Pharmacokinetic and Pharmacologic Characterization of the Dihydrotetrabenazine Isomers of Deutetrabenazine and Valbenazine

Valbenazine and deutetrabenazine are vesicular monoamine transporter 2 (VMAT2) inhibitors approved for tardive dyskinesia. The clinical activity of valbenazine is primarily attributed to its only dihydrotetrabenazine (HTBZ) metabolite, [+]‐α‐HTBZ. Deutetrabenazine is a deuterated form of tetrabenazine and is metabolized to four deuterated HTBZ metabolites: [+]‐α‐deuHTBZ, [+]‐β‐deuHTBZ, [−]‐α‐deuHTBZ, and [−]‐β‐deuHTBZ. An open‐label, crossover study characterized the pharmacokinetic profiles of the individual deuHBTZ metabolites, which have not been previously reported. VMAT2 inhibition and off‐target interactions of the deuHTBZ metabolites were evaluated using radioligand binding. The only valbenazine HTBZ metabolite, [+]‐α‐HTBZ, was a potent VMAT2 inhibitor, with negligible affinity for off‐target dopamine, serotonin, and adrenergic receptors. Following deutetrabenazine administration, [−]‐α‐deuHTBZ represented 66% of circulating deuHTBZ metabolites and was a relatively weak VMAT2 inhibitor with appreciable affinity for dopamine (D2S, D3) and serotonin (5‐HT1A, 5‐HT2B, 5‐HT7) receptors. [+]‐β‐deuHTBZ was the most abundant deuHTBZ metabolite that potently inhibited VMAT2, but it represented only 29% of total circulating deuHTBZ metabolites. The mean half‐life of [+]‐α‐HTBZ (22.2 hours) was ∼3× longer than that of [+]‐β‐deuHTBZ (7.7 hours). These findings are similar to studies with tetrabenazine, in that deutetrabenazine is metabolized to four deuHTBZ stereoisomers, the most abundant of which has negligible interaction with VMAT2 in vitro and appreciable affinity for several off‐target receptors. In contrast, valbenazine's single HTBZ metabolite is a potent VMAT2 inhibitor in vitro with no discernible off‐target activity. Determination of the effects of intrinsic/extrinsic variables on deutetrabenazine's safety/efficacy profile should incorporate assessment of the effects on all deuHTBZ metabolites.

of the pharmacologic activity of valbenazine. [7][8][9] In in vitro studies, both valbenazine and [+]-α-HTBZ were shown to be highly selective inhibitors of VMAT2, with [+]-α-HTBZ having a stronger binding affinity for VMAT2. In addition, valbenazine and [+]-α-HTBZ have negligible affinity for off-target receptors, including dopaminergic, serotonergic, and adrenergic receptors. 8 In pharmacokinetic studies with healthy subjects, valbenazine was rapidly absorbed and gradually eliminated, with a time to maximum concentration (t max ) of 0.5-1.0 hours and a half-life of 15-20 hours. 10 The active metabolite, [+]-α-HTBZ, was formed gradually (t max 4-8 hours), and had a half-life similar to valbenazine (16-23 hours), suggesting formation rate-limited clearance. This pharmacokinetic profile supports once-daily dosing of valbenazine. In addition, there is no requirement to administer valbenazine with food, as ingestion of a high-fat meal had no effect on the peak or total plasma exposure of [+]-α-HTBZ. Coadministration of valbenazine with paroxetine, a potent inhibitor of cytochrome P450 2D6 (CYP2D6), resulted in a 1.9-fold increase in total exposure to [+]-α-HTBZ, 11 thus the recommendation is to consider dose reduction based on tolerability for known CYP2D6 poor metabolizers or patients who are taking a strong CYP2D6 inhibitor.
