
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

71851
10.1038/s41598-024-71851-z
Article
A randomized placebo controlled trial demonstrates the effect of dl-methylephedrine on brain functions is weaker than that of pseudoephedrine
Sakayori Takeshi 1
Ikeda Yumiko 2
Arakawa Ryosuke 2
Nogami Tsuyoshi 1
Tateno Amane amtateno@nms.ac.jp

1
1 https://ror.org/00krab219 grid.410821.e 0000 0001 2173 8328 Department of Neuropsychiatry, Graduate School of Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo, 113-8603 Japan
2 https://ror.org/00krab219 grid.410821.e 0000 0001 2173 8328 Department of Pharmacology, Nippon Medical School, Tokyo, Japan
5 9 2024
5 9 2024
2024
14 2079329 3 2024
31 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Intellectual drug doping in athletics by using stimulants that affect central nervous system functions has been diversified. Stimulants are regulated by the World Anti-Doping Agency according to their levels of urinary concentration. Positron emission tomography could evaluate how stimulants affect central nervous system functions. We aimed to evaluate the effect of stimulants on brain function by examining the difference in brain dopamine transporter occupancy by PET after administration of dl-methylephedrine or pseudoephedrine at the clinical maximum daily dose. Four PET scans without and with drug administration (placebo, dl-methylephedrine 150 mg and pseudoephedrine 240 mg) were performed. The concentrations of dl-methylephedrine and pseudoephedrine in plasma and urine were measured. DAT occupancies in the striatum with placebo, dl-methylephedrine and pseudoephedrine were calculated by PET images. The urinary concentration of dl-methylephedrine (12.7 µg/mL) exceeded the prohibited concentration (10 µg/mL), but the DAT occupancy with dl-methylephedrine (6.1%) did not differ (p = 0.92) from that with placebo (6.2%). By contrast, although the urinary concentration of pseudoephedrine (144.8 µg/mL) was below the prohibited concentration (150 μg/mL), DAT occupancy with pseudoephedrine was 18.4%, which was higher than that with placebo (p = 0.009). At the maximum clinical dose, dl-methylephedrine was shown to have weaker effects on brain function than pseudoephedrine.

Subject terms

Biomarkers
Cognitive neuroscience
Reward
the Japan Sports Agency Ministry of Education, Culture, Sports, Science and Technology-JapanResearch and Development Project on Anti-doping Science issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Drug doping in athletics has been diversified, and the use of drugs aimed not only at purely athletic performance but also at intellectual performance has been expanding in recent years. Intellectual ability doping uses stimulants that eliminate fatigue, enhance attention, concentration, cognitive function, and improve the ability to handle complex performances1. Stimulants that affect central nervous system functions are listed as S6 stimulants on “The Prohibited List” provided by the World Anti-Doping Agency (WADA)2. Dopamine is a multifunctional neurotransmitter involved in the regulations of motor ability, learning ability, emotion, appetite, motivation and memory, and it is linked to the brain reward system3. Extracellular dopamine concentrations can be increased by stimulant-related inhibition or reversal of the monoamine reuptake transporters, of which the main one is the presynaptic dopamine transporter (DAT)4. Stimulants improve intellectual ability by increasing the levels of extracellular dopamine in the brain, including the reward and addiction pathways, by inhibiting DAT5.

dl-methylephedrine is a derivative of ephedrine, and pseudoephedrine is an optical isomer of ephedrine. Methamphetamine, synthesized from ephedrine, is a powerful modulator of the dopamine system, and it has a myriad of effects that influence dopamine release. It disrupts dopamine reuptake and packaging systems, likely through direct effects on DAT6,7. Thus, both dl-methylephedrine and pseudoephedrine are listed in S6 stimulants. The rate of ephedrine-related doping violations is considerable, as ephedrine-related substances are compounded in general medicines and in some nutritional supplements for relieving cold symptoms. Sine previous study reported, 2.8% of urine specimens from athletes contained at least one ephedrine-related substance, half of which exceeded the cutoff values (e.g. 44% for pseudoephedrine, 11% for dl-methylephedrine)8. Both dl-methylephedrine and pseudoephedrine are included in most drugs such as cold, rhinitis and cough medications, and these drugs are widely used as nonprescription drugs all over the world. Athletes should take great care when taking these medications because they may commit unintended doping violations9.

