and oil. Cannabinoids ms in published maps and institutio- reached higher systemic concentrations, also due to the fact that the acid precursors decarboxylated nal affiliations. to parent cannabinoids at high temperatures, and consequently, the physiological and subjective effects occurred earlier and resulted with higher intensity. No serious adverse effects were observed. Copyright: © 2021 by the authors. Li- Keywords: cannabis; cannabidiol; cannabidiol metabolism; medical cannabis censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- 1. Introduction tribution (CC BY) license (https:// Medical cannabis is formed by ∆9-tetrahydrocannabinol (THC), cannabidiol (CBD), creativecommons.org/licenses/by/ and their acidic precursors (∆-9-tetrahydrocannabinolic acid A—THCA and cannabidiolic 4.0/). Pharmaceuticals 2021, 14, 59. https://doi.org/10.3390/ph14010059 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 14 Pharmaceuticals 2021, 14, 59 1. Introduction 2 of 14 Medical cannabis is formed by Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and their acidic precursors (Δ-9-tetrahydrocannabinolic acid A—THCA and cannabidiolic acid—CBDA) acid—CBDA) at atdifferent differentproportions, proportions, together togetherwith other with minor other phytocannabinoids minor phytocannabinoids and terpenoids. [1–4].[1–4]. and terpenoids. Indeed, all these Indeed, components all these components havehavebeenbeen provedprovedessential for for essential anti-in- anti- inflammatory flammatory andand neuroprotective neuroprotective properties properties of effects of the the effects of medical of medical cannabis cannabis in the in the symp- symptomatic tomatic treatment treatment of neurogenic of neurogenic pain andpainseveral and several other other pathologies pathologies [5,6]. [5,6]. We recently investigated the disposition of THC, CBD, their precursors, and their principal metabolites in biological matrices of healthy individuals individuals treated with oral oral decoc- decoc- tion and oil, the most used medical cannabis herbal preparations [7,8]. We found that the aqueous preparation preparationwas wasgenerally generallybetter betterabsorbed absorbedthanthanthe theoil, even oil, evenif it if contained it contained a minor a mi- amount nor of THC, amount of THC,THCA-A, THCA-A, andandCBD. CBD.Consequently, Consequently, decoction decoction induced induced a greater feeling a greater feel- of hunger ing andand of hunger drowsiness drowsinessthan the the than oil preparation oil preparation [7].[7]. In any In any case, great case, interindividual great interindivid- variability ual in cannabinoids variability in cannabinoidskinetics and physiological kinetics and physiological and subjective effectseffects and subjective was observed, was ob- especially due to the difficult standardization of herbal preparations and served, especially due to the difficult standardization of herbal preparations and low ab- low absorption of oral administrations [9–12], as previously reported in different studies on sorption of oral administrations [9–12], as previously reported in different studies on adult adult and young patients and youngadministered with oral medical patients administered with oralcannabis medical [13,14]. cannabis [13,14]. Conversely, inhalation by vaporization is a promising application Conversely, inhalation by vaporization is a promising application mode mode for for medical medical cannabis due to the very rapid and good absorption of cannabinoids cannabis due to the very rapid and good absorption of cannabinoids after intrapulmonary after intrapulmonary administration [15,16]. administration [15,16]. Different Different instruments instruments for for vaporization vaporization have have been been validated validated and and applied with preliminary presented data providing optimal efficiency of delivery [17]. applied with preliminary presented data providing optimal efficiency of delivery [17]. Taking into Taking intoaccount accountthetheabove-reported above-reported evidence, evidence, weweinvestigated investigated the disposition and the disposition pharmacokinetics of THC, CBD, their acid precursors THCA and and pharmacokinetics of THC, CBD, their acid precursors THCA and CBDA, their prin- CBDA, their principal metabolites: 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), ∆-9- cipal metabolites: 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), THC-Glucuronide (THC-GLUC) and THC–COOH–Glucuronide (THC–COOH–GLUC) and Δ-9-THC-Glucuronide (THC-GLUC) and THC–COOH–Glucuronide (THC–COOH– CBD-7-oic acid (7-COOH-CBD), 7-hydroxy-hydroxyCBD (7-OH-CBD), 6-alpha-hydroxyCBD GLUC) and CBD-7-oic acid (7-COOH-CBD), 7-hydroxy-hydroxyCBD (7-OH-CBD), 6-al- (6-α-OH-CBD) and 6-beta-hydroxyCBD (6-β -OH-CBD) (Figures 1 and 2) in serum, oral pha-hydroxyCBD (6-α-OH-CBD) and 6-beta-hydroxyCBD (6-β -OH-CBD) (Figures 1 and fluid, and urine samples and acute pharmacological effects of individuals administered 2) in serum, oral fluid, and urine samples and acute pharmacological effects of individuals with vaporized medical cannabis. administered with vaporized medical cannabis. Figure 1. Figure Chemical structures 1. Chemical structures of ∆9-tetrahydrocannabinol (THC), of Δ9-tetrahydrocannabinol (THC), its its acidic acidic precursor, precursor, and and its its metabolites. metabolites. Pharmaceuticals 2021, 14, 59 3 of 14 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 14 Figure 2. Chemical Figure 2. Chemical structures structures of of cannabidiol cannabidiol (CBD), (CBD),its itsacidic acidicprecursor, precursor,and andits itsmetabolites. metabolites. 