In contrast to valbenazine, which only yields a single active HTBZ metabolite, deutetrabenazine is a deuterated form of tetrabenazine, thus deutetrabenazine is a racemic mixture of two enantiomers that are reduced to form four deuterated dihydrotetrabenazine (deuHTBZ) stereoisomers: [+]-α-deuHTBZ, [+]-β-deuHTBZ, [−]-α-deuHTBZ, and [−]-β-deuHTBZ ( Figure 1). In previous studies on the relative abundance and potency of four individual tetrabenazine HTBZ metabolites, [+]-β-HTBZ appeared to be the primary contributor to VMAT2 inhibition by tetrabenazine. 8,9 [−]-α-HTBZ, the other abundant tetrabenazine metabolite, had much lower VMAT2 inhibitory potency than [+]-β-HTBZ, but increased affinity for other CNS targets. 8,9 Despite the findings with tetrabenazine, as well as other studies demonstrating that isomers can have different pharmacokinetic and/or pharmacodynamic properties, [12][13][14] none of the available pharmacokinetic or pharmacologic data for deutetrabenazine have reported the circulating plasma levels of the individual deuHTBZ metabolites. [15][16][17] Instead, deuHTBZ exposures and pharmacokinetic parameters have been reported as the combination of total (α+β)-deuHTBZ or the sum of the two αand two β-deuHTBZ isomers. [15][16][17] In pharmacokinetic studies with healthy subjects, deutetrabenazine was rapidly absorbed and eliminated following oral administration. Total (α + β)-deuHTBZ had a t max of 3-4 hours and a half-life of 9-10 hours. 15,16 Based on this pharmacokinetic profile, deutetrabenazine should be administered twice daily. In addition, it should be taken with food, as exposure to total (α + β)-deuHTBZ was increased by ∼50% after a high-fat meal. 16 The affinities of the individual deuHTBZ isomers for VMAT2 have not been reported, and their selectivity profiles have not been published.
The effects of intrinsic (eg, CYP2D6 metabolizer status, hepatic impairment) or extrinsic (eg, CYP2D6 inhibitor coadministration) factors on the individual deuHTBZ isomers have not been reported. A recent study reported that coadministration of a potent CYP2D6 inhibitor differentially affected α-deuHTBZ (increases of 1.2-fold in maximum plasma concentration [C max ] and 1.8-fold in area under the curve [AUC 0-inf ]) and β-deuHTBZ (increases of 2.1fold in C max and 5.6-fold in AUC 0-inf ), but the effects on the individual [+] and [−] isomers were not reported. 17 The purpose of this study was to evaluate the in vitro binding profiles of the four deuHTBZ metabolites and [+]-α-HTBZ and quantify the pharmacokinetic profiles of the four individual deuHTBZ metabolites after deutetrabenazine administration in comparison with [+]-α-HTBZ after valbenazine administration. The effects of CYP2D6 metabolizer status on the exposures of the four deuHTBZ metabolites and [+]-α-HTBZ were also investigated. This is the first study to report the relative contribution of the individual [+] and [−] isomers of α-deuHTBZ and β-deuHTBZ to the pharmacologic activity of deutetrabenazine. Understanding the unique metabolic and pharmacodynamic characteristics of each deuHTBZ stereoisomer is critical to fully evaluate the relevance of factors that affect isomer exposure on the safety and efficacy profile of deutetrabenazine.
VMAT2 Inhibition Assays. Studies were conducted in human platelet homogenates to determine the ability of the four deuHTBZ metabolites and [+]-α-HTBZ to inhibit the binding of HTBZ to the VMAT2 transporter (Eurofins, New Taipei City, Taiwan). 18,19 Dilutions of the test compounds ranging from 1 nM to 3 μM were incubated for 60 minutes at 25°C with 2 nM [ 3 H]-HTBZ and an ∼0.175 mg aliquot of membrane protein from human platelet homogenates (Chinese Blood Services Foundation, Taipei City, Taiwan) prepared in modified HEPES buffer pH 8.0. Nonspecific binding was estimated in the presence of Ro4-1284 (10 μM). Membranes were filtered and washed, and concentrations of bound [ 3 H]-HTBZ were detected with scintillation counting.