In “The Prohibited List” by WADA, dl-methylephedrine and pseudoephedrine in S6 stimulants are regulated by the levels of urinary concentration. Prohibited urinary concentration of dl-methylephedrine and pseudoephedrine, which affect the central nervous system via DAT, are considerably different. In “The Prohibited List” by WADA, the prohibited urinary concentration of dl-methylephedrine is less than 10 µg/mL, the same as ephedrine, whereas that of pseudoephedrine is less than 150 μg/mL2. The prohibited urinary concentration of pseudoephedrine was 10 μg/mL as well as ephedrine and dl-methylephedrine before 2000, but was changed to 25 μg/mL in 2003 and is 150 μg/mL in 2010. Some studies have reported that pseudoephedrine has a dose-dependent enhancing effect on athletic performance10,11, but the review summarized that performance benefits at doses less than maximum daily dose of 240 mg are small and may be less than for permitted stimulants such as caffeine12. Then the urinary concentration of pseudoephedrine at its maximum daily dose was used as a reference for the prohibition value. On the other hand, the prohibited urinary concentration of dl-methylephedrine remains the same as ephedrine. This is because no studies have shown that dl-methylephedrine does not have any effect of improving athletic performance. We consider the prohibited urinary concentration of dl-methylephedrine is too strict compared to pseudoephedrine, since our previous study showed that even the minimum daily dose of 60 mg of dl-methylephedrine could exceed the prohibited value of 10 µg/mL, even though the effect on brain function was the same as those of placebo13.

Brain imaging studies are useful for showing how stimulants affect central nervous system functions, and the dopamine system can be evaluated by positron emission tomography (PET). It is possible to investigate the effect on brain function by using [18F] FE-PE2I, which has high affinity and selectivity especially for DAT14,15. Regarding pharmacological effects on the central nervous system, we have previously reported the effects of mazindol, modafinil and dl-methylephedrine on brain function using PET studies13,16,17.

In addition, functional magnetic resonance imaging (fMRI) can identify neural correlates of higher cognitive functions. fMRI measures neuronal activity in an indirect manner via the blood oxygenation level-dependent contrast, which arises from local changes in the ratio between oxygenated and deoxygenated hemoglobin owing to metabolic demands driven by neuronal activity18,19. fMRI has also been used to investigate the effects of central nervous system acting drugs on higher cognitive functions. For example, stimulants such as amphetamine and modafinil have been shown to increase activation in the nucleus accumbens for reward processing20,21. We have also evaluated how the neuronal networks responsible for various cognitive functions are modified under the effects of drugs by using fMRI22. Thus, fMRI as part of pharmacological intervention could assess the effects of drugs in improving cognitive functions and sport performance as well as enhance the underlying neural correlates.

The aim of this study was to evaluate the effect of stimulants on brain function by examining the difference in brain DAT occupancy by PET and brain activity by fMRI after administration of dl-methylephedrine or pseudoephedrine at the clinical maximum daily dose. Furthermore, by comparing the urinary concentrations of these two drugs and the extent of their effects on the brain, we examined whether it is appropriate for dl-methylephedrine to be regulated more strictly than pseudoephedrine in order to provide clear and objective evidence for managing drug doping.

Materials and methods

Subjects

We conducted this study from November 1, 2020 to February 28, 2022. Six healthy adult male volunteers were scheduled to undergo four PET scans one under no administration, three under the respective administration of placebo, dl-methylephedrine and pseudoephedrine, and also three fMRI scans under the administration of placebo, dl-methylephedrine and pseudoephedrine, respectively. One subject (case 3) was discontinued after two PET scans (no administration and placebo), and therefore another subject (case 7) was added to this study and three PET scans (no administration, dl-methylephedrine and pseudoephedrine) were performed.

No subject had a history of present and/or past psychiatric, neurological or physical disorders. All subjects had a Body Mass Index of 18.5 or more and less than 25, and none had a history of smoking. The subjects were forced to stop taking in caffeine and alcohol 48 h before the PET and fMRI scans.

