Hepatic drug‐metabolizing enzymes (DMEs) play critical roles in determining the pharmacokinetics and pharmacodynamics of numerous therapeutic agents. As such, noninvasive biomarkers capable of predicting DME expression in the liver have the potential to be used to personalize pharmacotherapy and improve drug treatment outcomes. In the present study, we quantified carboxylesterase 1 (CES1) protein concentrations in plasma samples collected during a methylphenidate pharmacokinetics study. CES1 is a prominent hepatic enzyme responsible for the metabolism of many medications containing small ester moieties, including methylphenidate. The results revealed a significant inverse correlation between plasma CES1 protein concentrations and the area under the concentration‐time curves (AUCs) of plasma d‐methylphenidate ( P = 0.014, r = −0.617). In addition, when plasma CES1 protein levels were normalized to the plasma concentrations of 24 liver‐enriched proteins to account for potential interindividual differences in hepatic protein release rate, the correlation was further improved ( P = 0.003, r = −0.703), suggesting that plasma CES1 protein could explain ~ 50% of the variability in d‐methylphenidate AUCs in the study participants. A physiologically‐based pharmacokinetic modeling simulation revealed that the CES1‐based individualized dosing strategy might significantly reduce d‐methylphenidate exposure variability in pediatric patients relative to conventional trial and error fixed dosing regimens. This proof‐of‐concept study indicates that the plasma protein of a hepatic DME may serve as a biomarker for predicting its metabolic function and the pharmacokinetics of its substrate drugs.

A tenable hypothesis is presented which explains disparities between older oral dl-MPH bioavailability data generated using chiral derivatization-gas chromatography versus more recent findings using chiral liquid chromatography. These disparities persist in current literature. The gas chromatographic methods found that the absolute bioavailability of d-MPH is 23% and that of l-MPH is 5% (i.e., 82% as the active d-isomer), while liquid chromatographic methods consistently report that approximately 99% of circulating MPH is d-MPH. Older methods used perfluoroacylated S-prolyl derivatizing agents which have a history of imprecision due to the susceptibility of the prolyl S-configuration to isomerize to the R-enantiomer. Accordingly, any R-prolyl impurity in the chiral derivatization reagent yields the (R,R,R)-MPH-prolyl diastereomer which, in being related as the opposite enantiomer of (S,S,S)-prolyl-MPH, co-elutes with l-(S,S)-MPH. This results in overestimation of the percent l-MPH at the expense of underestimating d-MPH. Unless compelling reasons exist to justify use of any chiral discriminators, less complex and less costly achiral analysis of plasma MPH appears appropriate for d-MPH quantitation since 99% exists as d-MPH. However, simultaneous plasma monitoring of d-MPH and l-MPH may be warranted when alterations in first-pass hepatic metabolism by carboxylesterase 1 (CES1) occurs. For example, (a) with transdermal dl-MPH delivery; (b) in cases of concomitant dl-MPH and a CES1 inhibitor, e.g., ethanol, which elevates l-MPH and d-MPH concentrations; (d) in forensic studies of intravenous or intranasal dl-MPH abuse; (e) were dl-MPH to be formulated as a free base sublingual product; or (f) as emerging advances in dl-MPH gene-dose effects warrant isomer correlations.

In 2000, the first biphasic modified‐release (MR) formulation of methylphenidate (MPH) was approved for the treatment of attention‐deficit/hyperactivity disorder (ADHD). An immediate‐release (IR) MPH pulse (22% of the dose) facilitates rapid onset of stimulant action, while the remaining MR portion of the dose provides for day‐long duration of efficacy. A wide array of oral MR‐MPH products has subsequently been approved that also allows for once‐daily dosing, though each product is characterized by distinctive exposure time courses. This review compares each member of the current MPH armamentarium to assist in the rational selection of a specific MPH regimen for the individualized treatment of patients with ADHD. The IR portion of biphasic MPH formulations now ranges from 15%, 20%, 22%, 25%, 30%, and 37% IR‐MPH, as well as a 50% IR‐MPH product whose distinctly pulsatile time course closely resembles that of the pre‐century “gold standard” twice‐daily IR‐MPH regimen. Further, transdermal, suspension, and orally disintegrating tablet products are now available to overcome any solid dosage form swallowing difficulties. Most of these formulations are racemic, though in 2001, a chiral switch drug IR‐dexmethylphenidate (dexMPH) was approved, followed by biphasic MR‐dexMPH (50% IR) in 2005. New U.S. Food and Drug Administration (FDA) partial area under the curve (pAUC) bioavailability metrics have improved discrimination between specific generic MR‐MPH products. This has resulted in two Orange Book MR‐MPH products being recoded from “AB” (i.e., meets necessary bioequivalence requirements) to “BX” (i.e., insufficient data to confirm bioequivalence). The metabolic drug interaction between MPH and alcohol, which increases MPH bioavailability, potentiates euphoric effects, and heightens abuse liability, is discussed. This review concludes with brief considerations of pharmacogenomic predictors of ADHD first‐line drug selection, carboxylesterase allelic variants influencing interindividual MPH metabolism, and novel MPH formulations in the regulatory pipeline.

