Increased Plasma Concentrations of 6-oxo-Methylphenidate in CES1 G134E Carriers Following a Single Oral Dose of Methylphenidate

Abstract

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder, with methylphenidate used as a first-line treatment. Methylphenidate is primarily hydrolyzed by carboxylesterase 1 (CES1) to inactive ritalinic acid, with minor oxidative metabolism producing active p-OH-methylphenidate and 6-oxo-methylphenidate lactam. The functional single-nucleotide polymorphism (SNP) in CES1, resulting in a glycine (G) to glutamic acid (E) substitution at 143 (G143E), is reported to significantly impair CES1 activity. However, limited clinical research has explored the pharmacokinetics of methylphenidate and its oxidation metabolites in ADHD therapeutics in G143E carriers.

Three G143E ADHD subjects were genotyped for the G143E variant, and four non-carriers were identified and enrolled in the pharmacokinetic study. Participants received a single oral dose of methylphenidate, and plasma concentrations of methylphenidate, 6-oxo-methylphenidate, and p-OH-methylphenidate were extracted and quantified. Pharmacokinetic data were analyzed, and in vitro incubation of 6-oxo-methylphenidate with G143E S9 has been conducted.

No significant differences were observed in the pharmacokinetics of methylphenidate. CES1 G143E carriers exhibited significantly elevated plasma concentrations of 6-oxo-methylphenidate, with a higher peak plasma concentration (C_max), area under the curve from time zero to infinity (AUC_0→∞), and longer half-life (T_1/2). Reduced function in in vitro studies suggested the impaired CES-mediated biotransformation of 6-oxo-methyphnidate to 6-oxo-ritalinic acid. These results provide pilot data on the substrate-dependent impact of the CES1 G143E variant. Whether or not the elevated concentrations of 6-oxo-methyphenidate contribute to the clinical activity of methylphenidate treatment remains a matter of speculation. Registry: ClinicalTrials.gov, TRN: NCT03781752, Registration date: 4-March-2018.

Graphic Abstract

Introduction

ADHD is a highly prevalent neurodevelopmental disorder both in the United States and globally. In 2023, an estimated 15.5 million U.S. adults (6.0%) had a current diagnosis of ADHD, with approximately half receiving the diagnosis at age 18 or older [1]. In 2022, an estimated 11.4% of U.S. children aged 3–17 years (approximately 7.1 million) had been diagnosed with ADHD by a healthcare provider [2]. Available since 1955, the psychostimulant dl-methylphenidate is the most commonly prescribed first-line medication for treatment of ADHD [3]. Following oral administration, in a reaction catalyzed by CES1, ~60 – 80% of a methylphenidate dose is de-esterified into its pharmacologically inactive and most abundant metabolite, ritalinic acid [4] (Figure 1). For oral administration of methylphenidate, the t_1/2 ranges from 2.5 to 5.4 hours [5].

Figure 1. Overview of the major metabolic pathways for methylphenidate [8].

The enzymes mediating this oxidative metabolism are unknown, but there has been some speculation that CYP3A4/5, CYP2D6, and CYP2B6 may be involved, but unconfirmed.

Because of extensive first-pass metabolism, the systemic exposure to the unchanged drug following oral dosing is low and variable [6]. Further, there is a marked degree of stereoselective metabolism, with the largely inactive l-methylphenidate isomer being preferentially metabolized such that an “In vivo resolution” of oral dl-methylphenidate results in only the active d-isomer being generally detected in the systemic circulation shortly after administration of the racemate [7]. Methylphenidate also undergoes limited oxidative metabolism via para-hydroxylation to form the active metabolites p-OH-methylphenidate and 6-oxo-methylphenidate. The enzymes mediating this oxidative metabolism are unknown, but there has been some speculation that CYP3A4/5, CYP2D6, and CYP2B6 may be involved, but unconfirmed [8]. Early studies suggest that p-OH-methylphenidate produces pharmacological activity as a stimulant comparable to or even higher than the parent compound [4], whereas 6-oxo-methylphenidate is the primary oxidized metabolite and accounts for up to 1.5% of total methylphenidate [9]. 6-oxo-methylphenidate exhibits greater lipid solubility than p-OH-methylphenidate [10]. In rats, the brain/plasma ratio is 2.2, indicating that 6-oxo-methylphenidate may cross the blood–brain barrier and may accumulate in the brain [10]. The biological activities of 6-oxo-methylphenidate remain largely uncharacterized [4, 11]. Following i.p. administration, 6-oxo-MPH has been shown to increase locomotor activity in rats, possibly through a nonspecific peripheral mechanism [10]. The biological activities and pharmacokinetics in humans, however, have yet to be fully investigated. Both p-OH-methylphenidate and 6-oxo-methylphenidate are subsequently hydrolyzed by CES1 into inactive metabolites [12] [9] (Figure 1). Recent uses of in vitro models of the hepatic metabolism of methylphenidate have been hindered by the unavailability of an authentic reference standard of the 6-oxo-methylphenidate metabolite [8].