Off-Target Radioligand Binding Assays. Radioligand binding assays were conducted to test the affinities of the four deuHTBZ metabolites on G protein-coupled receptors, including dopamine, serotonin, and adrenergic receptors. Dilutions of the test compounds ranging from 3 nM to 10 μM were mixed with membranes from CHO or human kidney (HEK-293) cell lysates expressing various receptors and 0.1-4 nM of the appropriate radioactive ligand. Dopamine D 1 , dopamine D 3 , serotonin 5-HT 2B , serotonin 5-HT 7 , adrenergic α 2A , and adrenergic α 2C were expressed in CHO cells. Dopamine D 2S , serotonin 5-HT 1A , serotonin 5-HT 2A , and serotonin 5-HT 2C were expressed in HEK-293 cells. Assay methods were the same as for VMAT2 binding, except that the test compounds were diluted in a buffer appropriate for each receptor subtype. All off-target receptor radioligand binding assays were performed at Eurofins (Eurofins Cerep, Celle-l'Evescault, France). Detailed methods for each of the receptors assayed for each radioligand in CHO or HEK-293 cell lines are described in Supporting Information Table S1. [20][21][22][23][24][25][26][27][28] Further information can also be found online. 29 Data Analysis. For the VMAT2 inhibition assays, IC 50 values were determined by a nonlinear, least squares regression analysis using MathIQ (ID Business Solutions Ltd., Guildford, Surrey, UK). For the offtarget binding assays, IC 50 values were determined by nonlinear regression analysis of the competition curves using software developed at Cerep (Hill software) and validated against SigmaPlot 4.0 (SPSS Inc., Chicago, Illinois). All IC 50 values were converted to K i using the radioligand concentration and its K d using the Cheng-Prusoff equation. 30 For competition experiments, nonspecific binding was fit as a variable using the plateau at the highest concentrations of competitor. The specific details of all the equations used in the data analysis for IC 50 , Hill slope, and K i can be found in the Supplemental Information under "Additional Analysis and Expression of Results."

Pharmacokinetics
Study Design and Subjects. The clinical study was conducted at Celerion, Inc. (Tempe, Arizona) in accordance with Good Clinical Practice guidelines and was approved by the Institutional Review Board at the study site. Written informed consent was obtained from all study subjects prior to study enrollment. The pharmacokinetics of valbenazine, [+]-α-HTBZ, and each of the four deuHTBZ metabolites were assessed in a single-center, phase 1, open-label, crossover study (NBI-98854-1723) following single-dose administration of valbenazine and deutetrabenazine. In previously published studies, plasma levels of deutetrabenazine were at or near the lower limit of detection and therefore could not be reliably estimated, 15,16 therefore the plasma concentrations of deutetrabenazine were not assessed in this study.
A total of 18 healthy males, aged 18-55 years, with a body mass index of 18.0-32.0 kg/m 2 , were randomized 1:1 to one of two treatment sequences as follows: • valbenazine 40 mg on day 1 and deutetrabenazine 24 mg on day 16 • deutetrabenazine 24 mg on day 1 and valbenazine 40 mg on day 16.
Valbenazine was administered as a single 40-mg capsule and deutetrabenazine as two 12-mg tablets. Both study drugs were administered under the fed state (30 minutes after the start of a standard breakfast).
Key exclusion criteria were as follows: predicted CYP2D6 poor or ultrarapid metabolizer (based on genotypic analysis of CYP2D6 status at screening); unstable medical condition, history of alcohol or drug abuse, long QT syndrome or cardiac arrhythmia, or history of suicidal ideation or behavior; use of any medication (prescription, over-the-counter, alternative, or investigational) within 30 days before baseline; prior exposure to valbenazine or deutetrabenazine; ingestion of grapefruit juice or products within 7 days of baseline; blood loss (≥500 mL) or donated blood within 56 days of baseline or plasma within 7 days of baseline; aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, or total bilirubin levels >1.5 times higher than the upper limit of normal at screening; and hemoglobin levels of <13.0 g/dL for males and <12.0 g/dL for females.
