Minocycline protects against neuronal mitochondrial dysfunction and cognition impairment.

The potential of minocycline to protect against methylphenidate­induced neurodegeneration has been extensively reported in the literature but the mechanism of action is still unknown. This study aims to determine the role of mitochondrial chain enzymes and redox homeostasis on the neuroprotective effects of minocycline in methylphenidate­induced neurodegeneration. Wistar adult male rats were randomly assigned to the seven experimental groups: Group 1 received saline solution; Group 2 received methylphenidate (10 mg/kg, i.p.); Groups 3, 4, 5, and 6 received methylphenidate and minocycline for 21 days; Group 7 received minocycline alone. Cognition was evaluated with the Morris water maze test. Activity of the hippocampal mitochondrial quadruple complexes I, II, III and IV, mitochondrial membrane potential, adenosine triphosphate (ATP) levels, total antioxidant capacity, and reactive oxygen species were determined. Treatment with minocycline inhibited methylphenidate­induced cognitive dysfunction. Minocycline treatment increased mitochondrial quadruple complex activities, mitochondrial membrane potential, total antioxidant capacity, and ATP levels in the dentate gyrus and cornu ammonis­1 (CA1) areas of the hippocampus. Minocycline is likely to confer neuroprotection against methylphenidate­induced neurodegeneration and cognition impairment by regulating mitochondrial activity and oxidative stress.

Oxytocin receptors mediate oxytocin potentiation of methylphenidate-induced stimulation of accumbens dopamine in rats.

While the illicit use and misuse of stimulants like cocaine and methylphenidate (MP) has increased, there remains no FDA-approved treatments for psychostimulant use disorders (PSUD). Oxytocin (OT) has shown promise as a potential pharmacotherapy for PSUD. Dopamine (DA) neurotransmission plays a significant role in PSUD. We have recently shown that OT blunts the reinforcing effects of MP but, surprisingly, enhanced MP-induced stimulation of DA levels. Such effects have been suggested as a result of activation of OT receptors or, alternatively, could be mediated by direct actions of OT on MP blockade of the DA transporter. Here, we employed fast scan cyclic voltammetry (FSCV) to investigate the effects of systemic OT on MP-induced changes in the dynamics of DA, phasic release and uptake, in the nucleus accumbens shell (NAS) of Sprague-Dawley rats. We also tested the systemic effects of an antagonist of OT receptors, atosiban, to counteract the OT enhancement of dopaminergic effects of MP under microdialysis procedures in the NAS in rats. Administration of OT alone (2 mg/kg; i.p.) did not significantly modify evoked NAS DA dynamics measured by FSCV, and when administered 10 min before MP (0.1, 0.3, 1.0 mg/kg; i.v.), OT did not potentiate MP-induced increases in phasic DA release and did not alter DA clearance rate, suggesting no direct interactions of OT with the MP-induced blockade of DA uptake. Also, OT alone did not elicit significant changes in tonic, extracellular NAS DA levels measured by microdialysis. However, consistent with previous studies, we observed that OT pretreatments (2 mg/kg; i.p.) potentiated MP-induced (0.1, 0.3, 1.0 mg/kg; i.v.) efflux of extracellular NAS DA levels. This effect was abolished when rats were pretreated with atosiban (2 mg/kg; i.p.), suggesting that OT receptors mediate this OT action. Overall, our results suggest that OT receptors mediated OT potentiation of MP-induced stimulation of extracellular NAS DA levels, likely driven by modulation of DA receptor signaling pathways, without affecting MP blockade of DAT.

Striatal D1 and D2 receptor availability are selectively associated with eye-blink rates after methylphenidate treatment.

Eye-blink rate has been proposed as a biomarker of the brain dopamine system, however, findings have not been consistent. This study assessed the relationship between blink rates, measured after oral placebo) (PL) and after a challenge with oral methylphenidate (MP; 60 mg) and striatal D1 receptor (D1R) (measured at baseline) and D2 receptor (D2R) availability (measured after PL and after MP) in healthy participants. PET measures of baseline D1R ([11C]NNC112) (BL-D1R) and D2R availability ([11C]raclopride) after PL (PL-D2R) and after MP (MP-D2R) were quantified in the striatum as non-displaceable binding potential. MP reduced the number of blinks and increased the time participants kept their eyes open. Correlations with dopamine receptors were only significant for the eye blink measures obtained after MP; being positive for BL-D1R in putamen and MP-D2R in caudate (PL-D2R were not significant). MP-induced changes in blink rates (PL minus MP) were negatively correlated with BL-D1R in caudate and putamen. Our findings suggest that eye blink measures obtained while stressing the dopamine system might provide a more sensitive behavioral biomarker of striatal D1R or D2R in healthy volunteers than that obtained at baseline or after placebo.