Background/Purpose Ethanol coadministered with immediate-release dl-methylphenidate (dl-MPH) or dexmethylphenidate (d-MPH) significantly increases the geomean maximum plasma concentration (Cmax) of d-MPH 22% and 15%, respectively, and elevates overall drug exposure and psychostimulant effects. We asked the question: Are these ethanol-MPH interactions based more fundamentally on (1) inhibition of postabsorption d-MPH metabolism or (2) acceleration of MPH formulation gastric dissolution by ethanol in the stomach? This was investigated using the pulsatile, distinctly biphasic, spheroidal oral drug absorption systems of dl-MPH and d-MPH. Methods In a randomized, 4-way crossover study, 14 healthy subjects received pulsatile dl-MPH (40 mg) or d-MPH (20 mg), with or without ethanol (0.6 g/kg), dosed 4 hours later. These 4 hours allowed the delayed-release second MPH pulse to reach a more distal region of the gut to preclude gastric biopharmaceutical influences. Plasma was analyzed using a highly sensitive chiral method. Subjective/physiological effects were recorded. Findings/Results Ethanol increased the second pulse of d-MPH Cmax for dl-MPH by 35% (P < 0.01) and the partial area under the plasma concentration curve from 4 to 8 hours by 25% (P < 0.05). The respective values for enantiopure d-MPH were 27% (P = 0.001) and 20% (P < 0.01). The carboxylesterase 1–mediated transesterification metabolite ethylphenidate served as a biomarker for coexposure. Ethanol significantly potentiated stimulant responses to either formulation. Implications/Conclusions These findings support drug dispositional interactions between ethanol and MPH as dominant over potential biopharmaceutical considerations. Understanding the pharmacology underlying the frequent coabuse of MPH-ethanol provides rational guidance in the selection of first-line pharmacotherapy for comorbid attention-deficit/hyperactivity disorder–alcohol use disorder.

Amphetamine (AMP), an indirectly acting psychostimulant approved for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults, is among the most long-standing therapeutic agents in all of clinical psychopharmacology. This review focuses on AMP absorption, metabolism, and elimination brought to bear on comparative pharmacokinetics in its various formulations. A comprehensive search of the published literature was conducted using MEDLINE (PubMed) and Google Scholar databases through April 2017 to retrieve all pertinent in vitro and human studies for review and synthesis. Additionally, Food and Drug Administration (FDA) databases were accessed for otherwise unavailable data when possible. Initially available as racemic (dl)-AMP, this drug was later supplanted by enantiopure (d)-AMPH or enantioenriched (75:25 dl)-AMP formulations; although racemic AMP returned as an approved drug to treat ADHD in 2014. Presently, there are several immediate-release (IR) formulations available, including d-AMP, dl-AMP, and mixed amphetamine salts, which are neither racemic nor the pure d-enantiomer (i.e., a 3:1 mixture of d-AMP and l-AMP). Furthermore, new modified-release AMP formulations, including an oral suspension and an orally disintegrating tablet, are now available. A lysine-bonded prodrug form of d-AMP also serves as a treatment option. Oral AMP is rapidly absorbed, with high absolute bioavailability, followed by extensive metabolism involving multiple enzymes. Some metabolic pathways exhibit stereoselective biotransformations favoring the l-isomer substrate. Drug exposure exhibits dose-proportional pharmacokinetics. Body weight is a fundamental determinant of differences in observed AMP plasma concentrations. IR formulations typically provide a Tmax from 2 to 3 hours. In replicated studies, children exhibit a shorter plasma T1/2 (∼7 hours) relative to adults (∼10 to 12 hours). There are few documented pharmacokinetic drug interactions of clinical significance beyond influences of drug-induced alteration of urinary pH. The array of AMP formulations addressed in this review offer flexibility in dosing, drug onset, and offset to assist in individualized pharmacotherapy of ADHD.