The study was approved by the Institutional Review Board of Nippon Medical School, and the Foundation certified by the Ministry of Health, Labour and Welfare in Japan. This study was conducted in accordance with the ethical principles based on the Declaration of Helsinki and in compliance with the Clinical Research Law of Japan, the enforcement regulations of the Law, and other relevant notices. Prior to the start of the study, the research investigator gave the research candidates a consent explanation document approved by the Institutional Review Board of Nippon Medical School, and the Foundation, and after providing sufficient explanation in writing and orally, written informed consent was obtained from all participants. This study was registered in Japan Registry of Clinical Trials (jRCTs031200205, 26/11/2020).

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Study design

This study was designed as a double-blind crossover-controlled trial.

Figure 1a shows the study design of PET. Four PET scans were performed for each participant with intervals of 6 days or more. The first PET scan was performed without any drug administration, and the second, third and fourth PET scans were performed with drug administrations 5 h and 2 h before each scan. The drug protocol was randomly assigned to include A, B, C in the second, third and fourth PET scans: (A) placebo 5 h before and dl-methylephedrine at 150 mg 2 h before, (B) pseudoephedrine at 240 mg 5 h before and placebo 2 h before, and (C) placebo 5 h before and 2 h before. This administration timing was set at 2 h for dl-methylephedrine and 5 h for pseudoephedrine according to the time to reach maximum drug plasma concentration (Tmax). Both doses are the maximum therapeutic daily doses. Placebo was 200 mg of lactose. Blood (venous blood) and urine tests were performed immediately before each PET scan, and the concentrations of dl-methylephedrine and pseudoephedrine were measured.Fig. 1 Study design of PET and fMRI. (a) PET 1 was performed without administration. The drug protocol was randomly assigned to include A, B and C in PET 2, 3 and 4 scans. A or B or C was taken 5 or 2 h before, and blood (venous plasma) and urine tests were performed immediately before each PET 2 or 3 or 4 scan. (b) During fMRI scans, participants performed the MID task. The drug protocol was randomly assigned to include A, B and C in fMRI 1, 2 and 3 scans. A or B or C was taken 5 or 2 h before, and blood (venous plasma) and urine tests were performed immediately before each fMRI 1 or 2 or 3 scan. After each fMRI scan, participants evaluated the VAS-effort. PET positron emission tomography, fMRI functional magnetic resonance imaging, MID monetary incentive delay, VAS visual analogue scale.

Figure 1b shows the study design of fMRI. Three fMRI scans were performed for each participant with intervals of 6 days or more. During the fMRI scans, participants performed the monetary incentive delay (MID) task. The interval between the PET and fMRI scans was at least 72 h. The first, second and third fMRI scans were performed with drug administrations 5 h and 2 h before each scan. The drug protocol and blood and urine tests were the same as for PET. After the fMRI scans, the participants completed a 100 mm visual analogue scale of their efforts on the MID task (VAS-effort).

Plasma and urinary concentrations

Venous blood samples were collected in tubes containing EDTA-2Na, and they were centrifuged at 3000 rpm for 10 min at 4 °C. Separated plasma samples were stored at − 80 °C until analysis. Plasma concentrations of dl-methylephedrine and pseudoephedrine were measured by a validated method using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) with a target lower quantification limit of 0.01 µg/mL (LSI Medience Corp., Japan). Urine samples were collected before the PET and fMRI scans and were stored at -80℃ until analysis. The urinary concentrations of dl-methylephedrine and pseudoephedrine were measured by LC–MS/MS (LSI Medience Corp., Japan).

Positron emission tomography

PET procedures

An Eminence SET-3000GCT-X (Shimadzu Corp., Japan) scanner system was used for all measurements, together with a head fixation device to minimize head movement. A 10-min transmission scan was performed to correct for attenuation. Dynamic PET scan was performed for 60 min after intravenous bolus injection of [18F]FE-PE2I. The injected radioactivity was 178.45–189.5 (mean 186.0 (SD 3.9)) MBq at baseline condition, 181.2–188.3 (184.9 (2.9)) MBq for placebo, 182.0–189.8 (185.2 (2.9)) MBq for dl-methylephedrine, and 183.6–189.8 (187.1 (2.0)) MBq for pseudoephedrine. Specific radioactivity was 316.13–960.84 (632.5 (235.6)) GBq/µmol at baseline condition, 620.64–1467.1 (907.7 (291.9)) GBq/µmol for placebo, 684.63–960.84 (797.2 (92.5)) GBq/µmol for dl-methylephedrine, and 620.64–1072.52 (813.6 (179.2)) GBq/µmol for pseudoephedrine.

