Introduction
According to a 2020 survey conducted by the United Nations Office on Drugs and Crime, amphetamine-type stimulants (ATS), along with marijuana and opioids [
1], are widely abused worldwide. The estimated number of ATS users aged 15–64 was reported to be approximately 34 million, which represents 0.7% of the world population. ATS seizures have continued to increase, reaching a high record of 525 tons, a 15% increase compared with the previous year. In particular, seizures owing to methamphetamine (METH), the most psychoactive ATS, have increased approximately fivefold in the last 10 years, and METH abuse is still a serious social problem [
2].
METH is taken up into presynaptic membranes via dopamine (DA) transporters and partially inhibits DA reuptake. It leads to an excessive release of newly generated DA into the synaptic cleft causing an increase in the DA concentration in the synaptic cleft and stimulation of DA receptors in the postsynaptic membrane [
3]. Continued use of METH induces functional changes in dopaminergic reward pathways, leading to drug addiction [
4]. In addition, METH increases serotonin and noradrenaline levels in the synaptic cleft and elicits symptoms caused by an excess of these monoamines [
4]. Low to moderate doses (5–30 mg) of METH have been reported to cause euphoria, elevated mood, tachycardia, hypertension, and increased body temperature, whereas frequent and high doses induce psychosis [
5].
METH can be abused in a variety of ways including intravenous injection, oral ingestion, snorting, and rectal administration [
6]. While intravenous injection creates a more intense and immediate “high” than other methods, it also leaves injection marks on the arm which increases the risk of being arrested. As a result, consumption through inhaling the smoke produced by heating METH crystals became widespread in the mid-1990s [
7], even among the younger generation [
8]. Smoking METH has become the most common route of intake in recent years [
9‐
11], because it leaves no injection marks and the user feels the effects immediately [
12,
13]. In Japan, prosecutions for possession and/or use of METH continue to be the most common drug offenses, and the amount of METH seized in recent years has remained high [
14], despite the emergence of a number of new psychoactive substances.
After absorption, a portion of METH is metabolized to amphetamine (AMP), and
p-hydroxymethamphetamine via aromatic ring hydroxylation, and
N-demethylation, mainly by CYP2D6 in the liver [
15,
16]. Approximately 20–50% of METH is excreted via urine in its unchanged form, 15% as
p-hydroxymethamphetamine, and 3% as AMP in the first 24 h [
13,
16,
17]. Therefore, the detection of METH and its metabolite AMP in biological samples such as blood and urine is necessary to prove METH use for prosecution.
Clinical studies in which subjects actively inhaled METH smoke indicated that METH was detected in the blood and urine of the subjects [
12,
13]. In some cases, METH was detected in the urine of persons who had not ingested it [
18‐
20]. These cases suggest that passive exposure to METH smoke may result in the excretion of METH and possibly its metabolites in urine. However, the difference in urinary concentrations between active and passive inhalation of METH smoke is not clear. As a result, suspects occasionally claimed in court that the detection of METH in their urine was the result of passive exposure to METH, and they were eventually acquitted in some cases [
21]. To prevent such confusion in court, clarification on the differences in urinary concentrations between active and passive inhalation of METH smoke is required. Clinical studies using METH are not legally and ethically allowed in Japan. Therefore, we investigated the possibility of using methoxyphenamine (MPA), which is a non-regulated and structural analog of METH, as a model drug for METH in mice [
22]. We found that mice exposed to METH or MPA smoke exhibited similar patterns of urinary excretion of the unchanged form. Although there were differences in the amount of metabolites excreted between the two drugs, similarities were observed in the course of excretion over time. In addition, urinary drug concentrations after simulating passive exposure to METH or MPA smoke were significantly lower than those after the active exposure in mice. These results indicate that MPA could be a useful model drug for the study of METH inhalation.
While MPA is administered orally to humans as a cough suppressant [
23], there are no reports of its administration by inhalation. In general, psychoactive drugs absorbed through the lungs are easily transported to the brain without first-pass effects and are, therefore, thought to act on the brain more quickly and profoundly than those administrated orally [
24,
25]. However, there is little information on the toxicity of MPA inhalation, particularly its effect on the central nervous system (CNS).
The purpose of this study was to clarify whether the pattern of urinary drug excretion differs between active and passive METH exposure, using MPA as a model drug. At the beginning of this study, we examined the effects of MPA inhalation on the CNS in mice to ensure the safety of the subsequent clinical study. We then examined urinary drug concentrations in subjects exposed actively and passively to MPA smoke.
Discussion
MPA is administered orally as a cough suppressant and has been used as a model drug for METH in clinical studies [
33,
34], but there are no reports of its inhalation in humans. In general, smoke inhalation of drugs may facilitate its transfer to the brain and affect the CNS. Although no adverse effects on the CNS have been observed when therapeutic doses of MPA are administered orally, the effects of inhalation are not clear. If inhalation of MPA caused toxicity, effects on the CNS would be observed. In our previous study, a temporary and slight decrease in body temperature and locomotor activity were observed in mice exposed to MPA smoke, suggesting that its inhalation may have a mild depressive effect on the CNS [
22]. Salbutamol, a β2-receptor agonist similar to MPA, has been reported to inhibit locomotor activity at high doses in rats and mice [
35,
36]. While measuring locomotor activity in the open field is a good method for evaluating CNS stimulants such as METH [
22,
37], it is not suitable for evaluating CNS depressants because mice acclimated to the environment are inactive and their body temperature tends to decrease [
38]. Therefore, in this study, locomotor activity was measured using the running wheel, which is thought to be a superior method for evaluating depression of the CNS [
30].