In humans, CES1 is the most abundant drug-metabolizing enzyme [13] and plays an important role in the hydrolysis of numerous ester- and amide-containing substrates, including endogenous compounds, toxins, and medications [14], and is responsible for approximately 80–95% of total hydrolytic activity in the human liver [13].

Extensive in vitro and clinical studies have been conducted to evaluate the impact of reduced CES1 activity due to drug-drug interactions and genetic polymorphisms [13, 15]. Substantial interindividual variability in CES1 expression and activity has been consistently reported in the literature, likely influenced by a combination of genetic and environmental factors [16]. The functional SNP in the CES1 gene, resulting in a glycine-to-glutamic acid substitution at position 143 (G143E, rs71647871), has been shown to significantly impair CES1 enzymatic activity [17]. In vitro studies have demonstrated that the G143E variant markedly reduces the hydrolysis of several CES1 substrates, including methylphenidate [17], oseltamivir [18], trandolapril [19], and remdesivir [20]. Clinical studies further support this finding, showing that individuals carrying the G143E variant exhibit impaired metabolism of CES1 substrates, clopidogrel [21]^, [22]. Despite these findings, pharmacogenomic investigations of G143E carriers remain limited, and the genetic and biological impact of G143E is not yet fully understood [23]. The minor allele frequency (MAF) of G143E (C>T) is approximately 0.014 in the global population (NCBI dbSNP), significantly reducing the likelihood of identifying homozygous individuals. Studies also suggest that the widely used fluorescence probe-based genotyping methods may fail to detect homozygous carriers, further complicating populational genetic investigations of G143E [24]. This knowledge gap regarding CES1 expression and activity holds important clinical implications, particularly for individualized dosing strategies involving CES1 substrates. A deeper understanding of the functional consequences of the G143E variant is desirable for advancing personalized therapeutic approaches in ADHD subjects treated with methylphenidate [15].

In this study, subjects carrying the G143E variant were identified through genotyping and administered a single weight-based oral dose of dl-methylphenidate. Plasma concentrations of methylphenidate and its primary oxidative metabolites, p-OH-methylphenidate and 6-oxo-methylphenidate, were measured and quantified, and the pharmacokinetics were further analyzed. To complement clinical measures, in vitro experiments were conducted using the S9 fraction derived from wild-type and G143E-expressing cell models. The current study provides new insights into the pharmacogenetic impact of the CES1 G143E variant. Since the pharmacological activity of the 6-oxo-methylphenidate has not been well-described, whether the elevated concentrations of the metabolite are of clinical importance with respect to the overall activity of methylphenidate treatment is an open question.

Result

Demographic Characteristics of Clinical Study Participants

Seven subjects were enrolled in the clinical study, including three heterozygous G143E carriers and four non-carrier controls. Among the G143E carriers, the mean age at the time of the clinical visit was 20 years, with an average body weight of 89.5 kg (Table 1). The drug was well tolerated at the administered dose, with no adverse effects reported.

Table 1.

Demographic Characteristics of Participants in Clinical Study.