2. Results 2. Results 2.1. 2.1. Subjects Subjects and and Study Study Design Design All All participants participants completed completed the the study study with with the theexclusion exclusionof ofone onemale malepresenting presentingdiffi- dif- ficulties culties inincoordinating coordinatinginhalation inhalation leading leading to to less less thanthan 25%25% vapor vapor absorption. absorption. For For this this rea- reason, son, 14 14 subjects, subjects, 12 12 menmenand and 2 women 2 women were were finally finally evaluated. evaluated. The 12 males presented a mean age of 21.6 ± The 12 males presented a mean age of 21.6 ± 1.0 years 1.0 years (range(range 20–24 years); mean weight 20–24 years); mean of ± 6.3 kg (range 58–85.5 kg), and mean height of 1.8 ± weight of 69.6 ± 6.3 kg (range 58–85.5 kg), and mean height of 1.8 ± 0.7 m (rangem). 69.6 0.7 m (range 1.7–1.9 The 1.7–1.9 2m). females were both 23 years old; weighed 59.1 and 62.0 kg, and were 1.6 The 2 females were both 23 years old; weighed 59.1 and 62.0 kg, and were 1.6 and 1.70 and 1.70 m high, respectively. The average m high, respectively. The onset of onset average cannabis use was use of cannabis ± 1.9 16.1was years 16.1 ± 1.9(range years 14–18 (rangeyears), 14–18 with a mean of 109 cannabis smoking sessions per year (range years), with a mean of 109 cannabis smoking sessions per year (range of 15–320 of 15–320 sessions) sessions)in the previous in the previousyear. In In year. addition, addition, 86% 86% participants participantsreported reportedprevious previoususe useofoforal oralcannabis cannabis (cookies, (cookies, brownies) and flowering tops smoking through the use of pipes, hookahs, or brownies) and flowering tops smoking through the use of pipes, hookahs, or aa bong. All the participants showed previous experience with tobacco, and bong. All the participants showed previous experience with tobacco, and 64.8% of subjects 64.8% of subjects were were regular regular smokers smokers and and alcohol alcohol users usersatatthe thetime timeofofthe thestudy, study,with witha amean mean daily daily useuseof of 1.2 ± 0.7 standard alcohol units (10 g alcohol)/day. Half of the participants had a 1.2 ± 0.7 standard alcohol units (10 g alcohol)/day. Half of the participants had a past his- past history of occasional hallucinogenic mushrooms or truffle consumption and 58% of tory of occasional hallucinogenic mushrooms or truffle consumption and 58% of different different psychostimulants (such as ecstasy-MDMA, amphetamines, and cocaine). psychostimulants (such as ecstasy-MDMA, amphetamines, and cocaine). The 14 subjects were administered 100 mg FM2 cannabis dried flowering tops contain- The 14 subjects were administered 100 mg FM2 cannabis dried flowering tops con- ing around 6 mg of total THC (also considering the acidic precursor THCA) and 8 mg of taining around 6 mg of total THC (also considering the acidic precursor THCA) and 8 mg total CBD (also considering the acidic precursor CBDA) vaporized by the Volcano vaporizer. of total CBD (also considering the acidic precursor CBDA) vaporized by the Volcano va- Calculating an inhalation bioavailability of approximately 10–30% [18], the systemically porizer. Calculating an inhalation bioavailability of approximately 10–30% [18], the sys- available amount of THC could be considered from 0.6 to 2 mg and CBD from 0.8 to 3 mg. temically available amount of THC could be considered from 0.6 to 2 mg and CBD from 0.8 to 2.2. 3 mg. Concentration-Time Profiles and Pharmacokinetics of THC, CBD, and Their Acidic Precursors in Serum and Oral Fluid after Vaporized Cannabis Administration Figure 3 shows the mean time-course of THC, CBD, THCA, and CBDA concentrations in serum and oral fluid following vaporized cannabis administration. Neither THC or CBD metabolites could be detected in this latter biological matrix. 2.2. Concentration-Time Profiles and Pharmacokinetics of THC, CBD, and Their Acidic Precursors in Serum and Oral Fluid after Vaporized Cannabis Administration Figure 3 shows the mean time-course of THC, CBD, THCA, and CBDA concentra- Pharmaceuticals 2021, 14, 59 4 of tions in serum and oral fluid following vaporized cannabis administration. Neither THC14 or CBD metabolites could be detected in this latter biological matrix. THCA concentration in serum and oral fluid THC concentration in serum and oral fluid 1 100 Concentration (ng/ml) Concentration (ng/ml) 10 0.1 1 0.01 0.1 0 1 2 3 4 6 8 0 1 2 3 4 6 8 Time (h) Time (h) THCA (serum) THC (serum) THCA (oral fluid) THC (oral fluid) CBDA concentration in serum and oral fluid CBD concentration in serum and oral fluid 100 1000 Concentration (ng/ml) Concentration (ng/ml) 100 10 10 1 1 0.1 0.1 0 1 2 3 4 6 8 0 1 2 3 4 6 8 Time (h) Time (h) CBD (serum) CBDA (serum) CBD (oral fluid) CBDA (oral fluid) Figure 3. Mean time-course of THC, CBD, and their acidic precursors (THCA and CBDA) concentrations (logarithmic scale) Figure 3. Mean time-course of THC, CBD, and their acidic precursors (THCA and CBDA) concentrations (logarithmic inscale) seruminand oraland serum fluid following oral the administration fluid following of vaporized the administration cannabis. of vaporized cannabis. THC peaked firstly in serum and then in oral fluid, with concentrations 20 times higher THC peaked firstly in serum and then in oral fluid, with concentrations 20 times in the first biological matrices and consequent higher AUCs (Figure 3 and Tables 1 and 2). higher in the first