Abstinence following intermittent methylphenidate exposure dose-dependently modifies brain glucose metabolism in the rat brain.

Methylphenidate (MP) is a psychostimulant chronically prescribed for the treatment of attention deficit hyperactivity disorder (ADHD). Additionally, MP users may take breaks from using the medication during "drug holidays," which may include short-term or long-term breaks from medication. The present study utilized fluorodeoxyglucose (FDG) positron emission tomography (PET) to analyze the effects of chronic oral MP use and abstinence on brain glucose metabolism (BGluM) in rats at two different doses: high dose (HD) and low dose (LD). The schedule of treatment was 3 weeks on-treatment and 1 week off-treatment for a period of 13 weeks, followed by an abstinence period of 4 total weeks. Results showed that chronic MP treatment using this schedule did not lead to significant changes in BGluM when comparing the control to HD MP groups. However, significant activation in BGluM was observed after periods of abstinence between control and HD MP rats in the following brain regions: the trigeminal nucleus, reticular nucleus, inferior olive, lemniscus, mesencephalic reticular formation, inferior colliculus, and several areas of the cerebellum. These brain regions and functional brain circuit play a role in facial sensory function, the auditory pathway, organizing connections between the thalamus and cortex, motor learning, auditory function, control over eye movement, auditory information integration, and both motor and cognitive functions. These results, when considered with previous studies, indicate that MP schedule of use may have differing effects on BGluM. BGluM following long-term MP use was dependent on MP dose and schedule of use in rats. This study was conducted in non-ADHD model rats with the aim to establish an understanding of the effects of MP itself, especially given the growing chronic off-label and prescribed use of MP. Further studies are needed for analysis of the drug's effects on an ADHD model.

Abstinence from Chronic Methylphenidate Exposure Modifies Cannabinoid Receptor 1 Levels in the Brain in a Dose-dependent Manner.

INTRODUCTION: Methylphenidate (MP) is a widely used psychostimulant prescribed for Attention Deficit Hyperactivity Disorder and is also used illicitly by healthy individuals. Chronic exposure to MP has been shown to affect physiology, behavior measures, and neurochemistry. METHODS: The present study examined its effect on the endocannabinoid system. Adolescent rats had daily oral access to either water (control), low dose MP (4/10 mg/kg), or high dose MP (30/60 mg/kg). After 13 weeks of exposure, half of the rats in each group were euthanized, with the remaining rats underwent a four-week- long abstinence period. Cannabinoid receptor 1 binding (CB1) was measured with in vitro autoradiography using [3H] SR141716A. RESULTS: Rats who underwent a 4-week abstinence period after exposure to chronic HD MP showed increased CB1 binding in several cortical and basal ganglia regions of the brain compared to rats with no abstinence period. In contrast to this, rats who underwent a 4-week abstinence period after exposure to chronic LD MP showed lower CB1 binding mainly in the basal ganglia regions and the hindlimb region of the somatosensory cortex compared to rats with no abstinence period. Following 4 weeks of drug abstinence, rats who were previously given HD MP showed higher [3H] SR141716A binding in many of the cortical and basal ganglia regions examined than rats given LD MP. These results highlight the biphasic effects of MP treatment on cannabinoid receptor levels. Abstinence from HD MP seemed to increase CB1 receptor levels, while abstinence from LD MP seemed to decrease CB1 levels. CONCLUSION: Given the prolific expression of cannabinoid receptors throughout the brain, many types of behaviors may be affected as a result of MP abstinence. Further research will be needed to help identify these behavioral changes.

The rate of dasotraline brain entry is slow following intravenous administration.