The postulate that twice the milligram/kilogram dose of dl-methylphenidate (dl-MPH) would result in equal exposure to d-MPH compared with half that milligram/kilogram dose of the chiral switch product dexmethylphenidate (d-MPH) was tested. Using a randomized, crossover study design, 12 men and 12 women received either immediate-release (IR) dl-MPH (0.3 mg/kg) or IR d-MPH (0.15 mg/kg). Relative bioavailability comparisons included partial area under the plasma concentration-time curves (pAUC0–3 h) for d-MPH. The pAUC0–3 h is a new regulatory metric presently only required for bioequivalence testing of a specific dl-MPH modified-release product. The geometric mean ratios for both the Cmax and area under the plasma concentration-time curve (AUC0–∞) were within the 90% confidence interval (CI) regulatory range of 0.8–1.25, indicating that these two drugs were bioequivalent in terms of d-MPH. However, the pAUC0–3 h geometric mean ratio for d-MPH after IR dl-MPH versus IR d-MPH was 0.76 (P < 0.001; 90% CI, 0.67–0.87), showing significantly less early exposure to the d-isomer than IR d-MPH. The 1-hour d-MPH concentration after dl-MPH was 56% of that after the enantiopure drug. The maximum d-MPH plasma concentration (Cmax) for dl-MPH was also significantly lower for dl-MPH (P < 0.05; CI, 1.02–1.19), whereas the AUC0–∞ ratio of 0.89 was not significantly different (P = 0.21; CI, 0.98–1.13). The AUC0–3 h difference reported here points to the potential limitations of using bioequivalence for sound predictions of dose-response relationships. Knowledge of the greater early exposure to d-MPH after the pure d-isomer drug compared with the racemate may contribute to drug individualization/optimization in the treatment of attention deficit hyperactivity disorder.

The potentiation of positive subjective responses to immediate-release dexmethylphenidate (d-MPH) or dl-methylphenidate (dl-MPH) by ethanol was investigated over the time course of maximal drug exposure after a single dose. In a 4-way, randomized, crossover study design, 12 men and 12 women normal volunteers received d-MPH (0.15 mg/kg) or dl-MPH (0.3 mg/kg) with or without ethanol (0.6 g/kg). Serial visual analog scales were used as surrogates for drug abuse liability ("high," "good," "like," "stimulated," and "any drug effect"). Combining pure d-MPH with ethanol significantly (P < 0.005) increased the area under the effect curves (AUC(0-5.25h)) of all 5 subscales. The dl-MPH-ethanol combination significantly (P < 0.05) increased these AUCs with the exception of like (P = 0.08). Effects of the pure d-MPH-ethanol combination exhibited delayed potentiation relative to dl-MPH-ethanol. A pharmacokinetic interaction between the l-isomer of dl-MPH and ethanol has previously been shown to increase early exposure to d-MPH. Administration of the pure isomer d-MPH precludes this absorption phase pharmacokinetic interaction with ethanol. This notwithstanding, the pure d-MPH-ethanol combination resulted in comparable, if not greater, cumulative stimulant potentiation than the dl-MPH-ethanol combination. These findings provide evidence of a pharmacodynamic component to d-MPH-ethanol synergistic interactions and carry implications for the rational drug individualization in the treatment of attention-deficit/hyperactivity disorder.