	Sex		Age (years)	Weight (kg)	Race	per kg Methylphenidate Dose (mg/kg)
G143E Carrier	Male		23	130.3	White	0.2*
	Male		18	67.9	White	0.3
	Male		18	70.3	Mixed	0.3
		Mean	20	89.5
		SD	3	35.4
Control	Male		17	57.2	White	0.3
	Male		20	98.9	Mixed	0.3
	Female		20	59.3	White	0.3
	Male		26	79.4	White	0.3
		Mean	21	73.7
		SD	4	19.6

^*The subject received a capped weight-based dosage of methylphenidate for safety considerations per protocol.

Pharmacokinetic analysis

No p-OH-methylphenidate was detected in plasma samples from any of the assessed subjects.

For methylphenidate, G143E carriers exhibited no significant difference between the G143E carriers and non-carriers for λz, Cmax, CL/F, and AUC_0→∞ (Figure 2, Table 2).

Figure 2. Pharmacokinetic analysis.

A, B: Plasma concentration of 6-oxo-methylphenidate versus time after administration, shown on a linear scale (A) and a semi-logarithmic scale (B). C, D: Plasma concentration of methylphenidate versus time after administration, shown on a linear scale (C) and a logarithmic scale (D). The G143E subject who received 0.2 mg/kg of methylphenidate is indicated with a dashed line.

Table 2.

Summary of pharmacokinetic analysis.

Non-compartmental analysis
Methylphenidate	λz (hour-1)	Cmax (ng/mL)	Tmax (hour)	T1/2 (hour)	CL/F (L/hour/kg)	AUC0→last (ng/mL·hour)	AUC0→∞ (ng/mL·hour)
G143E Carrier	0.17	14.60	2.00	4.02	2.74	50.38	109.54
Dose adjustment	0.17	26.10	0.50	2.43	1.89	83.73	105.64
	0.29	13.80	1.50	3.19	4.08	51.21	73.47
Geometric Mean	0.20	17.39		3.14	2.77		94.73
Geometric SD	1.33	1.42		1.29	1.47		1.25
Non-carrier	0.22	17.60	1.50	2.84	3.53	58.32	84.97
	0.24	9.38	2.00	3.04	6.47	31.04	46.35
	0.23	13.20	3.00	3.97	3.02	53.03	99.23
	0.23	19.60	1.50	3.08	2.81	75.41	106.77
Geometric Mean	0.23	13.44		3.34	3.73		80.37
Geometric SD	1.05	1.45		1.16	1.46		1.46
GMR of carrier/non-carrier	0.90	1.29		0.94	0.74		1.18
P-value	0.3143	0.3143		0.4286	0.2000		0.3143
6-oxo-Methylphenidate	λz (hour-1)	Cmax (ng/mL)	Tmax (hour)	T1/2 (hour)		AUC0→last (ng/mL·hour)	AUC0→∞ (ng/mL·hour)
G143E Carrier	0.18	2.67	2.00	3.92		11.24	21.59
Dose adjustment	0.19	4.10	0.50	3.71		10.08	14.92
	0.32	4.12	2.00	2.14		15.28	19.10
Geometric Mean	0.22	3.56	1.26	3.14		12.01	18.32
Geometric SD	1.40	1.28	2.23	1.40		1.24	1.21
Non-carrier	0.49	1.64	1.00	1.42		3.94	4.25
	0.53	1.85	1.50	1.31		5.18	5.67
	0.95	0.57	2.00	0.73		1.50	1.52
	0.33	1.53	1.50	2.11		4.64	5.39
Geometric Mean	0.53	1.18		1.26			3.75
Geometric SD	1.55	1.87		1.70			1.85
GMR of carrier/non-carrier	0.41	3.03		2.49			4.89
P-value	0.0286*	0.0286*		0.0286*			0.0286*

SD: Standard deviation. GMR: Geometric Mean Ratio.

^*: Significant difference with the Mann-Whitney test. Dose adjustment: G143E subjects who received 0.2 mg/kg body weight of methylphenidate.

For 6-oxo-methylphenidate, significant differences were observed in C_max, T_1/2, and AUC_0→∞ between G143E carriers and noncarriers (P < 0.05, Figure 2, Table 2).