biological matrices and consequent higher AUCs (Figure 3 and Tables 1 In addition, THC acidic precursor THCA firstly peaked in serum and then in oral fluid, and 2). In addition, THC acidic precursor THCA firstly peaked in serum and then in oral although maximum reached concentrations were similar in both matrices. Interestingly, fluid, although maximum reached concentrations were similar in both matrices. Interest- THCA-A-concentrations did not decline in oral fluid up to 6 h post-administration, thus ingly, THCA-A-concentrations did not decline in oral fluid up to 6 h post-administration, that AUCs in this matrix were 4 times higher than those in serum. thusCBD that disposition AUCs in thisinmatrix serumwere 4 times and oral fluidhigher than those in serum. was superimposable to that of THC with CBD disposition in serum and oral fluid was concentrations higher than those of THC, due to its superior superimposable to medical percentage in that of THC with cannabis concentrations higher than those of THC, due to its superior percentage in medical (Tables 1 and 2). CBDA kinetic profile in the above-reported biological matrices was similar can- tonabis that (Tables of CDB,1with and 2). CBDA serum kinetic profile concentrations in the above-reported one-third of those of CBDbiological and oralmatrices was fluid ones, similar to that of CDB, with serum concentrations three times those of CBD (Figure 3 and Tables 1 and 2). one-third of those of CBD and oral fluid ones, three times those of CBD (Figure 3 and Tables 1 and 2). Table 1. Pharmacokinetic parameters of THC, CBD, and their acidic precursors in oral fluid of healthy 2.3. Concentration-Time volunteers Profiles administered with 100 mgand Pharmacokinetics vaporized of THC Metabolites in Seum after medical cannabis. Vaporized Cannabis Administration All THC metabolites showed Pharmacokinetics Parameters a later onset with respect(mean to the± parent SD) compound in the absorption phase and a slower decrease Cmax (ng/mL) to baseline. Tmax (hour) AUC 0–8h AUC 0–24h (ng/mL·h) This was apparent, especially in the case of THC-COOH-GLUC, (ng/mL which ·h) the showed highest serum concentrations THC 1.29 ± 1.11among0.67 the(0.17–24.0) detected metabolites and a protracted 2.26 ± 2.32 elimina- 4.90 ± 5.22 THCA during the tion phase 24 ± 0.78 h 0.26 3.50 (0.17–8.0) time of collection ± 1.442). THC-COOH 3.26Table (Figure 4 and 7.79 ± 4.29 was the CBD 3.27 ± 2.77 0.67 (0.17–8.0) 4.88 ± 3.90 11.36 ± 8.88 CBDA 10.22 ± 8.49 0.17 (0.17–1.5) 8.79 ± 6.87 13.17 ± 12.60 Cmax , maximum concentration; Tmax , time needed to reach maximum concentration; AUC0–8h and AUC0–24h , area under the concentration–time curve at 18 and 24 h; SD, standard deviation. Pharmaceuticals 2021, 14, 59 5 of 14 Table 2. Pharmacokinetic parameters of THC, CBD, acidic precursors, and metabolites in serum of healthy volunteers administered with 100 mg vaporized medical cannabis. Pharmacokinetics Parameters (mean ± SD) AUC0–8h AUC0–24h Cmax (ng/mL) Tmax (hour) (ng/mL·h) (ng/mL·h) Ke (h−1 ) t1/2 (hour) MRT (hour) THC 24.92 ± 10.74 0.17 12.21 ± 4.11 15.91 ± 6.76 0.79 ± 0.56 1.75 ± 1.97 3.59 ± 4.06 THCA 0.95 ± 0.47 0.33 (0.17–8.0) 0.80 ± 0.1 1.49 ± 2.93 ND ND 3.37 ± 2.17 11-OH-THC 1.43 ± 0.66 0.33 (0.17–1.0) 1.67 ± 0.99 1.91 ± 1.17 0.83 ± 0.79 1.25 ± 0.65 3.05 ± 2.75 THC-COOH 12.08 ± 4.76 0.50 (0.33–2.0) 28.05 ± 16.71 50.23 ± 35.02 ND ND 6.73 ± 2.16 THC-COOH-GLUC 41.85 ± 26.81 3.0 (1.5–8.0) 213.83 ± 132.38 450.52 ± 322.36 0.06 ± 0.02 12.62 ± 3.79 8.55 ± 0.73 THC-GLUC 0.12 ± 0.11 0.5 (0.0–1.5) 0.09 ± 0.08 0.10 ± 0.09 ND ND 1.4 ± 1.14 CBD 93.17 ± 44.77 0.17 68.84 ± 18.25 88.42 ± 50.53 0.46 ± 0.30 3.45 ± 4.72 3.12 ± 2.68 CBDA 34.54 ± 21.54 0.17 (0.17–3.0) 36.90 ± 20.52 63.80 ± 23.21 1.02 ± 1.15 4.75 ± 7.29 7.76 ± 4.18 7-COOH-CBD 11.92 ± 6.29 0.67 (0.67–3.0) 55.38 ± 28.65 140.17 ± 78.63 ND ND 10.23 ± 1.83 7-OH-CBD 2.36 ± 1.02 0.33 (0.17–1.5) 3.28 ± 1.81 4.62 ± 2.82 0.77 ± 0.63 3.22 ± 5.10 4.71 ± 1.90 6α-OH-CBD 0.11 ± 0.11 1.75 (0.17–6.0) 0.22 ± 0.18 0.59 ± 0.94 ND ND 1.00 ± 0.00 6β-OH-CBD 0.08 ± 0.05 0.67 (0.17–24.0) 0.13 ± 0.09 0.25 ± 0.14 ND ND 8.80 ± 4.28 SD, standard deviation; Cmax , maximum concentration; Tmax , time needed to reach maximum concentration (hour, range); AUC0–8h and AU0–24h , area under the concentration–time curve at 8 and 24 h; Ke , elimination constant; t1/2 , elimination half-life. MRT, mean residence time from 0–24 h. ND: Not done. Pharmacokinetic parameters could not be calculated since the number of decreasing points to calculate these parameters was insufficient (less than 3). 2.3. Concentration-Time Profiles and Pharmacokinetics of THC Metabolites in Seum after Vaporized Cannabis Administration All THC metabolites showed a later onset with respect to the parent compound in the absorption phase and a slower decrease to baseline. This was apparent, especially in the case of THC-COOH-GLUC, which showed the highest serum concentrations among the detected metabolites and a protracted elimination phase during the 24 h time of collection (Figure 4 and Table 2). THC-COOH was the second most abundant THC metabolite, although with AUCs almost 10 times lower than those of THC-COOH-GLUC. Conversely, 11-OH-THC and THC-GLUC presented the lowest serum concentrations and a faster elimination from this biological matrix (Figure 2 and Table 2). 