PET data analysis

All PET images were co-registered to the individual MR images using the software package PMOD (version 4.1; PMOD Technologies Ltd, Switzerland). MR images were transformed into standard brain size and shape by anatomic standardization of the statistical parametric mapping (SPM8) system. All co-registered PET images were also transformed into standard brain size and shape using the same parameters as for the MR image standardization. Thus, brain images of all subjects had the same anatomic format23. Regions of interest (ROIs) were drawn on all anatomically standardized PET images with reference to the T1-weighted MR images. ROIs were defined for the striatum (caudate nucleus and putamen) and cerebellum.

Binding potential (BPND) was calculated by the simplified reference tissue model (SRTM) method. The cerebellum was used as reference region because of its negligible density of DAT24.

DAT occupancies by placebo, dl-methylephedrine and pseudoephedrine were calculated by the following equation: DAT occupancy (%) = (BPbaseline − BPdrug)/BPbaseline × 100. BPbaseline is BPND in the drug-free state, and BPdrug is BPND after administration of placebo, dl-methylephedrine or pseudoephedrine.

Statistics

Statistical analysis for the difference in DAT occupancy of the striatum between placebo, dl-methylephedrine and pseudoephedrine was performed by two-tailed t-test. In all analyses, p < 0.05 was considered significant.

Functional magnetic resonance imaging

MID task

The MID task was designed to assess neural responses to monetary reward anticipation25 and was slightly modified (Fig. 2)22. Participants performed two experimental sessions of 180 trials, with breaks. Each of the gain and loss cues was presented 18 times, and no response cue was presented 36 times in a pseudorandom order. Task difficulty was standardized to a hit rate of approximately 66% for each participant due to the application of an adaptive algorithm for the target duration.Fig. 2 Schema of MID task. (a) Each trial starts with an incentive cue. After a variable anticipation delay, participants have to press the button during target presentation. After another delay, feedback is presented. If participants could press the button during target presentation, they gained or did not lose the cued amount of money. If they could not press the button during target presentation, they lost or did not gain the cued amount of money. At the feedback period, participants were notified how much money they had gained or lost during the preceding trial and the total amount of money they had earned at that time. (b) All cues in the task are shown. Each cue indicates the possible monetary gain, loss of different amounts, or no response. MID monetary incentive delay.

fMRI procedures

MRI data were collected with an Intera Achieva 1.5 T Nova scanner using a standard head coil (Philips Electronics, The Netherlands). Functional images were acquired with the following parameters: repetition time (TR) = 2000 ms, echo time (TE) = 38 ms, flip angle = 90°, field of view (FOV) = 256 mm, matrix = 64 × 64. A total of 740 images were acquired from each participant with a T2*-weighted gradient-echo echo-planar imaging sequence.

fMRI data analysis

fMRI data analysis was performed using SPM12 (Wellcome Centre for Human Neuroimaging, UK). For preprocessing, the functional images were slice-time corrected, realigned, co-registered with the individual T1-weighted anatomical image, spatially normalized to the standard space defined by the Montreal Neurological Institute template, resampled to 2 mm isotropic voxels, and smoothed using an 8 mm full-width at half-maximum Gaussian kernel.

For subject-level statistical analyses, the functional images were analyzed using the general linear model for each participant. Hemodynamic responses to each event were modeled with a δ function convolved with a synthetic hemodynamic response function time-locked to the onset time of anticipation delay and feedback. The statistical parametric map for a contrast of the t statistic, i.e., reward anticipation (¥20 + ¥100 + ¥500 > ¥0), was calculated.

We set the nucleus accumbens (NAc) as an a priori ROI for reward anticipation26,27. The NAc ROI was anatomically defined by bilateral NAc templates using the Wake Forest University PickAtlas. The percent signal change within the ROI was calculated using MarsBaR. Statistical difference between drugs was analyzed with a one-way repeated measures analysis of variance (ANOVA) and reported at p < 0.05.

