8. Purchase Orders. All registrants involved in the distribution of mephedrone, methylone or MDPV must comply with the requirements of the Order Form in Part 1305 of Title 21 of the Code of Federal Regulations as of October 21, 2011. The result of this inhibition is an increased and prolonged concentration and postsynaptic effect resulting from dopaminergic and norepinephrine signals at dopamine and norepinephrine receptors on the receptor neuron. Serotonin also plays a role, although to a much lesser extent. This sudden increase in the concentration of neurotransmitters in the brain is thought to be responsible for the effect generated by MDPV. The first stimulant-type NPS that appeared in the United States were found in the so-called bath salt products that flooded the recreational drug market beginning in late 2010 . In early 2011, there was a dramatic increase in reports of bath salt poisoning in poison control centers and an influx of patients admitted to the emergency department with toxic exposures [10-12]. Bath salts consist of powders or crystals that are administered intranasally or orally to exert their psychoactive effects. Low doses of bath salts induce typical psychomotor effects such as increased energy and mood, but high doses or excessive consumption can cause severe symptoms such as hallucinations, psychosis, increased heart rate, high blood pressure and hyperthermia, often accompanied by combative or violent behavior [9, 13]. The most serious syndrome induced by bath salts is known as “excited delirium,” a constellation of symptoms such as high body temperature, delirium, restlessness, breakdown of muscle tissue, and kidney failure that sometimes lead to death [14, 15]. Forensic analysis of bath salt products conducted in 2010 and 2011 identified three main synthetic compounds: 4-methyl-N-methylcathinone (mephedrone), 3,4-methylenedioxy-N-methylcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV) (Spiller et al., 2011; [7, 12]).
These compounds are chemically similar to the natural substance cathinone, an amphetamine-like stimulant found in the khat plant Catha edulis. Legislation passed in 2013 placed mephedrone, methylone, and MDPV under permanent Schedule I control, making the drugs illegal in the United States . Figure 1 shows the chemical structures of bath salt cathinones in relation to the related compounds amphetamine and cathinone. The final order was published today in the Federal Register to draw public attention to this action. These chemicals are monitored for at least 12 months, with the possibility of a six-month extension. They are referred to as Schedule I substances, the most restrictive category under the Controlled Substances Act. The Schedule I designation is reserved for substances that have a high potential for abuse, currently have no use for treatment in the United States, and are not safe to be used for medically supervised use of the drug. The data reviewed to date suggest that hydroxylated metabolites of MDPV are unlikely to contribute to the in vivo effects of systemically administered MDPV, especially since these metabolites are present in conjugated form and are not “free” in circulation. Nevertheless, we investigated the possible biological activity of these metabolites, as our previous work has shown that the 3,4-dihydroxy metabolite of MDMA is bioactive . The effects of 3,4-catechol-PV and 4-OH-3-MeO-PV were first investigated in absorption inhibition tests for DAT, NET, and SERT. The data in Table 1 show that 3,4-catechol-PV is a potent absorption blocker in DATs (CI50 = 11 nM) and NETs (CI50 = 11 nM), while 4-OH-3-MeO-PV is much weaker in this regard. None of the metabolites show measurable activity in the inhibition of SERT, even at doses up to 10 μM.
The data on 3,4-catechol-PV presented in Table 1 are consistent with previous results from Meltzer et al. , who found that this compound is an inhibitor of absorption in ART and NETs, with similar efficacy to pyrovalerone . Next, we tested MDPV metabolites in the microdialysis paradigm to investigate possible in vivo effects. None of the metabolites affected dopamine or dialysate behaviour at intravenous doses of 0.1 and 0.3 mg/kg; the same doses of MDPV have robust effects on both parameters (see Fig. 2). Given the in vitro potency of 3,4-catechol-PV to DAT, we investigated the effects of higher doses of this metabolite in vivo. Figure 5 shows new data that i.v. Administration of 3 mg/kg 3,4-catechol-PV results in a small, albeit significant, increase in extracellular dopamine, but no change in walking ability. Taken together, the in vitro and in vivo results with 3,4-catechol-PV suggest that this compound may be too polar to easily cross the blood-brain barrier and achieve robust neurochemical effects. To support this hypothesis, the total polar area of 3,4-catechol-PV is 60.77 compared to 38.78 for MDPV.