Body temperature changes and locomotor activity in mice when compared with the control conditions were not significantly different after inhalation of MPA with air concentrations simulating active inhalation or oral administration of the corresponding doses. In addition, body temperature changes and locomotor activity were similar between the 1 mg/kg p.o. group, which is equivalent to the human therapeutic dose, and the 5 mg/kg p.o. group, which is equivalent to inhalation under the present experimental conditions, and no CNS toxicity was observed with the increased dose. Body temperature changes and locomotor activity between the inhalation and 5 mg/kg p.o. groups were similar, and there were no differences in toxicity between inhalation and oral administration. Taken together, it is clear that MPA inhalation has little effect on the CNS, similar to oral administration, at least up to a dose equivalent to 5 mg/kg. Based on these findings, we considered MPA inhalation in humans to be feasible and conducted a clinical study.
Similar to METH, MPA is metabolized mainly by CYP2D6 [
39,
40], undergoing
O-demethylation and aromatic ring hydroxylation to produce ODMP and 5-hydroxymethoxyphenamine [
41‐
43]. In our previous study, the total urinary excretion rate of METH and AMP was similar to that of MPA and ODMP after mice were exposed to smoke generated by heating METH or MPA, and the urinary excretion patterns were also similar [
22]. Therefore, in this study, urinary concentrations of MPA and ODMP were measured after inhalation of MPA to estimate the changes in urinary concentrations of METH after its inhalation. The maximum urinary concentration ratio of MPA to ODMP was approximately 6:1 in the 1st and 2nd periods in this study (Fig.
4). The maximum urinary concentration ratio of METH to AMP is reported to be approximately 7:1 when 30 mg of METH (as hydrochloride salt) was heated in a glass pipe and the smoke generated was actively inhaled [
12]. Therefore, it is indicated that the concentration ratio of MPA and ODMP excreted in the urine after inhalation is resemble to that of METH and AMP [
12].
When MPA is administered orally to humans, the urinary excretion rate of the unchanged form up to 24 h is reported to be 18–31% [
42,
43], which values are very close to the case of METH administered orally where 18–27% were excreted [
16]. When METH is actively inhaled, the urinary excretion rate up to 72 h is reported to be approximately 37% [
12], which is comparable to the urinary excretion rate when METH was administered orally [
16]. The urinary excretion rate of MPA in mice during 24 h after active inhalation was about 20% [
22], which is comparable to the urinary excretion rate when MPA was administered orally to humans [
42,
43]. These reports suggest that the urinary excretion rates of the unchanged form after inhalation of MPA and METH are comparable. In this study, it was difficult to determine the amount of MPA inhaled by the subjects and it was not possible to calculate the exact urinary excretion rate. However, we observed similar drug concentrations in the air of the exposure chamber when 50 mg of METH or MPA was heated [
22], suggesting that there is no difference in the vaporization of the two drugs. If METH was inhaled in the same way as MPA and the urine volume was similar, the urinary drug concentrations would be comparable. Considering these previous reports and the results obtained in this study, we conclude that MPA is a good model drug for studying urinary pharmacokinetics, including metabolites, in the human inhalation study. It was also indicated that it is possible to estimate the urinary METH concentrations of people who smoked the drug passively and actively from the results of this clinical study by substituting the urinary concentrations of MPA with METH.
The highest urinary METH concentrations reported was approximately 4500 ng/mL, and a concentration of approximately 1500 ng/mL was observed during 24–48 h after active inhalation of METH [
12]. Since MPA concentrations of the air measured at the inhalation position of subjects in the 2nd period were as high as expected (Fig. S1), urinary MPA were estimated to be comparable to METH concentrations previously reported. However, urinary MPA concentrations were significantly lower than reported METH concentrations where METH smoke was inhaled. This is the first in human study where MPA was administered by inhalation, the smoke was inhaled through the nose rather than the mouth for safety reasons. The nose has a lower maximum inspiratory flow rate than the mouth [
44], and therefore, the inhalation volume in the present study would be lower than that of the previous report in which METH was inhaled through the mouth. Although the subjects were asked to inhale as actively as possible, subjects complained that MPA smoke had a distinct and irritating smell, which might make it difficult to inhale through the nose actively. The active inhalation environment in this study was different from the actual abuse environment, and thus could result in lower urinary concentrations of MPA compared to METH.
Currently, the cutoff values or the detection limit for METH screening kits such as AccuSign and Signify ER often used for the screening assays are 500 or 1000 ng/mL. We revealed in this study that the maximum urinary concentrations of MPA and ODMP after passive inhalation of MPA were significantly lower than those after active inhalation in humans, at about 1/58 and 1/56 the amount, respectively (Fig.
4). Subjects with relatively low urine volume and high urinary MPA concentrations in the 1st period of this study, which simulated the passive inhalation condition, the maximum urinary MPA concentration at 0–4 h was 25.9 ng/mL, which was well below the cutoff value. There should be no substantial difference between passive exposure from the adjacent METH abuser and the passive inhalation conditions of MPA in this study. Even if there is some variation in urine volume among subjects, urinary concentrations are not expected to exceed the cutoff value. In the case of METH abusers, on the contrary, it is expected to detect METH well above the cutoff value for a longer time compared to the active inhalation of MPA shown in this study.
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