Statistics of behavioral data

The mean reaction time, mean hit rate and mean VAS-effort were calculated. Statistical differences between drugs were analyzed with a one-way repeated measures ANOVA and reported at p < 0.05.

Results

Among the seven subjects, pseudoephedrine was detected in the urinary concentration test of one subject (case 4) under administration of dl-methylephedrine or placebo. All data of this subject were excluded from the analysis concerning PET and fMRI from the point of view of data reliability. Thus, six (cases 1, 2, 3, 5, 6, 7) healthy adult male volunteers (age range 27–39 years, mean age 31.8 years (SD 4.1)) participated in the PET arm of this study. In summary, the data of administration of placebo, dl-methylephedrine and pseudoephedrine gave N = 5; the data of case 3 and case 7 were combined. On the other hand, five (case 1, 2, 3, 5, 6) healthy adult male volunteers (age range 27–38, mean age 32.4 years (SD 4.3)) participated in the fMRI arm of this study.

Table 1 shows the PET data of six subjects concerning DAT occupancy in the striatum with placebo, dl-methylephedrine and pseudoephedrine, and the blood and urinary concentrations of dl-methylephedrine and pseudoephedrine. The mean plasma concentration of dl-methylephedrine 150 mg and pseudoephedrine 240 mg was 0.62 µg/mL and 0.82 µg/mL, respectively. The mean urinary concentration of dl-methylephedrine 150 mg and pseudoephedrine 240 mg was 12.7 µg/mL and 144.8 µg/mL, respectively. The mean DAT occupancies in the striatum with placebo, dl-methylephedrine and pseudoephedrine measured by [18F]FE-PE2I were 6.2%, 6.1% and 18.4%, respectively. There was no significant difference between placebo and dl-methylephedrine (p = 0.92). On the other hand, there were significant differences between dl-methylephedrine and pseudoephedrine (p = 0.028) and between placebo and pseudoephedrine (p = 0.009).Table 1 DAT occupancy in the striatum with placebo, dl-methylephedrine and pseudoephedrine, and blood and urinary concentrations of dl-methylephedrine and pseudoephedrine.

Case	DAT occupancy (%)	Blood concentration (ng/mL)	Urinary concentration (μg/mL)	
Placebo	dl-Methylephedrine
150 mg	Pseudoephedrine
240 mg	dl-Methylephedrine
150 mg	Pseudoephedrine
240 mg	dl-Methylephedrine
150 mg	Pseudoephedrine
240 mg	
1	− 1.1	2.1	16.8	0.61	0.835	9.51	54.76	
2	5.5	10.7	14.6	0.69	0.77	19.1	135.67	
3	10.8							
5	5.9	15.3	28.8	0.72	0.95	24.2	82.58	
6	9.7	8.0	17.6	0.76	0.90	6.28	269.23	
7		0.5.7	14.4	0.33	0.64	4.26	181.66	
Mean (SD)	6.2 (0.04)	6.1 (0.07)	18.4 (0.05)	0.62 (0.15)	0.82 (0.11)	12.7 (7.68)	144.8 (76.03)	
DAT dopamine transporter, SD standard deviation.

Figure 3a shows [18F]FE-PE2I PET images without and with administrations of placebo, dl-methylephedrine and pseudoephedrine in case 6.Fig. 3 [18F]FE-PE2I PET images and NAc activation in reward anticipation. (a) The respective images showed without administration, and with administration of placebo, dl-methylephedrine and pseudoephedrine of case 6. (b) ROI of bilateral NAc is shown in red. (c) There was no difference in percent signal change in the NAc ROI during reward anticipation (gain and loss anticipation versus no-gain and no-loss anticipation) between placebo, dl-methylephedrine and pseudoephedrine. NAc nucleus accumbens, ROI region of interest.

Table 2 shows the reaction time, hit rate during the MID task, and subjective VAS-effort after fMRI scans under administrations of each of placebo, dl-methylephedrine and pseudoephedrine. One-way repeated measures ANOVA revealed no significant main effect of drug treatment on reaction time (p = 0.271), hit rate (p = 0.178) or VAS-effort (p = 0.915).Table 2 Reaction time, hit rate, VAS-effort and signal change in NAc ROI with placebo, dl-methylephedrine and pseudoephedrine.