2.4. Concentration-Time Profiles and Pharmacokinetics of CBD Metabolites in Serum after Vaporized Cannabis Administration Similarly to what was observed in the case of THC metabolites, CBD metabolites exhibited a delayed onset and decreased to baseline in relation to those of the parent cannabinoid (Figure 5 and Table 1). The carboxy metabolite, 7-COOH-CBD, showed the highest serum concentrations among the detected metabolites and a protracted elimination phase during the 24 h time of collection (Figure 3 and Table 1). 7-OH-CBD was the second most abundant CBD metabolite with AUCs more than 20 times lower than those of 7-COOH-CBD. Finally, 6-α-OH-CBD and 6-β-OH-CBD presented the lowest serum concentrations (Figure 3 and Table 1). 2.5. Urinary Excretion of THC and CBD Metabolites after Vaporized Cannabis Administration Following the administration of vaporized medical cannabis, only THC-COOH-GLUC, THC-GLUC, and THC-COOH could be detected in urine, whereas 11-OH-THC was not detected in urine samples as it is mainly excreted in stool and only present at low concen- trations in urine as a glucuronide conjugate, whose reference standard was not currently commercially available (Figure 6). In agreement with greater systemic bioavailability, THC- COOH-GLUC was the significantly most excreted THC metabolite. Likely, in agreement with serum time course, 7-COOH-CBD was the highly eliminated CBD metabolite followed by 7-OH-CBD and by minimal amounts of 6-α-OH-CBD and 6-β-OH-CBD (Figure 6). Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 14 second most abundant THC metabolite, although with AUCs almost 10 times lower than Pharmaceuticals 2021, 14, 59 those of THC-COOH-GLUC. Conversely, 11-OH-THC and THC-GLUC presented 6the of 14 lowest serum concentrations and a faster elimination from this biological matrix (Figure 2 and Table 2). 11-OH-THC concentration in serum THC-COOH concentration in serum 1.2 15 Concentration (ng/ml) Concentration (ng/ml) 0.9 10 0.6 5 0.3 0.0 0 0 1 2 3 4 6 8 24 0 1 2 3 4 6 8 24 Time (h) Time (h) 11-OH-THC (serum) THC-COOH (serum) THC-COOH glucuronide concentration in serum THC glucuronide concentration in serum 50 0.15 40 Concentration (ng/ml) Concentration (ng/ml) 0.10 30 20 0.05 10 0 0.00 0 1 2 3 4 6 8 24 0 1 2 3 4 6 8 24 Time (h) Time (h) THC-COOH glucuronide (serum) THC glucuronide (serum) Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 14 Figure Figure 4. Mean 4. Mean time-course time-course of THC of THC metabolites metabolites concentrations concentrations in serum in serum following following the the administration administration of vaporized of vaporized can- cannabis. nabis. 7-COOH-CBD concentration in serum 7-OH-CBD concentration in serum 2.4. Concentration-Time Profiles and Pharmacokinetics of CBD Metabolites in Serum after 14 2.8 Vaporized Cannabis Administration 12 Concentration (ng/ml) Concentration (ng/ml) 10 Similarly to what was observed in2.1the case of THC metabolites, CBD metabolites ex- hibited a delayed onset and decreased to baseline in relation to those of the parent canna- 8 binoid (Figure 5 and Table 1). 1.4 6 The carboxy metabolite, 7-COOH-CBD, showed the highest serum concentrations 4 among the detected metabolites and a 0.7 protracted elimination phase during the 24 h time 2 of collection (Figure 3 and Table 1). 7-OH-CBD was the second most abundant CBD me- 0 0.0 0 1 2 3 4tabolite6 with 8 AUCs 24 more than 20 times lower 0 1 than 2 3 those 4 of6 7-COOH-CBD. 8 24 Finally, 6-α- Time (h) OH-CBD and 6-β-OH-CBD presented the lowest serum Time (h) concentrations (Figure 3 and Ta- 7-COOH-CBD (serum) 7-OH-CBD (serum) ble 1). 6-α-OH-CBD concentration in serum 6-β-OH-CBD concentration in serum 0.09 0.06 Concentration (ng/ml) Concentration (ng/ml) 0.06 0.04 0.03 0.02 0.00 0.00 0 1 2 3 4 6 8 24 0 1 2 3 4 6 8 24 Time (h) Time (h) 6-α-OH-CBD (serum) 6-β-OH-CBD (serum) Figure 5. Mean Figure time-course 5. Mean of CBD time-course metabolites of CBD concentrations metabolites in serum concentrations following in serum the the following administration of vaporized administration cannabis. of vaporized can- nabis. Table 1. Pharmacokinetic parameters of THC, CBD, and their acidic precursors in oral fluid of healthy volunteers admin- istered with 100 mg vaporized medical cannabis. Pharmacokinetics Parameters (mean ± SD) Cmax (ng/mL) Tmax (hour) AUC0–8h (ng/mL·h) AUC0–24h (ng/mL·h) Pharmaceuticals 2021, 14, 59 7 of 14 Figure 6. The total amount of THC-GLUC, THC-COOH, THC-COOH-GLUC, and 6-α-OH-CBD, 6-β -OH-CBD, 7-OH-CBD, 7-COOH-CBD excreted in 24 h urine samples following the administration of vaporized medical cannabis. 2.6. Physiological Measures and Subjective Effects The administration of vaporized medical cannabis induced prototypical effects associ- ated with the administration of cannabis or THC in humans (Table 3 and Figure 4) [19,20]. Very mild changes in blood pressure and a significant increase in heart rate, with a peak effect of +25 beats per min were observed at 10 min and persisted at 20 and 40 min post- administration were observed. The scores of some visual analog scales (VAS) feelings of wellbeing as intensity, high, and good effects during almost 1–2 h, increased with peak effects at 10 min post-inhalation. Similar increases in the euphoria-ARCI-MBG scale and amphetamine life effects-ARCI-A were detected 2 h post-administration. Somnolence increased significantly during 3 h with a peak effect of 43 mm in the drowsiness-VAS scale and in the sedation-ARCI-PCAG scale. Hunger sensation increased from 20 min to 4 h. Other effects appeared only at one or two evaluation times (Table 3 and Figure 7). Whereas time courses of hearth rate, feelings of intensity, and high overlapped those of THC and CBD, variations in feelings of hunger, drowsiness, euphoria, and