We review the pharmaceutical science of ethylphenidate (EPH) in the contexts of drug discovery, drug interactions, biomarker for dl-methylphenidate (MPH)-ethanol exposure, potentiation of dl-MPH abuse liability, contemporary "designer drug," pertinence to the newer transdermal and chiral switch MPH formulations, as well as problematic internal standard. d-EPH selectively targets the dopamine transporter, whereas d-MPH exhibits equipotent actions at dopamine and norepinephrine transporters. This selectivity carries implications for the advancement of tailored attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy in the era of genome-based diagnostics. Abuse of dl-MPH often involves ethanol coabuse. Carboxylesterase 1 enantioselectively transesterifies l-MPH with ethanol to yield l-EPH accompanied by significantly increased early exposure to d-MPH and rapid potentiation of euphoria. The pharmacokinetic component of this drug interaction can largely be avoided using dexmethylphenidate (dexMPH). This notwithstanding, maximal potentiated euphoria occurs following dexMPH-ethanol. C57BL/6 mice model dl-MPH-ethanol interactions: an otherwise depressive dose of ethanol synergistically increases dl-MPH stimulation; a substimulatory dose of dl-MPH potentiates a low, stimulatory dose of ethanol; ethanol elevates blood, brain, and urinary d-MPH concentrations while forming l-EPH. Integration of EPH preclinical neuropharmacology with clinical studies of MPH-ethanol interactions provides a translational approach toward advancement of ADHD personalized medicine and management of comorbid alcohol use disorder.

OBJECTIVEThe most widely utilized pharmacological treatment of attention-deficit/hyperactivity disorder (ADHD) is the psychostimulant methylphenidate (MPH). Most MPH formulations consist of the racemic mixture of d-threo-(R, R)-MPH and l-threo-(S, S)-MPH isomers. MPH is characterized by its low bioavailability and short half-life (2-3 hours). Additionally, significant inter-individual variability in MPH pharmacokinetics has been consistently documented. Accordingly, efforts have been directed at developing alternatives to MPH as therapeutic agents. A wide range of MPH analogues (dl-α-[2-piperidyl]-phenylacetic acid esters) have been synthesized with the dopamine transporter (DAT) and norepinephrine transporter (NET) as principle neuropharmacological targets. The present study investigated the metabolic profiles and pharmacological activity of the isopropyl ester derivative of MPH, dl-isopropylphenidate (IPH), both in vitro and in vivo.METHODSThe synthesis, monoaminergic transporter binding, cellular uptake profiles, and assessment of metabolic hydrolysis and transesterification in the presence of ethanol are described using MPH as a comparator. Additionally, an in vivo assessment of IPH stimulant effects (vs. saline) in rats was performed with locomotor activity as a pharmacodynamic outcome.RESULTSIPH displayed unique pharmacological characteristics including greater DAT than NET binding and cellular uptake activity, and greater resistance to hydrolysis and transesterification via carboxylesterase 1 relative to MPH. Further, sustained psychostimulant properties offer the prospect of an enhanced duration of action.CONCLUSIONSOur findings are consistent with IPH exhibiting attributes distinguishing it from MPH and warranting further study and development of IPH as a novel psychotherapeutic agent.