	Mean (SD)	ANOVA (p-value)	
Placebo	dl-Methylephedrine
150 mg	Pseudoephedrine
240 mg	
Reaction time (ms)	232.9 (8.9)	220.9 (31.1)	214.6 (23.5)	0.271	
Hit rate (%)	62.0 (3.7)	63.5 (3.0)	64.5 (0.6)	0.178	
VAS-effort (mm)	66.2 (21.8)	68.6 (20.2)	69.2 (19.8)	0.915	
Signal change in NAc ROI (%)	0.065 (0.063)	0.063 (0.053)	0.065 (0.066)	0.995	
ANOVA analysis of variance, NAc nucleus accumbens, ROI region of interest, SD standard deviation, VAS visual analogue scale.

We examined the percent signal changes within bilateral NAc ROIs during reward anticipation (Fig. 3b). One-way repeated ANOVA revealed no significant main effect of drug treatment in the signal changes (p = 0.995) (Fig. 3c, Table 2). This result suggests that there is no effect of methylephedrine on brain activation associated with reward processing, although the mean urine concentration of dl-methylephedrine was greater than the prohibited threshold value (10 µg/mL).

Discussion

This is the first pharmacological PET and fMRI study to evaluate the effects of taking the maximum therapeutic daily dose of dl-methylephedrine (150 mg) and pseudoephedrine (240 mg) on brain function. The urinary concentration of dl-methylephedrine (12.7 µg/mL) exceeded the prohibited concentration (10 µg/mL), but DAT occupancy with dl-methylephedrine (6.1%) did not differ from that with placebo (6.2%). In contrast, the urinary concentration of pseudoephedrine (144.8 µg/mL) was below the prohibited concentration (150 μg/mL), and the DAT occupancy with pseudoephedrine was 18.4%, which was higher than that with placebo. Even the DAT occupancy of dl-methylephedrine case, which had the highest urinary concentrations above the regulatory limits, was equal to or lower than the occupancy of pseudoephedrine cases, which had urinary concentrations below the prohibited concentration.

Various previous studies reported DAT occupancies of prohibited or monitored stimulants. Regarding prohibited stimulants, DAT occupancies in the striatum of modafinil 200 mg and 300 mg measured by [18F]FE-PE2I were reported as 51.4% and 56.9%, respectively16. The DAT occupancies in the striatum of methylphenidate at clinical doses of 10–60 mg measured by [11C]cocaine were reported to be 40–74%28. DAT occupancy in the striatum with mazindol 1.5 mg measured by [18F]FE-PE2I was approximately 25%29. As for the monitored stimulant bupropion, DAT occupancy in the striatum by [99mTc]-TRODAT-1 SPECT was approximately 21%30. In addition, it has been reported that there is a positive correlation between DAT occupancy and extracellular dopamine concentration31. It is considered that a higher DAT occupancy leads to an increase not only in addiction but also in cognitive enhancement32.

Moreover, previous studies have reported the positive effects of prohibited stimulants by WADA, such as amphetamine and modafinil, on reward processing using the MID task21,33. These drugs increased brain activation in the nucleus accumbens for anticipation of monetary incentives, and they also increased subjective eagerness or positive arousal in response to monetary incentive cues.

dl-methylephedrine is rapidly absorbed following oral administration, crosses the blood–brain barrier, and exerts a central nervous system stimulant effect34. The plasma concentration of dl-methylephedrine reached its maximum level at 2–8 h after administration, and it showed a low inter-individual variability. The peak urinary concentration of dl-methylephedrine occurred 12 h after administration (Tmax) and its maximum urinary concentration was 5.9–21.3 μg/mL (Cmax)35. dl-methylephedrine is regulated by urinary concentrations according to “The Prohibited List”, although the urinary excretion of dl-methylephedrine can be strongly affected by urinary pH and/or urinary volume, and the urinary concentration may not reflect the psychoactive level of dl-methylephedrine in circulation35. We have already reported that a single dose of dl-methylephedrine 60 mg has the same effect on brain function as a placebo13. The results of the present study showed that dl-methylephedrine had the pharmacological effect of inhibiting DAT, but even when its urinary concentration exceeded the prohibited level, its DAT occupancy was lower than that of pseudoephedrine, which does not exceed the prohibited level. This suggested that a single dose of dl-methylephedrine 150 mg, the maximum daily dose, does not have a significant effect on the dopamine system. The fMRI results also showed that dl-methylephedrine did not alter brain activity. Thus, considering the regulation of doping that stimulates brain function, the current regulated urinary concentration of dl-methylephedrine (less than 10 µg/mL) that limits the use of the maximum therapeutic daily dose of dl-methylephedrine (150 mg) might not be appropriate.