amphetamine-like effects presented a slower onset and a slower decrease to baseline similar to that of THC and CBD metabolites No psychotic effects or other serious side effects were observed. Table 3. Physiological and subjective effects in healthy volunteers administered with 100 mg vaporized medical cannabis. Effects Emax (mean ± SD) Tmax (Median-Range) AUC0–8h (mean ± SD) Systolic blood pressure, mm Hg −6.57 ± 16.37 1.34 (0.17–6.0) −46.14 ± 54.18 Diastolic blood pressure, mm Hg 2.93 ± 14.12 0.84 (0.17–6.0) −16.04 ± 46.15 Heart rate, bpm 25.64 ± 23.23 0.17 (0.17–8.0) 10.97 ± 55.62 VAS Intensity, mm 42.86 ± 22.45 0.17 (0.17–0.67) 44.20 ± 35.69 VAS High, mm 43.79 ± 22.87 0.17 (0.17–0.33) 42.18 ± 32.15 VAS Good effects, mm 33.00 ± 22.38 0.33 (0.0–1.0) 37.34 ± 30.49 VAS Bad effects, mm 13.00 ± 19.42 0.17 (0.0–0.33) 7.14 ± 12.94 VAS Appetite, mm 47.36 ± 27.42 2.0 (0.0–3.0) 141.31 ± 113.40 VAS Drowsiness, mm 39.00 ± 28.25 1.5 (0.17–3.0) 76.83 ± 59.86 VAS Dizziness, mm 19.64 ± 22.38 0.17 (0.0–1.0) 10.06 ± 13.36 VAS Confusion, mm 11.79 ± 16.04 0.17 (0.0–0.67) 4.27 ± 5.90 VAS Nausea, mm 1.57 ± 4.85 0.0 (0.0–0.67) 0.80 ± 2.80 VAS Vomit, mm 1.43 ± 5.35 0.0 (0.0–0.33) 0.46 ± 1.72 VAS Anxiety, mm 7.0 ± 14.82 0.0 (0.0–0.33) 4.66 ± 10.86 ARCI-PCAG, score 5.71 ± 2.02 0.33 (0.33–3.0) 12.25 ± 8.82 ARCI-MBG, score 4.21 ± 4.58 0.33 (0.0–2.0) 8.26 ± 11.61 ARCI-LSD, score 2.14 ± 2.54 0.33 (0.33–1.5) 0.81 ± 3.46 ARCI-BG, score −1.00 ± 2.57 0.67 (0.33–4.0) 0.03 ± 5.54 ARCI-A, score 3.00 ± 2.39 0.33 (0.0–1.5) 7.63 ± 8.45 VESSPA-S, score 0.90 ± 0.53 1.0 (1.0) 2.88 ± 2.11 VESSPA-SA, score 0.69 ± 0.31 1.0 (1.0–2.0) 1.86 ± 1.17 1 Pharmaceuticals 2021, 14, 59 8 of 14 Table 3. Cont. Effects Emax (mean ± SD) Tmax (Median-Range) AUC0–8h (mean ± SD) VESSPA-CP, score 0.02 ± 0.09 0.0 (0.0–1.0) 0.02 ± 0.09 VESSPA-PCS, score 0.64 ± 1.08 1.0 (0.0–2.0) 2.69 ± 4.75 VESSPA-AE, score 0.36 ± 0.65 0.0 (0.0–8.0) 1.19 ± 2.35 VESSPA-SP, score 0.46 ± 0.75 1.0 (0.0–2.0) 0.83 ± 0.99 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 14 Emax = peak effects; Tmax = time needed to reach maximum effects; AUC0–8h = area under the curve from 0 to 8 h; SD (standard deviation); VAS (Visual analog scales); ARCI (Addiction Research Center Inventory); VESSPA-SEE (Evaluation of subjective effects of substances with abuse potential). Heart rate VAS Intensity VAS High 40 50 50 beats per minute (bpm) 30 40 40 20 30 30 mm mm 10 20 20 0 10 10 -10 0 0 0 1 2 3 4 6 8 0 1 2 3 4 6 8 0 1 2 3 4 6 8 Time (h) Time (h) Time (h) VAS Hunger VAS Drowsiness ARCI-PCAG 50 50 6 5 40 40 4 30 30 3 Score mm mm 20 20 2 1 10 10 0 0 0 -1 0 1 2 3 4 6 8 0 1 2 3 4 6 8 0 1 2 3 4 6 8 Time (h) Time (h) Time (h) Figure 7. Mean time-course of physiological and subjective mostly modified effects in healthy volunteers administered Figure 7. Mean time-course of physiological and subjective mostly modified effects in healthy volunteers administered with 100 mg vaporized cannabis. with 100 mg vaporized cannabis. Table 3. Physiological and 3. Discussion subjective effects in healthy volunteers administered with 100 mg vaporized medical cannabis. This is the first study on disposition THC, CBD, their acidic precursors, and their Effects Emax (mean ± SD) Tmax (Median-Range) AUC0–8h (mean ± SD) metabolites in serum, oral fluid, and urine samples of healthy adult volunteers after Systolic blood pressure,controlled mm Hg administration−6.57 ± 16.37 1.34 (0.17–6.0) of vaporized medical cannabis containing−46.14THC ±and 54.18 CBD in Diastolic blood pressure, mm Hg similar percentages. 2.93 ± 14.12 0.84 (0.17–6.0) −16.04 ± 46.15 Heart rate, bpm 25.64several As a matter of fact, ± 23.23previous studies 0.17 (0.17–8.0) investigated cannabinoids10.97disposition ± 55.62 in VAS Intensity, mm 42.86 blood, oral fluid, thus as ±urinary 22.45 excretion0.17 (0.17–0.67) following vaporized of metabolites 44.20 ± 35.69 cannabis VAS High,mmadministration, but 43.79 ± 22.87 strain selected the cannabis 0.17 (0.17–0.33) for considered trials was 42.18 the±recreational 32.15 one, VAS Good effects, mm mainly containing THC 33.00 ± 22.38at percentages ranging from 0.33 (0.0–1.0) 2.9% to 6.7% and negligible 37.34 ± 30.49 amounts VAS Bad effects, mm of CBD [19–22]. Nevertheless, 13.00 ± 19.42 results obtained 0.17 (0.0–0.33)for THC and its 7.14 ± 12.94 are in metabolites VAS Appetite, mm agreement with those below 47.36 summarized. 2.0 (0.0–3.0) ± 27.42 141.31 ± 113.40 VAS Drowsiness, mm This experiment with vaporized 39.00 ± 28.25 medical cannabis 1.5 (0.17–3.0)has been the third 76.83part of a study ± 59.86 VAS Dizziness, mm on the disposition of THC, CBD, 19.64 ± 22.38 their acidic precursors, 0.17 (0.0–1.0) and their metabolites following 10.06 ± 13.36 the administration of the three most common formulations of medical cannabis: Decoction, VAS Confusion, mm 11.79 ± 16.04 0.17 (0.0–0.67) 4.27 ± 5.90 oil, and vaporized flowering tops [7,8]. In this regard, it is worth noticing that: VAS Nausea, mm 1.57 ± 4.85 0.0 (0.0–0.67) 0.80 ± 2.80 VAS Vomit, mm • Pharmacokinetics of cannabinoids, 1.43 ± 5.35 their0.0 precursors, (0.0–0.33)and their metabolites 0.46 ±in1.72 biological fluids of healthy individuals treated with the three different formulations all showed VAS Anxiety, mm 7.0 ± 14.82 0.0 (0.0–0.33) 4.66 ± 10.86 great interindividual variability, likely due to the difficult standardization of herbal ARCI-PCAG, score 5.71 ± 2.02 0.33 (0.33–3.0) 12.25 ± 8.82 ARCI-MBG, score 4.21 ± 4.58 0.33 (0.0–2.0) 8.26 ± 11.61 ARCI-LSD, score 2.14 ± 2.54 0.33 (0.33–1.5) 0.81 ± 3.46 ARCI-BG, score −1.00 ± 2.57 0.67 (0.33–4.0) 0.03 ± 5.54 ARCI-A, score 3.00 ± 2.39 0.33 (0.0–1.5) 7.63 ± 8.45 VESSPA-S, score 0.90 ± 0.53 1.0 (1.0) 2.88 ± 2.11 Pharmaceuticals 2021, 14, 59 9 of 14 preparations in the case of decoction and oil and different inhalation rates in case of the vaporized formulation; • A 60 times higher bioavailability and a significantly faster concentration peak of THC was observed in the case of medical cannabis inhalation vs. oral formulations, corresponding to a more than 60 times lower availability of THCA, due to acidic precursor decarboxylation during the vaporization process; • The proportion between THC metabolites was similar in the three different prepara- tions, with THC-COOH-GLUC being always the most formed metabolite followed by THC-CCOH. • Oral fluid did not appear a suitable biological matrix alternative to serum for cannabi- noids monitoring following vaporized medical cannabis administration since oral fluid concentrations did not reflect the ones measured in serum. • A more than 90 times higher bioavailability and a significantly faster concentration peak of CBD was observed in the case of medical cannabis inhalation vs. oral formula- tions, corresponding to a more than 3 times lower availability of CBDA in comparison with that of cannabis decoction due to acidic precursor decarboxylation during the va- porization process. Conversely, the bioavailability of CBDA in cannabis oil as similar to that of vaporized cannabis. • Whereas minimal salivary excretion did not discriminate different methods of med- ical cannabis administration, urinary extraction of both THC and CBD metabolites reflected the different parent compound’s availability following the three kinds of medical cannabis formulations. Our results are quite comparable to those observed in a previous study, in which 200 mg of medical cannabis containing 6.6% THC and 8% CBD were administered to relieve chronic pain in patients with fibromyalgia [23]. THC and CBD Cmax of 76 and 80 ng/mL, respectively, was found in comparison to 24.92 and 93.17 ng/mL obtained in our study, in which medical cannabis dose was half. With respect to pharmacological effects, an Emax in a high feeling of 45 mm at 30 min in the previous study reflected our maximum score of 43.79 mm at 20 min. It has to be said that the present study shows some limitations: The inclusion of healthy subjects instead of treated patients, the relatively low dose administered in comparison to those recommended for medical cannabis administration, the difficult interpretation of the subjective effects since a placebo condition was not administered, and the fact that the volunteers were almost exclusively males, preventing the evaluation of possible differences between genders. 4. Materials and Method 4.1. Subjects Enrolment Fifteen subjects, 13 men and 2 women, were initially recruited. The subjects were recruited by the “word of mouth” method and the database of volunteers who had partici- pated in previous studies at the Clinical Pharmacology Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Spain. The participants were informed of the characteristics of the study and signed informed consent. Eligibility criteria included social or recreational cannabis use in the past 12 months (monthly to weekly use, without criteria of cannabis use disorder) and vaporized cannabis use at least once in their life. Exclusion criteria were history of any serious medical or psychopathological conditions, including substance use disorder (with the exception of nicotine, based on diagnosis and statistical criteria for DSM-5 mental disorders), pregnancy and lactation, chronic medicines use, and serious adverse reactions associated with cannabis use. Within 3 weeks prior to inclusion in the study, participants underwent a general medi- cal examination, which included blood tests, urinalysis, and a 12-lead electrocardiogram (ECG). In addition, they completed a training session to become familiar with the study procedures and questionnaires. The study protocol was approved by the local Human Pharmaceuticals 2021, 14, 59 10 of 14 Research Ethics Committee (CEIC-HGTiP, Badalona, Spain), and the study was carried out in accordance with the Declaration of Helsinki and Spanish laws on clinical research. 4.2. Preparation and Inhalation Procedure for Vaporized Cannabis A standardized medicinal cannabis FM2 flower cup extract containing 3.4% ± 0.7% THC, 2.8% ± 0.8% THCA-A, 2.7% ± 0.5% CBD, and 6.2% ± 1.1% CBDA (plus minor phytocannabinoids with percentages below 0.5%) was provided by the Italian Military Pharmaceutical Chemical Factory in Florence with the authorization of the Italian and Spanish Medicines Agencies [1,2]. Considering that at high temperatures, the acidic precursors THCA and CBDA are decarboxylated to THC and CBD, the total content of these latter compounds can be calculated adjusting for the differing molecular weight of the cannabinoids and carboxylic conjugative components of each cannabinoid: total % THC: %THC + (0.877%*THCA) = 5.8% total % CBD: %CBD + (0.877%*CBDA) = 8.1% The FM2 preparation was administered by inhalation/vaporization using the Volcano vaporizer. The preparation of vaporized cannabis was carried out in accordance with the recommendations of the Volcano Vaporizer System (https://keytocannabis.com/volcano- classic-vaporizer-review-how-to-use-it-and-why-you-need-one/). For the preparation of inhaled vaporized cannabis, a 100 mg dose of FM2 cannabis administered via the Volcano vaporizer (containing around 6 mg of THC and 8 mg of CBD) was