The data for the subject who received the capped methylphenidate dose (30mg) is presented separately in Table 2.

In vitro incubation

For wild-type CES1, substrate inhibition was observed at higher substrate concentrations. Fitting the data to the substrate inhibition model (Eq. 2) yielded the following parameter estimates: Vmax = 19.14 ± 0.91 units, Km = 18.69 ± 2.00 μM, and CL_int = 1.02 unit/μM. Km: substrate concentration at half of the maximum reaction velocity, and Vmax: maximum reaction velocity. The intrinsic clearance (CL_int) was calculated by Vmax/km. The G143E variant exhibited markedly reduced enzymatic activity, insufficient to reach Km even at the highest substrate concentration tested (Figure 3).

Figure 3. In vitro incubation of 6-oxo-methylphenidate with wild-type CES1 S9 and G143E S9.

Nonlinear regression was performed using Eq. 2. Each data point represents the average of duplicate assays (n=2).

Discussion

The current studies provide additional insight into the metabolism and disposition of methylphenidate. As the most commonly prescribed medication for ADHD, methylphenidate is known to exhibit substantial interindividual variability in therapeutic response [25]. Given that CES1 is the primary enzyme responsible for methylphenidate metabolism, genetic polymorphisms in CES1, particularly within its coding regions, are of significant clinical interest [17]. The synthesis of the commercially unavailable 6-oxo-methylphenidate metabolite permitted us to conduct a novel assessment of the influence of the CES1 G143E variant on the pharmacokinetics of methylphenidate and its primary oxidized metabolite, 6-oxo-methylphenidate, in ADHD subjects carrying the G143E variant. The noncompartmental analysis results for methylphenidate are consistent with previously reported values [5]. No significant differences were observed in the pharmacokinetics of methylphenidate between G143E carriers and non-carriers. However, G143E carriers exhibited a marked increase in C_max, T_1/2, and AUC_0→∞ for 6-oxo-methylphenidate by GMR of 3.03, 2.49, and 4.89, respectively. This finding was further supported by in vitro incubation studies using G143E S9 (Figure 3), as reduced function in in vitro studies suggested the impaired/reduced CES-mediated biotransformation of 6-oxo-methyphnidate to 6-oxo-ritalinic acid. Previous reports indicate that individuals heterozygous for the G143E variant exhibit elevated plasma concentrations of methylphenidate following a single oral dose [26]. However, the current study did not observe a similar elevation in methylphenidate concentrations among G143E carriers. The reason for this difference remains unclear and may be attributable to the following factors. The previous study [26] included a larger sample size (n = 16) and an extended sampling duration (33 hours post-administration), which allowed differences in methylphenidate pharmacokinetics among G143E carriers to be more clearly identified. In small sample size pharmacokinetic studies of methylphenidate, a more substantial change in CES1 activity may be required to observe a clinically meaningful distinction for heterozygous carriers. In cases of complete loss of CES1 function, differences in probe substrate levels are more detectable clinically [17][27].