The following is a brief background and account of the psychostimulant ‘designer drug’ and curious methylphenidate-ethanol transesterification metabolite, ethylphenidate (EPH; Fig 1). That is ethylphenidate with an ‘E’, not it's well known homolog, methylphenidate (MPH; Ritalin®, Concerta®, others) – the first-line agent for the treatment of attention-deficit/hyperactivity disorder (ADHD). Note that the only structural difference resides in the ester where EPH has an ethyl group rather than the methyl group in MPH.dl-EPH is described chemically as (RR/SS)-ethyl phenyl(piperidin-2-yl)acetate or ritalinic acid ethyl ester. It has been characterized in the literature as the racemic crystalline hydrochloride salt and as its separate enantiomers.123 Interestingly, EPH holds unique pharmacological significance in both legal and illicit arenas which will be touched upon in this paper.The earliest investigation of EPH in the biomedical literature was reported over 50 years ago by Portoghese and Malspeis, who found EPH to be 80% as potent as MPH in antagonizing sedation in mice. Beyond this report, little to any information was published until Schweri and associates reported that EPH was ~50% as potent as MPH in inhibiting dopamine uptake in rat striatal synaptosomes.14 However, due to the structural similarity of EPH to MPH, it was frequently used as an internal standard for MPH pharmacokinetic studies from the 1970s–1990s. Analytical methods incorporating EPH as an internal standard became problematic once it became known that EPH was also a MPH metabolite. Contemporary analytical methods generally incorporate deuterated MPH as an internal standard.5In the late 1970s, the cocaine-ethanol transesterification pathway, which yields the active metabolite cocaethylene (ethyl cocaine or benzoylecgonine ethyl ester), emerged as a precedent for a methyl ester containing drug to be metabolically transformed into an ethyl ester.6 This unique drug interaction eventually became the focus of numerous investigations. This peculiar bioconversion requires a catalytic enzyme to extract two separate drugs from the bloodstream, and covalently bond them. In the context of cocaine-ethanol co-ingestion and potential toxicity, plasma cocaethylene concentrations can exceed those of the parent drug cocaine and this metabolite appears to be cardiotoxic.78We hypothesized the potential for EPH formation in individuals who co-abused MPH and alcohol, or even in adult ADHD patients who consume modest amounts of alcohol while being treated with MPH. There were, however, some practical impediments to testing this in humans. It was appealing to speculate that EPH would be formed in humans in view of both MPH and cocaine sharing a common binding site of stimulant action, i.e., the dopamine transporter (DAT).9 In 1997, utilizing a rat liver preparation, Bourland and co-workers reported that MPH may serve as a substrate for ethanol transesterification, as could the ester-containing drug meperidine which, like MPH, is otherwise primarily metabolized by hydrolysis.10 In this approximate time frame (i.e. late 1990's) we received two sets of postmortem blood and tissue samples recovered from unrelated overdose victims whose case histories had documented multi-drug ingestion, including large quantities of MPH along with evidence of ethanol consumption. These samples held the potential to demonstrate that humans metabolically generate EPH from MPH and ethanol. Following the in-house synthesis of an authentic reference standard of EPH, we confirmed by liquid chromatography–mass spectrometry (LC-MS) the novel detection of EPH formed in vivo in these human samples.11 It was not suggested that EPH factored into the toxicology of either fatality, as it was not part of the initial toxicological report and its presence would not have been suspected or assayed for. These were multi-drug ingestion cases and other medications and ethanol explained the fatalities.Although the above findings confirmed that EPH could be formed in humans, questions remained. Due to the history of these biological samples, the circumstances surrounding their collection (i.e., autopsy) and the unknown MPH-ethanol doses, it remained to be established whether EPH formation occurs using typical therapeutic MPH doses (10–20mg) and moderate ethanol consumption. Subsequently, our group conducted a normal volunteer study in which 6 healthy volunteer subjects were administered a single fixed 20 mg dose of MPH followed 30 min later by a weight-based dose (0.6 g/kg) of ethanol.12 The 20mg dose of MPH was chosen as it most closely approximated a commonly studied 0.3mg/kg weight based dose which would be taken by a 70kg individual. The dose of ethanol chosen in the study was within the range of dosing parameters being utilized in analogous cocaethyelene studies, and approximated a double vodka (80 proof) and orange juice in a 70 Kg subject. Using a non-enantiospecific LC-MS/MS assay, EPH was detected in both blood and urine samples in every subject. Although concentrations were low relative to MPH, this provided a proof-of-concept regarding EPH formation under simulated clinical drug utilization.To advance our investigations, the National Institutes of Health supported our more comprehensive and enantiospecific study of the pharmacokinetics and pharmacodynamics of MPH-ethanol interactions.13 dl-MPH (0.3 mg/kg) was administered orally 30 min before ethanol (0.6gm/kg), 30 min after ethanol (0.6gm/kg), or without ethanol, in a randomized, normal subject three-way crossover study of 10 men and 10 women. Ethanol (1) elevated the Cmax and AUC of d-MPH 40% and 25%, respectively; (2) the transesterification pathway yielded approximately 10 times more l-EPH than d-EPH; and (3) ethanol significantly increased positive subjective effects of MPH. Further, a novel MPH poor metabolizer was discovered. Plasma concentrations of l-MPH reached ~70 times that found in normal metabolizers and, distinct in this individual, no EPH was formed.14 Although it had long been recognized that MPH underwent substantial stereoselective metabolism favoring the de-esterification (and deactivation) of l-MPH over d-MPH, Sun and associates15 identified the specific carboxylesterase CES1A1 as the enzyme responsible for the first-pass, stereoselective metabolism of MPH. CES1A1 is also now known to catalyze the transesterification reaction. In concordance with this enantioselective hydrolysis of l-MPH to the metabolite ritalinic acid (Fig 1), it follows that transesterification of MPH also favors l-MPH as the substrate, and indeed the vast majority of EPH detected as the l-EPH isomer.13We are also interested in developing novel therapeutic agents. Though Schweri and coworkers reported evidence of lower potency for EPH compared to MPH in inhibiting DA uptake in synaptosomes from a dopaminergic region of rat brain, d-EPH exhibits approximately the same low nanomolar DAT inhibition as d-MPH when tested in a more substrate-specific assay using DAT-transfected human embryonic kidney cells.4 Perhaps importantly, d -EPH was found to be only 10% as potent as d-MPH at inhibiting the norepinephrine transporter.3 This dopaminergic selectivity of EPH relative to MPH offers potential for exploitation in drug discovery. Consistent with EPH DAT selectivity relative to MPH, we have now shown that isopropylphenidate (the isopropyl ester homolog of MPH) is DAT selective and offers potential as a novel therapeutic agent.16Recognizing the structural similarity of EPH to MPH (Fig 1), and its aforementioned effects on monoamines, the issue is raised – ‘Is EPH subject to abuse?’ Although EPH is not explicitly controlled in the US presently, it could possibly be considered an analog of MPH, a Schedule II substance covered under the Federal Analog Act, in that it is ‘substantially the same structure as a Schedule II drug, i.e., MPH’.2 Two recent reviews of so-called ‘Legal Highs’ and designer drugs have indicated that EPH is being sold online as an illicit stimulant or cognitive enhancer which is sometimes called ‘nopaine’.1718 Indeed, discussion rooms and accounts of EPH use as a ‘legal high’ abound on the internet- as do vendors selling EPH in gram quantities. As evidence of a wider concern of EPH use and abuse, EPH is now being included in MS assays designed to screen for and detect many other designer drugs which are of growing concern.19 Although pure EPH can be purchased for research use by legitimate vendors which supply analytical standards and research biochemicals, our laboratory recently received a sample of EPH purchased from an internet vendor, and purported to be EPH hydrochloride. Using a validated LC-MS/MS assay established in our laboratory we were able to determine that the substance was indeed racemic (dl-EPH), and of high purity.5 This finding suggests that EPH purchased in this way, could be readily ingested by almost any individual such that they were effectively ‘dosed’ far in excess of any amount metabolically formed during the ethanol-MPH transesterification pathway. The toxicological implications are unknown.However, there are further concerns. For those individual users not finding the EPH ‘experience’ to be to their liking, they could easily convert EPH to MPH using readily available methanol and other items stocked in their local hardware store- at far less effort than routinely employed by clandestine ‘chemists’ manufacturing methamphetamine for instance. Alternatively, criminally enterprising non-users may engage in MPH manufacture using the EPH precursor, one synthetic step removed from MPH, to supply crystalline MPH hydrochloride for intranasal, intravenous or oral abuse.We have chronicled the evolution of EPH over the past several decades. First as a member of a structure-activity series, then as an internal standard, followed by it serving as a MPH-ethanol biomarker, and now as a potential therapeutic agent with a burgeoning designer drug of abuse presence.