used. Calculating an inhalation bioavailability of approximately 10–30% [24], the available dose exposure dose would be from 0.6 to 2 mg THC and from 0.8 to 3 mg CBD. The procedure for cannabis administration consisted of weighing 100 mg of FM2 cannabis and placing it in the filling chamber. At that point, the vaporizer was set at 225 ◦ C, and the pump was turned on. After the plant material was heated, the valve balloon was fitted, and steam was allowed to flow into the valve balloon for approximately 45 s. For the vaporized cannabis inhalation, participants had to place their lips on the mouthpiece of the balloon and to inhale the vapor through a deep breath of about 5 s, hold their breath for 5–10 s before exhaling it for an additional 5 s. The subjects then rested for about 10 s and the above-mentioned process was repeated every 30 s until the vapor contained in the balloon ended. Theoretically, participants made an average of 20 inhalations in about 10 min. During this procedure, the subjects remained seated in a chair and could not speak. 4.3. Study Design The study was open-label, non-randomized, and single-blind since the participants knew they would be administered with medical cannabis, but they were not aware of administered doses. The subjects entered the Clinical Research Unit (UPIC) at Hospital Universitari Germans Trias i Pujol, Badalona, Spain at approximately 13:00 h (01:00 p.m.). Upon arrival, participants were asked about any substance/drug or event that could have affected their participation in the study. Subjects had been fasting from food for the previous 3 h, from water for the previous 2 h, in addition to abstinence from alcohol for the preceding 48 h, from coffee, tea, cola, cacao for the previous 24 h, from tobacco smoking for 1 h before. Most importantly, they had not used cannabis from 5 days before the start of the experimental session up to 24 h nor any other psychotropic drug from the screening visit to the end of the study. Before starting the experimental sessions, a urine sample was taken for the on-site drug test (Instant-View® , Multipanel 10 Test Drug Screen, Alfa Scientific Designs Inc., Poway, CA, USA), and alcohol was tested in exhaled air (Dräger alcotest 5820, Dräger, Germany). If a participant resulted positive for drugs or alcohol, he/she was excluded from the experiment. The participants stayed in a quiet and comfortable room in the Hospital’s Clinical Research Unit (UPIC) throughout the session. Tobacco smoking was not allowed. Vaporized cannabis was administered between 14:00 and 14:30 h (02:00–2:30 p.m.), and the experimental session had a duration of 8 h following administration. At the end of Pharmaceuticals 2021, 14, 59 11 of 14 cannabis inhalation, the participants’ mouth was washed twice with water to avoid oral contamination in the oral fluid collection. The participants received the treatment with 100 mL of bottled water. Two and 5 h after the administration, subjects received a snack and a meal, respectively. After 4–7 days of each experimental session, a final medical control including physical examination, vital signs, electrocardiogram, blood and urine tests, and collection of adverse events, was conducted. 4.4. Physiological and Subjective Effects Measurement Non-invasive systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), oral temperature (T), and subjective effects were repeatedly recorded 45 and 15 min before and 10, 20, 40 min and 1, 1.5, 2, 3, 4, 6, 8, and 24 h after vaporized cannabis admin- istration. Physiological parameters were monitored using a vital sign monitor (Philips SureSigns VM4 monitors, Phillips). Subjective pharmacological effects were measured using the visual analog scales (VAS), the 49-item-short form of the Addiction Research Center Inventory (ARCI), the valuation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) questionnaires, and the Positive and Negative Syndrome Scale (PANSS). The VAS scales used were a set of 15 items (100 mm, from “not at all” to “ex- tremely”) to rate the intensity of the following: Intensity; high; good effects; bad effects; appetite (hunger); drowsiness; dizziness; confusion; nausea; vomiting; anxiety; aggressive- ness; visual hallucinations with lights and spots; visual hallucinations with animals, things, insects, or people; auditory hallucinations. The VAS scales were assessed at −45 (baseline), 10, 20, 40 min, and at 1, 1.5, 2, 3, 4, 6, 8, and 24 h after administration. The ARCI is a true/false 49-item questionnaire, which is a sensitive instrument for determining subjective drug effects and consists of 5 subscales (ARCI-PCAG-sedation, ARCI-MBG-euphoria, ARCI-LSD- dysphoria and somatic symptoms, ARCI-BG-intellectual efficiency, ARCI-A-amphetamine-like effect) [25,26]. This was administered at the same times as VAS, except for the 10 min evaluation. The VESSPA-SE questionnaire measured changes in subjective effects caused by a number of drugs. It included 6 subscales: Sedation (S), psychosomatic anxiety (ANX), changes in perception (CP), pleasure and sociability (SOC), activity and energy (ACT), and psychotic symptoms (PS) [27,28]. It was administered at −45 min (baseline) and at 1, 2, and 8 h after administration. The PANSS for the evaluation of psychotic symptoms was scored before and 8 h after substance administration. 