To date, the present research is the first report to highlight differences in 6-oxo-methylphenidate pharmacokinetics associated with the G143E variant. Limited literature has been published on the role of 6-oxo-methylphenidate, including its potential CNS activity and its potential contribution to both therapeutic and/or adverse effects. 6-oxo-methylphenidate exhibits greater lipid solubility than p-OH-methylphenidate (overall ranking: methylphenidate > 6-oxo-methylphenidate > p-OH-methylphenidate) [10]. In rats, its brain/plasma ratio is 2.2, suggesting that 6-oxo-methylphenidate can cross the blood–brain barrier and potentially accumulate in the brain [10]. Nonetheless, its biological activities remain largely uncharacterized [4, 11]. In animal studies with rats, i.p. administration, but not i.v. administration, 6-oxo-methylphenidate increased locomotor activity, “possibly via a nonspecific peripheral effect”, although the underlying mechanism remains unclear [10]. As suggested by the findings of the current study, 6-oxo-methylphenidate appears to be more sensitive to CES1 activity loss. The pharmacokinetics difference of 6-oxo-methylphenidate demonstrates a substrate-dependent reduction in CES1-mediated metabolism in G143E carriers. In homozygous carriers, different substrates exhibit varying sensitivity to changes in CES1 activity. A similar substrate-dependent difference for G143E has also been reported elsewhere [28]. This finding may have implications in the treatment of ADHD when additional psychopharmacological treatments are employed and polypharmacy results. Reduced CES1 activity, whether resulting from genetic polymorphisms or drug–drug interactions, may alter the metabolism of other CES1 substrate therapeutics as well as their active metabolites that may be sensitive to variations in CES1 expression or activity. In ADHD treatment, the primary therapeutic agents methylphenidate and dexmethylphenidate are known as CES1 substrates [29][30]. Coadministration of methylphenidate with other psychotropic drugs, including antipsychotics and antidepressants, is not uncommon but could potentially lead to clinically relevant interactions through CES1 inhibition [31]. For example, the antipsychotic agent aripiprazole and the antidepressant fluoxetine have both been shown to inhibit CES1 activity in vitro. Moreover, exposure to medical or recreational cannabis introduces an additional risk, as cannabinoids have also been shown to inhibit CES1, further increasing the potential for drug–drug interactions [32]. Further studies are warranted to identify compounds that are particularly sensitive to CES1 activity variations and to better understand the clinical implications of CES1 polymorphisms.

Regarding the pharmacogenetics of methylphenidate, the current study demonstrated that individuals heterozygous for the G143E variant exhibit a significant pharmacokinetic difference in the metabolite 6-oxo-methyphenidate. In addition, the pharmacological activity of 6-oxo-methylphenidate remains to be investigated, and it is unclear whether its elevation has any impact on the clinical efficacy or adverse effects of methylphenidate in ADHD treatment. The current findings highlight the importance of further exploration of the metabolism of methylphenidate and the physiological and pharmacological significance of 6-oxo-methylphenidate. Moreover, homozygous G143E carriers have rarely been identified in the literature to date [24] [27], although previous research has shown that complete CES1 deficiency [17] and homozygosity for G143E are not lethal [27]. This rarity is partly due to the methodological limitations, as previous studies have highlighted potential issues with current genotyping techniques, the inability of certain fluorescent probes to distinguish between heterozygous and homozygous carriers [24]. However, the low MAF of G143E may reflect underlying biological selection, potentially due to developmental lethality associated with homozygosity. The possibility that G143E homozygosity confers deleterious effects cannot be ruled out.

Regarding the single subject who received the capped dosage of methylphenidate, as λz, t½, and CL/F are dose-independent parameters, dose adjustment would only reduce AUC and C_max. However, CES1 G143E patients receiving the capped dosage still have higher AUC and C_max of 6-oxo-methyphinidate compared with control patients on the standard dosage, which further strengthens the findings.

There are several limitations to the current study. In the present study, only 6-oxo-methylphenidate, p-OH-methylphenidate, and methylphenidate were analyzed due to technical limitations. The current extraction method is capable of extracting only these two metabolites and the parent compound methylphenidate, because of the polarity differences between methylphenidate metabolites and ritalinic acid metabolites. Once the ester bond is cleaved, the resulting phenolic acid derivatives are significantly more acidic. Effective extraction of downstream metabolites—ritalinic acid, 6-oxo-ritalinic acid, and OH-ritalinic acid—would require a lower pH extraction method. Future research should be done on developing further techniques to measure these additional metabolites, including the synthesis of standards if commercial sources are unavailable. These approaches enable a more comprehensive pharmacokinetic profile and allow for a better evaluation of the impact of G143E variants on methylphenidate metabolism and clinical outcomes. Additionally, only a limited number of participants could be recruited for clinical testing. Ideally, additional time points would have been collected to better capture the elimination phase. However, due to practical constraints, the current study was limited to 6 hours post methylphenidate administration. To strengthen these findings, future studies with larger sample sizes and extended sampling durations are warranted. In addition, the present study genotyped only the G143E variant, without assessing CES1 copy number. In conclusion, no significant differences were observed in the pharmacokinetics of methylphenidate. p-OH-methylphenidate was not detected in plasma samples. CES1 G143E carriers exhibited significantly elevated plasma concentrations of 6-oxo-methylphenidate compared to non-carriers. In vitro incubation of 6-oxo-methylphenidate demonstrated significantly lower intrinsic clearance in G143E S9 fractions relative to the wild type. These results provide novel insights into the substrate-dependent impact of the CES1 G143E variant and suggest a potential need for personalized dosing strategies in methylphenidate administration, as well as for the administration of other drugs metabolized by CES1. Nevertheless, since the potential pharmacological effect of 6-oxo-methylphenidate has not been thoroughly investigated, the significance of elevated concentrations of the metabolite is uncertain with respect to the overall clinical effects of methylphenidate.