Milk thistle (Silybum marianum) extracts, one of the most widely used dietary supplements, contain a mixture of six major flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin) and other components. However, the pharmacokinetics of the free individual flavonolignans have been only partially investigated in humans. Furthermore, antioxidant effects of the extract, which may underlie the basis of many therapeutic effects, have not been thoroughly assessed. The present study evaluated the pharmacokinetics of the six major flavonolignans in healthy volunteers receiving single doses of either one (175 mg), two (350 mg), or three (525 mg) milk thistle capsule(s) on three separate study visits. Additionally, the steady-state pharmacokinetic parameters were determined after the subjects were administered one capsule three times daily for 28 consecutive days. Our results demonstrated that all six flavonolignans were rapidly absorbed and eliminated. In order of abundance, the exposure to free flavonolignans was greatest for silybin A followed by silybin B, isosilybin B, isosilybin A, silychristin, and silydianin. The systemic exposure to these compounds appeared linear and dose proportional. The disposition of flavonolignans was stereoselective, as evidenced by the apparent clearance of silybin B, which was significantly greater than silybin A, whereas the apparent clearance of isosilybin B was significantly lower than isosilybin A. The concentrations of urinary 8-epi-prostaglandin F2α, a commonly used biomarker of oxidative status in humans, were considerably decreased in study subjects after a 28-day exposure to the extract (1.3 ± 0.9 versus 0.8 ± 0.9 ng/mg creatinine) but failed to reach statistical significance (P = 0.076).