RATIONALE: Drugs that rapidly increase dopamine levels have an increased risk of abuse. Dasotraline (DAS) is a dopamine and norepinephrine reuptake inhibitor characterized by slow oral absorption with low potential for abuse. However, it remains unclear whether intravenous (i.v.) administration would facilitate the rapid elevation of dopamine levels associated with stimulant drugs. OBJECTIVE: To assess the kinetics of DAS across the blood-brain barrier and time to onset of dopamine transporters (DAT) inhibition. METHODS: We compared the onset of DAT occupancy and the associated elevation of synaptic dopamine levels in rhesus monkey following i.v. administration of DAS or methylphenidate (MPH) using positron emission tomography (PET). Brain entry times were estimated by reductions in [18F]-FE-PE2I binding to DAT in rhesus monkeys. Elevations of synaptic dopamine were estimated by reductions in [11C]-Raclopride binding to D2 receptors. RESULTS: Intravenous administration of DAS (0.1 and 0.2 mg/kg) resulted in striatal DAT occupancies of 54% and 68%, respectively; i.v. administered MPH (0.1 and 0.5 mg/kg) achieved occupancies of 69% and 88% respectively. Brain entry times of DAS (22 and 15 min, respectively) were longer than for MPH (3 and 2 min). Elevations in synaptic dopamine were similar for both DAS and MPH however the time for half-maximal displacement by MPH (t = 23 min) was 4-fold more rapid than for DAS (t = 88 min). CONCLUSIONS: These results demonstrate that the pharmacodynamics effects of DAS on DAT occupancy and synaptic dopamine levels are more gradual in onset than those of MPH even with i.v. administration that is favored by recreational drug abusers.

Dopaminergic loss of cyclin-dependent kinase-like 5 recapitulates methylphenidate-remediable hyperlocomotion in mouse model of CDKL5 deficiency disorder.

Cyclin-dependent kinase-like 5 (CDKL5), a serine-threonine kinase encoded by an X-linked gene, is highly expressed in the mammalian forebrain. Mutations in this gene cause CDKL5 deficiency disorder, a neurodevelopmental encephalopathy characterized by early-onset seizures, motor dysfunction, and intellectual disability. We previously found that mice lacking CDKL5 exhibit hyperlocomotion and increased impulsivity, resembling the core symptoms in attention-deficit hyperactivity disorder (ADHD). Here, we report the potential neural mechanisms and treatment for hyperlocomotion induced by CDKL5 deficiency. Our results showed that loss of CDKL5 decreases the proportion of phosphorylated dopamine transporter (DAT) in the rostral striatum, leading to increased levels of extracellular dopamine and hyperlocomotion. Administration of methylphenidate (MPH), a DAT inhibitor clinically effective to improve symptoms in ADHD, significantly alleviated the hyperlocomotion phenotype in Cdkl5 null mice. In addition, the improved behavioral effects of MPH were accompanied by a region-specific restoration of phosphorylated dopamine- and cAMP-regulated phosphoprotein Mr 32 kDa, a key signaling protein for striatal motor output. Finally, mice carrying a Cdkl5 deletion selectively in DAT-expressing dopaminergic neurons, but not dopamine receptive neurons, recapitulated the hyperlocomotion phenotype found in Cdkl5 null mice. Our findings suggest that CDKL5 is essential to control locomotor behavior by regulating region-specific dopamine content and phosphorylation of dopamine signaling proteins in the striatum. The direct, as well as indirect, target proteins regulated by CDKL5 may play a key role in movement control and the therapeutic development for hyperactivity disorders.

Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate.

Loss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1-120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1-120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management.

Use of UPLC-HRMS/MS for In Vitro and In Vivo Metabolite Identification of Three Methylphenidate-derived New Psychoactive Substances.

The distribution of so-called new psychoactive substances (NPS) as substitute for common drug of abuse was steadily increasing in the last years, but knowledge about their toxicodynamic and toxicokinetic properties is lacking. However, a comprehensive knowledge of their toxicokinetics, particularly their metabolism, is crucial for developing reliable screening procedures and to verify their intake, e.g., in case of intoxications. The aim of this study was therefore to tentatively identify the metabolites of the methylphenidate-derived NPS isopropylphenidate (isopropyl 2-phenyl-2-(2-piperidyl) acetate, IPH), 4-fluoromethylphenidate (methyl 2-(4-fluorophenyl)-2-(piperidin-2-yl) acetate, 4-FMPH) and 3,4-dichloromethylphenidate (methyl 2-(3,4-dichlorophenyl)-2-(piperidin-2-yl) acetate, 3,4-CTMP) using different in vivo and in vitro techniques and ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS/MS). Urine samples of male rats were analyzed, and the transfer to human metabolism was done by using pooled human S9 fraction (pS9), which contains the microsomal fraction of liver homogenisate as well as its cytosol. UHPLC-HRMS/MS analysis of rat urine revealed 17 metabolites for IPH (14 phase I and 3 phase II metabolites), 13 metabolites were found for 4-FMPH (12 phase I metabolites and 1 phase II metabolite) and 7 phase I metabolites and no phase II metabolites were found for 3,4-CTMP. pS9 incubations additionally indicated that all investigated substances were primarily hydrolyzed, resulting in the corresponding carboxy metabolites. Finally, these carboxy metabolites should be used as additional analytical targets besides the parent compounds for comprehensive mass spectrometry-based screening procedures.