The 3,4-catechol-PV results presented here serve as a reminder that the derivation of the drug`s mechanism of action should not be based solely on the results of in vitro profiling of the transporter/receptor. In our laboratory, we are interested in studying the in vivo pharmacokinetics and metabolism of MDPV in rats due to the lack of data from controlled drug administration studies in humans. We have previously evaluated the pharmacodynamic and pharmacokinetic parameters of MDMA in rats [96, 97] and used similar methods to investigate the effects of MDPV . As a first step, Anizan et al.  developed a fully validated analytical method for the simultaneous detection and quantification of MDPV, 3,4-catechol-PV and 4-OH-3-MeO-PV using LCHRMS. The method involves hydrolysis of the sample to split conjugated 3,4-catechol PV and 4-OH-3-MeO-PV into their free forms, followed by protein precipitation prior to analysis. The detection limits are 0.1 μg/L and the linear range is 0.25 to 1,000 μg/L. The high sensitivity of the test is essential to quantify the low concentrations of analytes in the small plasma volume of catheterized rats. To investigate the pharmacokinetics of MDPV and its metabolites, Anizan et al.  HC administered doses of MDPV (0.5, 1.2 mg/kg) to rats with surgically implanted intravenous catheters. The rats were placed in chambers equipped with photographic beams to measure motion parameters and connected to a fastening system that allowed free movement in the chamber.
The IV catheters were attached to an extension tube threaded into the rope to allow a stress-free blood sample without disturbing the animal. Repeated blood samples (300 μL) were taken through the catheter at different times before and after injection. Blood samples were centrifuged and plasma samples were analyzed for MDPV, 3,4-catechol-PV and 4-OH-3-MeO-PV using the LC-HRMS methods described above. With this strategy, we were able to simultaneously obtain pharmacodynamic measures (i.e. walking ability and stereotypy) and circulating concentrations of MDPV and its metabolites. The topic list contains a list of index terms (topic list) for each CFR exhibit number cited in the document title. The terms form a common vocabulary for indexing all organizations` regulatory documents and form the basis of the “CFR” created by the CAO. If you have any questions for the organization that published the current document, please contact the organization directly. 6. Recordings. All registrants handling mephedrone, methylone or MDPV shall maintain records in accordance with sections 1304.03, 1304.04, 1304.21, 1304.22 and 1304.23 of 21 of the Code of Federal Regulations.
Current DEA licensees have thirty (30) calendar days from the effective date of this Final Order to comply with all record-keeping requirements. Self-administration of drugs is considered a “gold-standard” behavioural test for determining the addictive potential of drugs, since most drugs administered by laboratory animals are abused by humans [74, this volume] . In the rat self-administration paradigm, animals equipped with surgically implanted intravenous catheters are trained to press levers or poke their noses to receive intravenous injections of drugs administered via a computerized infusion pump. A number of studies have shown that rats self-administer mephedrone [44, 76, 77] and methylone [78-80], suggesting that these drugs are prone to abuse. With respect to MPV, Aarde et al.  reported that MPVC is readily self-administered from rats at intravenous exercise doses of 0.01 to 0.5 mg/kg and that the drug is more effective than methamphetamine, a known stimulant of abuse. Watterson et al.  found similar results in rats that self-administered the VCPM, and also showed that the amount of drug administered shows a sharp escalation when rats are granted prolonged access to the drug. Schindler et al.  directly compared the acquisition of IV self-administration behaviour for MDPV (0.05 mg/kg) and methylone (0.5 mg/kg) in rats.
It was found that self-administration of MDPV is acquired quickly in the first days of training, while the development of methylone self-administration takes much longer. In addition, the number of infusions per session for MDPV is significantly higher than for methylone. Based on the neurochemical effects of MDPV and methylone already mentioned, it is tempting to speculate that the serotonergic effects of methylone serve to counteract the positive reinforcing effects of this drug compared to MDPV. Consistent with this idea, Bonano et al.  showed that MDPV is much more effective than methylone in facilitating intracranial self-stimulation (ICSS) in rats, an indicator of amplification of drug effects.