4.5. Biological Samples Collection Serum (2 mL) from whole blood was collected from 15 min (zero time) prior and 10 min, 20 min, 40 min, 1, 1.5, 2, 3, 4, 6, 8, and 24 h after vaporized cannabis administration. Oral fluid samples were collected using a Salivette device (Sarsted, Nümbrecht, Germany) at the same time points. Before oral fluid collection, participants had to wash their mouth with two glasses of water. Urine was collected 15 min prior to administration (baseline time) and then between 0–2 h, 2–4 h, 6–8 h, and 8–24 h intervals. After collection, all biological samples were stored at −30 ◦ C until analysis. 4.6. Determination of Cannabinoids, Acidic Precursors, and Metabolites in Biological Samples THCA, THC, THC-COOH, 11-OH-THC, THC-GLUC, THC–COOH–GLUC, CBDA, and CBD were quantified in the collected biological matrices by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) equipped with an electrospray ionization source operating in positive ion mode as previously reported [29]. With respect to CBD metabolites, 6-α-OH-CBD, 6-β -OH-CBD, 7-OH-CBD, 7-COOH-CBD, a specific UHPLC-MS-MS assay recently developed and validated [30]. For both assays, limits of quantification ranged from 0.05 to 0.2 ng/mL. with average inter/intra-day accuracy and precision <15% [29,30]. Pharmaceuticals 2021, 14, 59 12 of 14 4.7. Statistical Analysis Maximum concentration (Cmax ), time needed to reach maximum concentrations (Tmax ), area under the concentration-time curve at 10 and 24 h (AUC0–10h , AUC0–24h ), elimination half-life (t1/2 ), elimination constant (Ke , calculated with at least 3 sample points), mean residence time (MRT as the area under the concentration times time ver- sus time curve-AUMC0–24h divided by AUC0–24h ) for THC, CBD, their acidic precur- sors, and their metabolites in serum and oral fluid were determined using Pharma- cokinetic Functions for Microsoft Excel (Joel Usansky, Atul Desai, and Diane Tang-Liu, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA, USA at https://www.coursehero.com/file/30859156/pkfdoc/). Maximum effects (Emax) were determined and the areas under the curve of the effects (AUC0–8h ) were calculated using the trapezoidal rule. To evaluate the physiological (SBP, DBP, HR, and T) and subjective effects (VAS, ARCI, VESSPA) along time in comparison to baseline, a one-way repeated measures ANOVA, with time as a factor, was done to evaluate the time-course of the effects. When the time condition was statistically significant, a Dunnett multiple comparison post hoc test was conducted to compare the different time points with baseline. 5. Conclusions Notwithstanding the listed limitations, the results obtained in this study are relevant and in agreement with those already presented on the issue of oral cannabis preparations: The pharmacokinetics of cannabinoids, their precursors, and their metabolites in biological fluids of individuals treated with medical cannabis preparations showed high interindi- vidual variability, especially due to the difficult standardization of herbal preparations. Vaporized medical cannabis was earlier absorbed into the organism, cannabinoids reached higher systemic concentrations, also due to the fact that the acid precursors decarboxylated to parent cannabinoids at high temperatures. Consequently, the physiological and subjec- tive effects occurred earlier and were higher. In no case did adverse effects appear, although the doses administered had always been low. Greater clinical trials, including both healthy volunteers and patients suffering from pathologies treated with medical cannabis and a larger number of women, and studying clinical and side effects of chronic administration will complement these significant preliminary observations. Author Contributions: Conceptualization, R.P., M.F., S.P., and F.P.B.; methodology, A.P.P.-A., G.M., M.G., L.P., E.P., C.P.-M., and S.M.; resources, R.P., M.F., F.P.B.; data curation, A.P.P.-A., G.M., M.G., L.P., E.P., C.P.-M., and S.M.; writing—original draft preparation, R.P., M.F., L.P., S.P. and F.P.B.; writing— review and editing, All authors. All authors have read and agreed to the published version of the manuscript. Funding: The investigation was partially carried out with funding from Presidency of the Ministers Council, Department of Antidrug Policy and from Department of Excellence SBSP, University “Politecnica delle Marche” of Ancona; and partially funded by grants from the Instituto de Salud Carlos III (ISCIII, Fondo Investigación sanitaria (FIS)-Fondo Europeo de Desarrollo Regional (FEDER), Grant Numbers: FIS PI14/00715 and PI20/00879, ISCIII-Red de Trastornos Adictivos RTA Grant Number: RD16/0017/0003, ISCIII-Plataforma de Unidades de Investigación y Ensayos Clínicos- SCREN, Grant Number: PT 17/0017/0016; AGAUR Gencat Suport Grups Recerca, Grant Number: 2017 SGR 316; Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional Sobre Drogas-PNSD, Grant Number 2015I054). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Human Research Ethics Committee (Comité de Ética de la Investigación) del Hospital UniversitarioGermans Trias i Pujol (CEI-HUGTiP, protocol code PI-17-163 and date of approval 21-July-2017). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available within the article or on request from the corresponding author. Pharmaceuticals 2021, 14, 59 13 of 14 Acknowledgments: The authors thank Laura Martucci, Simonetta Di Carlo and Antonella Bacosi, Michele Sciotti, and Josuè Gottardi, for technical assistance. Conflicts of Interest: There are no potential conflict of interest or any financial or personal relation- ships with other people or organizations that could inappropriately bias conduct and findings of this study. References 1. 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