Biodegradation of ritalinic acid by Nocardioides sp. - Novel imidazole-based alkaloid metabolite as a potential marker in sewage epidemiology.

The consumption of methylphenidate, a nootropic drug used to improve mental performance, is becoming increasingly serious. Methylphenidate is metabolized in human liver to ritalinic acid, which has been commonly detected in sewage and surface waters. Additionally, ritalinic acid serves as a biomarker in sewage epidemiology studies. Thus knowledge of the stability and microbial degradation pathways of ritalinic acid is essential for proper estimation of methylphenidate consumption. In the study reported here, we describe the fast formation of a previously unknown, dead-end metabolite of ritalinic acid by Nocardioides sp. strain MW5. HRMS and 2D NMR analyses allowed precisely identification of the compound as an imidazole-based alkaloid cation with chemical formula 11-[3-(formylamino)propyl]-1,2,3,4,6,7,8,9-octahydrodipyrido[1,2-a:1',2'-c]imidazole-5-ium. In experiments, Nocardioides sp. strain MW5 transformed 34% of ritalinic acid into this metabolite, while 52% was mineralized into CO2. Alkaloid was not biodegraded during the OECD 301 F test. This study provides new insight into the environmental fate of methylphenidate and its metabolites. The data collected are essential for assessing nootropic drug consumption by sewage epidemiology and should lead to a better understanding of microbial degradation of ritalinic acid.

Role of homeostatic feedback mechanisms in modulating methylphenidate actions on phasic dopamine signaling in the striatum of awake behaving rats.

Methylphenidate is an established treatment for attention-deficit hyperactivity disorder that also has abuse potential. Both properties may relate to blocking dopamine and norepinephrine reuptake. We measured the effects of methylphenidate on dopamine dynamics in freely moving rats. Methylphenidate alone had no effect on the amplitude of phasic responses to cues or reward. However, when administered with the D2 receptor antagonist raclopride, methylphenidate increased dopamine responses, while raclopride alone had no effect. Using brain slices of substantia nigra or striatum, we confirmed that methylphenidate effects on firing rate of nigral dopamine neurons and dopamine release from terminals are constrained by negative feedback. A computational model using physiologically relevant parameters revealed that actions of methylphenidate on norepinephrine and dopamine transporters, and the effects of changes in tonic dopamine levels on D2 receptors, are necessary and sufficient to account for the experimental findings. In addition, non-linear fitting of the model to the data from freely moving animals revealed that methylphenidate significantly slowed the initial cue response dynamics. These results show that homeostatic regulation of dopamine release in the face of changing tonic levels of extracellular dopamine should be taken into account to understand the therapeutic benefits and abuse potential of methylphenidate.

Electroencephalography Functional Networks Reveal Global Effects of Methylphenidate in Youth with Attention Deficit/Hyperactivity Disorder.

Methylphenidate (MPH) is the leading drug for treatment of attention deficit/hyperactivity disorder (ADHD), yet its underlying neuronal mechanisms are still unclear. Here, we use a dynamical brain networks approach to explore the effects of cognitive effort and MPH on ADHD subjects. Electroencephalography data were recorded from 19 ADHD subjects and 18 controls during a Go/No-Go Task. ADHD subjects completed the task twice a day over 2 days. The second session was administered post-ingestion of placebo/MPH (alternately). Controls performed two tasks in 1 day. The data were divided into 300 ms windows from -300 pre-stimulus until 1200 ms post-stimulus. Brain networks were constructed per subject and window, from which network metrics were extracted and compared across the experimental conditions. We identified an immediate shift of global connectivity and of network segregation after the stimulus for both groups, followed by a gradual return to baseline. Decreased global connectivity was found to be 400-700 ms post-stimulus in ADHD compared with controls, and it was normalized post-MPH. An increase of the networks' segregation occurred post-placebo at 100-400 and 400-700 ms post-stimulus, yet it was inhibited post-MPH. These global alterations resulted mainly from changes in task-relevant frontal and parietal regions. The networks of medicated ADHD subjects and controls exhibited a more significant and lasting change, relative to baseline, compared with those of nonmedicated ADHD. These results suggest impaired network flexibility in ADHD, corrected by MPH.