A single dose of pseudoephedrine 240 mg may have affected DAT, as DAT occupancy with pseudoephedrine in this study (18.4%) was greater than the value of the test–retest variability of BPND with [18F]FE-PE2I for the striatum36. This was as expected from the research result that the effect of pseudoephedrine on athletic performance was dose-dependent11. It is not yet clear to what extent (i.e., what percentage) inhibition of DAT causes changes in brain function. Modafinil, regulated in the S6 category and which we have previously shown to sufficiently inhibit DAT and stimulate the reward system, has been reported to improve memory and attention37, and it is assumed that this effect on brain function is via DAT inhibition38. Based on previous reports that DAT inhibitors improve athletic performance, pseudoephedrine may improve athletic performance through increased DAT occupancy39. However, because pseudoephedrine even at the maximum clinical dose has less of an effect on athletic performance than permitted stimulants such as caffeine12,40, in 2010 WADA stipulated urinary concentration limits that do not restrict its use as therapeutic doses. In the present study, pseudoephedrine inhibited DAT even though it was below the regulated urinary concentration. However, fMRI results indicated that pseudoephedrine 240 mg does not have enough effect to influence brain function, which is consistent with WADA's guideline that this dose is not regulated in competition.

The main limitation of this study is that it does not investigate the effects on the peripheral nervous system. It is also necessary to evaluate the effect on motor function via the peripheral nervous system, since dl-methylephedrine has a sympathetic stimulating effect not only on the central nervous system but also on the peripheral nervous system. We are planning additional studies to examine the effects of dl-methylephedrine on motor function. Another limitation is that all participants in this study were male. Gender differences in dopamine function, including the reward system, have been reported, and further discussion may be needed to determine whether the results of this study can be generalized.

In conclusion, the present study revealed that the average of urinary concentration of pseudoephedrine did not exceed the prohibited urinary concentration (150 µg/mL) when taking the maximum therapeutic daily dose of pseudoephedrine 240 mg, whereas the average of urinary concentration of dl-methylephedrine exceeded the prohibited urinary concentration (10 µg/mL) when taking the maximum therapeutic daily dose of dl-methylephedrine 150 mg. However, the effect of dl-methylephedrine on DAT was less than that of pseudoephedrine. This study showed that dl-methylephedrine had a weaker effect on brain function than pseudoephedrine at the maximum clinical dose. Considering the regulation of doping by stimulating the action of brain function, it seems necessary to consider setting new regulation values that allow the use of dl-methylephedrine as a therapeutic drug.

Acknowledgements

We thank Minoru Sakurai, Koji Nagaya, Koji Kanaya, Satoe Aoyama, Hiroki Nosaka, Miho Hiramatsu, Yuka Naoi and Kazuyoshi Honjo (Clinical Imaging Center for Healthcare, Nippon Medical School) for their assistance in performing MRI and PET examinations. We also thank Yuya Ise (Department of Pharmaceutical Services, Nippon Medical School Hospital) for his assistance with drug preparations.

Author contributions

T.S., R.A. and A.T. designed the study and wrote the protocol. T.S., R.A., T.N., Y.I. and A.T. recruited the patients and made psychiatric evaluations. T.S., Y.I. and R.A. participated in the data analysis. T.S. wrote the first draft of the manuscript. T.S., R.A. and A.T. had discussions and corrected the manuscript. All authors contributed to and have approved the final manuscript.

Funding

This study was funded by the Japan Sports Agency Ministry of Education, Culture, Sports, Science and Technology-Japan (Grant no. Research and Development Project on Anti-doping Science).

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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