Materials and Methods

Chemicals

dl-methylphenidate HCl was purchased from Cerilliant Corp. (Round Rock, TX, USA). Phenacetin was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). LC-MS grade water and methanol were purchased from Birch Biotech (Morgantown, PA, USA). p-OH-methylphenidate and 6-oxo-ritalinic acid were synthesized, and (dl)-threo-ritalinic acid was previously provided by Kennerly S. Patrick, PhD (deceased). Iodine (I₂, solid, purity ≥ 99.8%) was purchased from Sigma Aldrich. Sodium bicarbonate (NaHCO₃, solid, purity ≥ 99.7%) and sodium thiosulfate (solid, purity 98.5%) were acquired from Acros Organics. Anhydrous tetrahydrofuran (THF, purity > 99.5%) was sourced from TCI chemicals.

Synthesis of 6-oxo-methylphenidate

The chemical synthesis is outlined in Scheme 1. A round-bottom flask was charged with 100 mg of (dl)-threo-ritalinic acid (1), followed by adding 2 mL of 2N HCl in methanol, and the reaction mixture was heated at 55 °C for 45 minutes. The HCl/methanol was evaporated to complete dryness using a rotary evaporator. The reaction product was confirmed to be methylphenidate (2) through LC-MS and NMR. The regio- and chemo-selective oxidation of Cα-H in the piperidine ring of methylphenidate was achieved using iodine to obtain the 6-oxo-methylphenidate [33]. Iodine (1.605 mM, 7.5 eq.) was transferred to a round-bottom flask containing a mixture of methylphenidate 2 (50 mg, 0.214 mM) and sodium bicarbonate (2.143 mM, 10 eq.) in THF/H₂O (8.8:3.5). The mixture was stirred gently at room temperature. The product formation was checked at intervals of 3, 6, 9, and 20 hrs using LC-MS. After 20 hrs, the reaction mixture was pipetted into equal volumes of saturated aqueous solutions of sodium thiosulfate and sodium bicarbonate. The product was then extracted with dichloromethane, and the collected organic layer was dried over anhydrous sodium sulfate before being concentrated under reduced pressure. The crude product was purified using silica gel column chromatography with a gradient of hexanes and ethyl acetate, followed by ethyl acetate and methanol, yielding 6-oxo-methylphenidate (3) as a white solid (4.5 mg). ¹H NMR (600 MHz, Methanol-d₄) δ 7.41–7.26 (m, 5H), 4.08 (ddd, J = 10.0, 7.2, 5.1 Hz, 1H), 3.76 (d, J = 10.1 Hz, 1H), 3.70 (s, 3H), 2.37–2.24 (m, 2H), 1.90–1.79 (m, 1H), 1.65 (m, 1H), 1.55 (m, 1H), 1.37–1.31 (m, 1H).¹³C NMR (151 MHz, Methanol-d₄) δ 175.42, 174.54, 137.10, 130.21, 129.58, 129.25, 58.48, 55.65, 52.97, 32.07, 25.94, 19.25. MS (ESI⁺): m/z = 248.51 [M+H]⁺.

Scheme 1. Synthesis of 6-oxo-methylphenidate.