Stability and uptake of methylphenidate and ritalinic acid in nine-spine stickleback (Pungitius pungitius) and water louse (Asellus aquaticus).

The presence of human pharmaceuticals in the environment has garnered significant research attention because these compounds may exert therapeutic effects on exposed wildlife. Yet, for many compounds, there is still little research documenting their stability in the water column and uptake in organism tissues. Here, we measured the uptake and stability of methylphenidate (Ritalin®, a frequently prescribed central nervous system stimulant) and its primary metabolite, ritalinic acid, in (1) water only or (2) with nine-spine stickleback and water louse. Methylphenidate degraded to ritalinic acid in both studies faster at a higher temperature (20 °C versus 10 °C), with concentrations of ritalinic acid surpassing methylphenidate after 48-100 h, depending on temperature. The concentration of methylphenidate in stickleback was highest at the first sampling point (60 min), while the concentration in water louse tissues reached comparatively higher levels and peaked after ~ 6 days. Neither stickleback nor water louse took up ritalinic acid in tissues despite being present in the water column. Our findings provide valuable data for use in future risk assessment of methylphenidate and will aid in the design of studies aimed at measuring any ecotoxicological effects on, for example, the behaviour or physiology of aquatic organisms.

L- and D-threo ethylphenidate concentrations, pharmacokinetics, and pharmacodynamics in horses.

Ethylphenidate is a psychostimulant and analog of the commonly prescribed compound, methylphenidate (Ritalin®). There are a limited number of studies describing the disposition and pharmacologic/toxicological effects of ethylphenidate in any species. The abuse potential in equine athletes along with the limited data available regarding administration in horses necessitates further study. The objectives of the current study were to describe drug concentrations, develop an analytical method that could be used to regulate its use, and describe the pharmacodynamic effects of ethylphenidate in horses. To that end, 12 horses were randomized into 3 dose groups (intravenous: 10 mg or 40 mg, oral: 40 mg). Ethylphenidate was administered and blood and urine samples were collected prior to and for up to 72 hours post drug administration. Concentrations of D-threo ethylphenidate and the metabolite ritalinic acid were measured using Liquid Chromatography-tandem Mass Spectrometry. L-threo ethylphenidate concentrations were estimated from D-threo ethylphenidate concentrations. Serum concentrations of ethylphenidate were below detectable levels by 8, 18, and 12 hours following intravenous administration of 10 mg and 40 mg and oral administration of 40 mg, respectively. Ritalinic acid was non-detectable at 72 hours in the group that received a 10-mg intravenous and 40-mg oral dose of ethylphenidate. Ritalinic acid concentrations were below the LOQ at 72 hours following intravenous administration of 40 mg of ethylphenidate. While the number of animals per dose group were small, no stimulatory behavior or significant changes in heart rate were noted. Untoward effects including gastrointestinal adverse effects were noted in all dose groups.

Interindividual and Intraindividual Variation of Methylphenidate Concentrations in Serum and Saliva of Patients With Attention-Deficit/Hyperactivity Disorder.