Prior research indicates methylphenidate (MPH) and alcohol (ethanol, EtOH) interact to significantly affect responses humans and mice. The present studies tested the hypothesis that MPH and EtOH interact to potentiate ethanol-related behaviors in mice.

Enantioselective hydrolysis of oral racemic methylphenidate (dl-MPH) by carboxylesterase 1 (CES1) limits the absolute bioavailability of the pharmacologically active d-MPH isomer to approximately 30% and that of the inactive l-MPH to only 1–2%. Coadministration of dl-MPH with ethanol results in elevated d-MPH plasma concentrations accompanied by CES1-mediated enantioselective transesterification of l-MPH to l-ethylphenidate (EPH). The present study tested the hypothesis that administration of the pure isomer dexmethylphenidate (d-MPH) will overcome the influence of ethanol on d-MPH absorption by eliminating competitive CES1-mediated presystemic metabolism of l-MPH to l-EPH. Twenty-four healthy volunteers received dl-MPH (0.3 mg/kg) or d-MPH (0.15 mg/kg), with or without ethanol (0.6 g/kg). During the absorption phase of dl-MPH, concomitant ethanol significantly elevated d-MPH plasma concentrations (44–99%; P < 0.005). Furthermore, immediately following the ethanol drink the subjective effects of "high," "good," "like," "stimulated," and overall "effect" were significantly potentiated (P ≤ 0.01). Plasma l-EPH concentrations exceeded those of l-MPH. Ethanol combined with pure d-MPH did not elevate plasma d-MPH concentrations during the absorption phase, and the ethanol-induced potentiation of subjective effects was delayed relative to dl-MPH-ethanol. These findings are consistent with l-MPH competitively inhibiting presystemic CES1 metabolism of d-MPH. Ethanol increased the d-MPH area under the curve (AUC)0-inf by 21% following dl-MPH (P < 0.001) and 14% for d-MPH (P = 0.001). In men receiving d-MPH-ethanol, the d-MPH absorption partial AUC0.5–2 hours was 2.1 times greater and the time to maximum concentration (Tmax) occurred 1.1 hours earlier than in women, consistent with an increased rate of d-MPH absorption reducing hepatic extraction. More rapid absorption of d-MPH carries implications for increased abuse liability.

In humans, concomitant dl-methylphenidate (dl-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite dl-ethylphenidate (dl-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between d- and l-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic–pharmacodynamic correlations regarding the MPH–ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of d- and l-MPH as well as d- and l-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025ng/ml for each isomer when extracting 0.5ml plasma aliquots. Calibration curves were linear over the range from 0.025ng/ml to 25ng/ml for all analytes (r2>0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed.

PurposeMany abusers of dl-methylphenidate co-abuse ethanol. The present animal study examined behavioral effects of oral or transdermal dl-methylphenidate in combination with a high, depressive dose of ethanol to model co-abuse.MethodsLocomotor activity of C57BL/6J mice was recorded for 3h following dosing with either oral dl-methylphenidate (7.5mg/kg) or transdermal dl-methylphenidate (Daytrana®;1/4 of a 12.5cm2 patch; mean dose 7.5mg/kg), with or without oral ethanol (3g/kg). Brains were enantiospecifically analyzed for the isomers of methylphenidate and the transesterification metabolite ethylphenidate.ResultsAn otherwise depressive dose of ethanol significantly potentiated oral dl-methylphenidate induced increases in total distance traveled for the first 100min (p<0.05). Transdermal dl-methylphenidate increased total distance traveled after a latency of 80min, though this effect was not potentiated by concomitant ethanol. Mean 3h brain d-methylphenidate concentrations were significantly elevated by ethanol in both the oral (65% increase) and transdermal (88% increase) groups. The corresponding l-ethylphenidate concentrations were 10ng/g and 130ng/g.ConclusionsStimulant induced motor activity in rodents may correlate with abuse liability. Potentiation of dl-methylphenidate motor effects by concomitant ethanol carries implications regarding increased abuse potential of dl-methylphenidate when combined with ethanol.