Clinical Study

DNA was extracted using the DNA Extraction and Purification Kit (ThermoFisher Scientific inc., Waltham, MA) from oral swabs or blood collected from subjects. Recruited ADHD subjects were genotyped for the G143E variant using TaqMan Real-Time PCR assays (ThermoFisher Scientific inc., Waltham, MA) targeting the CES1 SNP rs71647871. During the screening phase, 481 subjects were genotyped, and the minor allele frequency was 0.01. Three G143E carriers and four non-carriers were subsequently enrolled in the clinical study. Seven subjects from two separate study sites completed written informed consent, and the study was approved by the Institutional Review Boards of the Icahn School of Medicine at Mount Sinai (coordinating site), Cincinnati Children’s Hospital, and the University of Florida were enrolled in the clinical study, including three heterozygous G143E carriers identified through genotyping and four noncarriers (ClinicalTrials.gov: NCT03781752). All participants were ADHD subjects who had not received methylphenidate treatment for over a year at the time of the study. Each participant was admitted to a research unit, had an intravenous catheter placed, and then received a single oral dose of methylphenidate at 0.3 mg/kg oral solution (2 mg/mL) under fasting conditions, except for one higher-weight G143E carrier, whose dose was capped at 30 mg per the protocol’s safety design to ensure the total did not exceed the recommended maximum single-dose limit. As a result, the weight-based dose for this participant was 0.2 mg/kg. Serial plasma samples were collected at nine time points: pre-dose (0 hour), and at 0.5, 1, 1.5, 2, 3, 4, 5, and 6 hours post-administration. All plasma samples were stored at −80 °C until analysis.

Plasma Preparation

A liquid–liquid extraction method for 6-oxo-methylphenidate, p-OH-methylphenidate, and methylphenidate was adapted from previously described protocols with modifications [34]. Briefly, 0.2 mL of plasma was mixed with 0.1 mL of 10 mM KH₂PO₄ buffer (pH 4.4), where the pH was optimized based on preliminary recovery experiments using spiked plasma samples. Phenacetin was added to the buffer as an internal standard at a final concentration of 1 μg/mL. The mixture was then extracted with 1 mL of a butyl chloride/acetonitrile solution (4:1, v/v). The organic phase was transferred to clean glass tubes and evaporated to dryness under a gentle stream of nitrogen. The dried residue was reconstituted in 100 μL initial mobile phase (50:50 methanol:water with 0.1% formic acid) for subsequent LC-MS/MS analysis.

Preparation of Cell S9 Fractions Containing Wild-Type and G143E CES1

Wild-type and G143E S9 fractions were prepared as previously described [17]. In brief, a human embryonic kidney cell line (Flp-In-293, Invitrogen, Carlsbad, CA) stably expressing WT and CES1 G143E was cultured in Dulbecco’s modified Eagle medium with 10% FBS and 100 mg/ml hygromycin. Upon reaching approximately 90% confluency, cells were then harvested in PBS and sonicated for 10 seconds. The mixture was then centrifuged at 9000 g for 30 min to isolate the S9 fraction, which was then stored at −70 °C until use. The total protein concentrations in S9 fractions were determined using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific inc., Waltham, MA).

In vitro Assay Conditions

The in vitro incubation procedure followed a previously published method [20]. Each 100 μL reaction mixture contained 6-oxo-methylphenidate at various concentrations (1.6, 3.1, 6.3, 12.5, 25.0, and 50.0 μg/mL) in phosphate-buffered saline (pH = 7.4). To initiate the reaction, 10 μL of either wild-type or G143E S9 fraction was added, resulting in a final S9 protein concentration of 40 μg/mL. Incubations were carried out in a water bath at 37 °C for 1 hr. The reactions were terminated by adding 50 μL of acetonitrile containing phenacetin (1 μg/mL) as an internal standard. Phenacetin has been widely used as an internal standard in bioanalytical methods when deuterated analogs are unavailable. Phenacetin exhibited comparable retention times to all analytes in the present study. It is not a clinically used drug and has no endogenous sources. Samples were then centrifuged at 2,000 × g for 10 minutes to precipitate proteins, and the resulting supernatant was collected for analysis of 6-oxo-ritalinic acid.