BACKGROUND: Therapeutic drug monitoring is becoming increasingly important in psychiatric therapy, especially in children. However, for several reasons, it cannot yet be implemented as a daily routine in clinical or outpatient settings. To evaluate new, noninvasive procedures, blood and saliva (oral fluid) samples were collected from patients with attention-deficit/hyperactivity disorder (ADHD) who were also being administered methylphenidate (MPH). The study's main purposes were to correlate MPH concentrations in serum and saliva between subjects and to analyze intraindividual variation of serum concentration. METHODS: Thirty-six patients with ADHD (27 children and 9 adults) on MPH medication were included for drug analysis. MPH and its major metabolite ritalinic acid were quantified using liquid chromatography-tandem mass spectrometry measurements. The following correlations were investigated: (1) between drug concentrations in serum and saliva, and (2) between pH value and saliva to serum concentration ratio. Furthermore, the mean intraindividual MPH-concentration fluctuation in saliva under constant frame conditions was analyzed. RESULTS: After quantification, MPH concentrations were approximately 5 times higher in the saliva than in the serum, whereas the concentrations of ritalinic acid were much lower in saliva. We found significant correlations between concentrations of MPH in serum and saliva (r = 0.51, P < 0.05). Saliva MPH measures, compared with serum, were pH-dependent (r = -0.56, P < 0.01). Daily coefficient of variance of saliva concentration in children taking constant medication was 27.3% (11%-42%), whereas the coefficient of variance for the ratio of saliva to serum was 122% (2%-2060%). CONCLUSIONS: Our data indicate that the interindividual variation in saliva to serum concentrations is rather high, whereas the intraindividual variation is fairly low, as already shown in the literature for repeated citalopram serum measurements. Saliva may well serve as an alternative matrix for therapeutic drug monitoring of MPH in patients with ADHD, especially for follow-up examinations. Future research should focus on analyzing the relationship between drug levels in saliva and clinical effects as well as on understanding the mechanisms that generate saliva drug concentrations. These are essential steps before potential clinical use.

Bacterial isolates degrading ritalinic acid-human metabolite of neuro enhancer methylphenidate.

The consumption of nootropic drugs has increased tremendously in the last decade, though the studies on their environmental fate are still scarce. Nootropics are bioactive compounds designed to alter human's physiology therefore the adverse effects towards wildlife can be expected. In order to understand their environmental impact, the knowledge on their transformation pathways is necessary. Methylphenidate belongs to the most prescribed neuro-enhancers and is among the most favored smart drugs used in non-medical situations. It is metabolized in human liver and excreted as ritalinic acid. Here, we showed for the first time that ritalinic acid can be biodegraded and used as a sole carbon and nitrogen source by various microbial strains originating from different environmental samples. Five axenic strains were isolated and identified as: Arthrobacter sp. strain MW1, MW2 and MW3, Phycicoccus sp. strain MW4 and Nocardioides sp. strain MW5. Our research provides the first insight into the metabolism of ritalinic acid and suggests that it may differ depending on the strain and growth conditions, especially on availability of nitrogen. The isolates obtained in this study can serve as model organisms in further studies on the catabolism of ritalinic acid and methylphenidate but potentially also other compounds with similar structures. Our findings have important implication for the sewage epidemiology. We demonstrated that ritalinic acid is subject to quick and efficient biodegradation thus its use as a stable biomarker should be reconsidered.

Biotransformation of ritalinic acid by laccase in the presence of mediator TEMPO.

Methylphenidate is widely used as a medication for the treatment of attention deficit hyperactivity disorder (ADHD) in children. Less than 1% of methylphenidate is excreted unchanged in urine, while 80% of an oral dose is excreted as ritalinic acid (which is reportedly poorly degradable). This study aims to investigate the biotransformation of ritalinic acid by free and immobilized enzymes. The influence of various laccase mediators on biotransformation efficiency has been tested. Formation of the main transformation products has been monitored and their potential structures suggested. The effective transformation of ritalinic acid was observed only in the presence of 2,2,6,6-tetramethylpiperidine 1-oxyl mediator (TEMPO). The most effective enzyme was the laccase of T. versicolor 159. The main transformation product was an N-methyl derivative of ritalinic acid. Ritalinic acid was also reduced to aldehyde and alcohol, and a broad spectrum of intermediate complexes with oxoammonium ion of TEMPO were detected. This is the first time the biotransformation of ritalinic acid has been investigated in detail.

Methylphenidate and Atomoxetine-Responsive Prefrontal Cortical Genetic Overlaps in "Impulsive" SHR/NCrl and Wistar Rats.