Methylphenidate (MPH) therapy for attention-deficit/hyperactivity disorder is common in children and adults. Concerns regarding abuse of MPH prompted studies to better understand its pharmacology. We used an established drug discrimination task to determine whether MPH could be discriminated by C57BL/6J (B6) mice. B6 mice learned to discriminate cues produced by racemic MPH (dl-MPH 5.0 mg/kg) or half the dose of pure d-isomer (2.5 mg/kg), and dose–response tests established appropriate reductions in discrimination with declining dose. Importantly, the two drug forms generalized to each other completely in substitution tests; consistent with reports that the l-isomer is pharmacodynamically inactive. An additional experiment indicated that lower doses (1 and 2 mg/kg) of dl-MPH did not support acquisition of MPH discrimination despite extensive training. Mice acquired discrimination of dl-MPH only when the dose was increased to 4 mg/kg. Thus, although these lower doses increased drug lever responding in mice trained on the higher dose, their stimuli were not sufficient to support acquisition of the discrimination task. These findings correspond to earlier studies conducted in our laboratory on threshold doses needed to produce stimulatory effects of motor activity inB6 mice. These preclinical findings provide insight intothe relative potency, and by extension, efficacy of dl-MPH versus d-MPH doses.

We tested the hypothesis that C57BL/6J mice will model human metabolic interactions between dl-methylphenidate (MPH) and ethanol, placing an emphasis on the MPH transdermal system (MTS). Specifically, we asked: (1) will ethanol increase d-MPH biological concentrations, (2) will MTS facilitate the systemic bioavailability of l-MPH, and (3) will l-MPH enantioselectively interact with ethanol to yield l-ethylphenidate (l-EPH)? Mice were dosed with MTS (¼ of a 12.5 cm(2) patch on shaved skin) or a comparable oral dl-MPH dose (7.5 mg/kg), with or without ethanol (3.0 g/kg), and then placed in metabolic cages for 3 h. MPH and EPH isomer concentrations in blood, brain, and urine were analyzed by gas chromatographic-mass spectrometry monitoring of N-(S)-prolylpiperidyl fragments. As in humans, MTS greatly facilitated the absorption of l-MPH in this mouse strain. Similarly, ethanol led to the enantioselective formation of l-EPH and to an elevation in d-MPH concentrations with both MTS and oral MPH. Although only guarded comparisons between MTS and oral MPH can be made due to route-dependent drug absorption rate differences, MTS was associated with significant MPH-ethanol interactions. Ethanol-mediated increases in circulating concentrations of d-MPH carry toxicological and abuse liability implications should this animal model hold for ethanol-consuming attention-deficit hyperactivity disorder patients or coabusers.

The concomitant use of alcohol (EtOH) and the psychotherapeutic agent dl-methylphenidate (MPH) has risen as a consequence of an increase in ADHD diagnoses within the drinking age population. It was recently found that the combination of MPH and EtOH increases the self-report of pleasurable feelings relative to MPH alone. This finding raises concerns regarding the combined abuse liability for these two widely used drugs. The present behavioral study reports on the development of an adult male C57BL/6J (B6) mouse model to further characterize this MPH–EtOH interaction. We examined the effects of MPH on EtOH consumption in a limited access paradigm and EtOH stimulation of locomotor activity. B6 mice consumed about 2 g/kg EtOH daily and MPH dose-dependently reduced drinking. The most effective dose of MPH was 1.25 mg/kg, which produced a 41% decrease in drinking and had no effect on locomotor activity. However, when the 1.25 mg/kg dose of MPH was combined with a stimulatory dose of ethanol (1.75 g/kg) by intraperitoneal injection, there was a significantly enhanced stimulation of locomotor activity. The drug combination increased activity compared to the vehicle or MPH injections by 45% and increased the activity relative to EtOH alone by an additional 25%. The results of the EtOH and MPH interactions observed with the mouse model appear to be behaviorally relevant and suggest several converging mechanisms that may underlie MPH–EtOH interactions.