Bioanalysis

The LC-MS/MS method is a modification of a previously described assay [34]. In brief, the analysis was performed on a Shimadzu HPLC system (Shimadzu, Tokyo, Japan) including two pumps (LC-10ATvp), an autosampler (SIL-10AD-vp), and a system controller (SCL-10Avp) coupled to an Applied Biosystems-Sciex API 3000 triple quadrupole mass spectrometer (Foster City, CA, USA). In brief, 5 μL of the samples is injected for chromatographic separation. A reverse-phase method was used with a Synergi 4μ Fusion-RP 80A C18 column (100 × 2 mm) with a flow rate of 0.300 mL/min. The mobile phase consisted of 50% methanol and 50% water with 0.1% formic acid, over a total run time of 7 minutes. The following MS parameters were used: curtain gas, 14 psi; nebulizer gas, 14 psi; collisionally activated dissociation gas, 4 psi; TurboIonSpray (IS) voltage, 4500 V; entrance potential, 10 V; and source temperature, 400 °C. The declustering potential, focusing potential, collision energy, and collision cell exit potential were set as follows: For POM (m/z 250.16 → 83.8): 26 V, 50 V, 45 eV, and 6 V, respectively. For 6-oxo-methylphenidate (m/z 246.3 → 97.6): 31 V, 50 V, 27 eV, and 18 V, respectively. For methylphenidate (m/z 234.0 → 84.0): 31 V, 50 V, 27 eV, and 18 V, respectively. For 6-oxo-ratalinic acid (m/z 234.1 → 97.9), 26 V, 50 V, 57 eV, 10 V, respectively. For phenacetin (IS, m/z 180.1 → 110.1): 30 V, 30 eV, and 15 V. The dwell time for all transitions was 23 ms.

Pooled plasma spiked with standards was processed identically to the study samples and used for calibration. Standard curves were prepared using a 7-point calibration, with concentration ranges of 0.01 – 1.50 ng/mL for 6-oxo-methylphenidate, 0.30 – 20.00 ng/mL for OH-methylphenidate, and 0.20 – 30.00 ng/mL for methylphenidate. All calibration curves had an r² value greater than 0.98. The lower limits of quantitation (LLOQ) were 0.01 ng/mL for 6-oxo-methylphenidate, 0.30 ng/mL for OH-methylphenidate, and 0.20 ng/mL for methylphenidate. Quality control samples were prepared at three concentrations (0.375, 0.75, and 1.5 ng/mL for 6-oxo-methylphenidate; 7.5, 15, and 30 ng/mL for methylphenidate), with all accuracy values within ± 25%. All samples from all subjects were analyzed in a continuous run. The calibration curve and quality control samples were injected twice, at both the beginning and end of the 24-hour analytical sequence, with no significant differences observed between the quality control results at these two injections.

Statistical analysis

MATLAB SimBiology (Natick, MA) was used for noncompartmental analysis. Peak concentration (C_max) and the time to C_max (T_max) were reported as observed. The terminal elimination rate constant (λz) was estimated by linear least-squares regression of the terminal portion of the plasma concentration (on natural logarithmic scale)-time curve, and the elimination half-life (T_1/2) was then calculated using the formula T_1/2 = ln2/λz. The area under the plasma concentration-time curve from time 0 to infinity (AUC_0→∞) and AUC_0→last were calculated according to the linear trapezoidal rule. The apparent clearance (CL/F) was calculated using the formula dose/AUC_0→∞. The Mann-Whitney test (one-tailed) has been performed to determine the significance between the G143E carrier and non-carrier.

GraphPad PRISM 9 (San Diego, CA) was used for In vitro evaluation data analysis, and Michaelis-Menten models with substrate inhibition were used as previously described (Eq. 2) [35].

$$V=\frac{Vmax\left[S\right]}{Km+\left[S\right]+\frac{{\left[S\right]}^2}{Km}}$$ [Eq. 2]

Data points are: [S]: substrate concentration, and V: reaction velocity. Iterated variables are: Km: substrate concentration at half of the maximum reaction velocity, and Vmax: maximum reaction velocity. The intrinsic clearance (CL_int) was calculated by Vmax/km.