Impulsivity, the predisposition to act prematurely without foresight, is associated with a number of neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). Identifying genetic underpinnings of impulsive behavior may help decipher the complex etiology and neurobiological factors of disorders marked by impulsivity. To identify potential genetic factors of impulsivity, we examined common differentially expressed genes (DEGs) in the prefrontal cortex (PFC) of adolescent SHR/NCrl and Wistar rats, which showed marked decrease in preference for the large but delayed reward, compared with WKY/NCrl rats, in the delay discounting task. Of these DEGs, we examined drug-responsive transcripts whose mRNA levels were altered following treatment (in SHR/NCrl and Wistar rats) with drugs that alleviate impulsivity, namely, the ADHD medications methylphenidate and atomoxetine. Prefrontal cortical genetic overlaps between SHR/NCrl and Wistar rats in comparison with WKY/NCrl included genes associated with transcription (e.g., Btg2, Fos, Nr4a2), synaptic plasticity (e.g., Arc, Homer2), and neuron apoptosis (Grik2, Nmnat1). Treatment with methylphenidate and/or atomoxetine increased choice of the large, delayed reward in SHR/NCrl and Wistar rats and changed, in varying degrees, mRNA levels of Nr4a2, Btg2, and Homer2, genes with previously described roles in neuropsychiatric disorders characterized by impulsivity. While further studies are required, we dissected potential genetic factors that may influence impulsivity by identifying genetic overlaps in the PFC of "impulsive" SHR/NCrl and Wistar rats. Notably, these are also drug-responsive transcripts which may be studied further as biomarkers to predict response to ADHD drugs, and as potential targets for the development of treatments to improve impulsivity.

Methylphenidate disintegration from oral formulations for intravenous use by experienced substance users.

BACKGROUND AND AIMS: Methylphenidate (MPH) is a prescription stimulant used to treat attention-deficit hyperactivity disorder. MPH is currently the preferred substance among most intravenous (i.v.) substance users in Iceland. Four types of MPH preparations were available in Iceland at the time of study: Immediate-release (IR), sustained-release (SR), osmotic controlled-release oral delivery (OROS) tablet and osmotic-controlled release (OCR). MPH OROS has previously been rated the least desirable by i.v. users and we hypothesized that this was associated with difficulty of disintegrating MPH from OROS formulation. The aim of the study was to measure the amount of MPH and the viscosity of the disintegrated solutions that were made from the four MPH formulations by four i.v.-users and non-users. METHODS: A convenience sample of four i.v. substance users and 12 non-users. Non-users imitated the methods applied by experienced i.v. substance users for disintegrated MPH formulations. RESULTS: Both groups managed to disintegrate over 50% of MPH from IR and SR formulations but only 20% from OROS (p<0.0001). The viscosity of the disintegrated MPH was significantly higher for MPH OROS and MPH OCR and the preparation was significantly more time-consuming than for the other MPH samples. No differences were observed between users and non-users. CONCLUSIONS: To our knowledge, this is the first investigation of viscosity and the amount of disintegrated MPH from prescription drugs for i.v. use. The results indicate that the ease of disintegration, amount of MPH and viscosity may explain the difference in popularity for i.v. use between different MPH formulations.

Ultraviolet/ultrasound-activated persulfate for degradation of drug by zinc selenide quantum dots: Catalysis and microbiology study.

In this study, wet chemical method used for ZnSe quantum dots (QDs) and characterized by, UV-vis, photoluminescence spectroscopy, X-ray diffraction and transmission electron microscopy. The crystallites size of ZnSe QDs was 4.0nm. The average diameters of ZnSe QDs were 3.0-5.3nm. Ritalin was degraded using the UV/ZnSe QDs/persulfate process. The several parameters investigated for the influence of Rtialin degradation were the temperature, the persulfate concentration, and the initial Ritalin concentration. The values of optimum parameters ware room temperature, concentration persulfate 5mmol/L and initial Ritalin concentration 0.09mmol/L. Comparative analyses showed the maximum degradation of Ritalin was found for ZnSe/persulfate under ultra-visible and ultra-sonic irradiation process. Comparative analysis showed the maximum degradation of Ritalin was found for ZnSe/persulfate under ultra-visible and ultra-sonic irradiation process. The values of first-order rate constants from degradation of Ritalin at 25°C were 0.96×10-2, 1.09×10-2, 1.59×10-2 and 2.19×10-2 for US/PS, UV/PS, ZnSe/US/PS and ZnSe/UV/PS system, respectively. The antibacterial activity evaluation against two bacterials, including Gram-positive bacteria Staphylococcus aureus (ATCC 43300), Bacillus megaterium (ATCC 14581) and Gram-negative bacteria Pseudomonas aeruginosa (ATCC 27853), Micrococcus luteus (ATCC 4698) was considered. It was found that the MIC values for the antibacterial assay in the presence of ZnSe QDs were around 0.30mM with 64.0, 66.0, 79.2, and 83.5% inhibition for the S. aureus, B. megaterium, P. aeruginosa and M. luteus bacterial strains, respectively. Then, results show that the ZnSe QDs have antibacterial activity.