The plasma concentrations of the four deuHTBZ isomers were quantified by ultra-high performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) analysis. Sample processing was performed by means of liquid-liquid extraction followed by a derivatization followed by a second liquid-liquid extraction. Separation between isomers and interfering endogenous compounds was achieved by LC-MS/MS using a CORTECS UPLC C18+ (2.1 × 100 mm, 1.6 μm) column and eluted as a gradient using 0.1% formic acid in water as mobile phase A and acetonitrile:methanol (75:25 v/v) as mobile phase B. A Triple Quad 6500 mass spectrometer equipped with a turbo ion spray source was used for detection in positive ion mode. For quantification of the deuterium-labeled analytes, the nondeuterated analogs served as internal standards. Quantification was based on multiple reaction monitoring (MRM) of the transitions of m/z 506.3 → 280.4 for analytes (all four analytes have the same mass and appear in the same transition) and m/z 500.3 → 302.3 for internal standards. A linear calibration curve was used ranging from 0.250 to 125 ng/mL for all analytes with a 1/x 2 weighting factor. The lower limit of quantification was 0.250 ng/mL for each of the deuterated isomers. Across quality control samples for all isomers, inter-and intra-run precision (CV%) was ≤10.7% and accuracy (% bias) ranged from −6.6% to 7.4%.
A triple quadrupole mass spectrometer (Sciex API 5000) equipped with Turbo Ion Spray source was used in the positive ion mode. Quantitation was based on MRM of the transitions of m/z 419.3 → 205.3 for valbenazine, 422.3 → 208.3 for 13 C-labeled valbenazine (internal standard), 320.3 → 165.3 for [+]˗α˗HTBZ, and m/z 323.3 → 167.3 for a 13 C-labeled structural analog that served as the internal standard of [+]˗α˗HTBZ. A linear calibration curve was used ranging from 1.00 to 1000 ng/mL for valbenazine and 0.100 to 100 ng/mL for the [+]˗α˗HTBZ metabolite with a 1/x 2 weighting factor. Across quality control samples for both valbenazine and its [+]˗α˗HTBZ metabolite, inter-and intra-run precision (CV%) was ≤9.2% and accuracy (% bias) ranged from −4.3% to 3.0%.
Pharmacokinetic Parameters and Analysis. Pharmacokinetic parameters were determined using noncompartmental analysis with Phoenix WinNonlin v8.2 software (Certara USA, Inc., Princeton, New Jersey) and summarized descriptively. The pharmacokinetic parameters assessed included the following: maximum concentration (C max ), time to maximum plasma concentration (t max ), apparent terminal half-life (t 1/2 ), delay time between time of dosing and time of appearance of  All assays were performed in one to three independent experiments and in duplicate; n = number of independent experiments performed. Shaded cells indicate relatively higher binding (ie, lower K i ). deuHTBZ, deuterated dihydrotetrabenazine. a n = 1 experiment in duplicate for this assay.
measurable analyte (t lag ), area under the curve from time 0 to the last measurable concentration (AUC 0-tlast ), and area under the curve from time 0 to infinity (AUC 0-inf ).

Pharmacokinetics
Of 18 male subjects who enrolled and completed the pharmacokinetic study, most were white (66.7%) and classified as extensive CYP2D6 metabolizers (61.1%); mean age was 37.5 years (Table S2). Following administration of deutetrabenazine, all four deuHTBZ metabolites were quantifiable in plasma. C max of the deuHTBZ metabolites was attained within 4.0 hours, and plasma concentrations declined thereafter with an apparent t 1/2 ranging from 5.2 to 12.3 hours (Table 3 and Figure 2). The most abundant of the deuHTBZ metabolites was [−]-α-deuHTBZ, which accounted for 66% of the total deuHTBZ exposure based on AUC 0-inf (Figure 2). The [+]-β-deuHTBZ metabolite accounted for 29% of total deuHTBZ exposure.
In this study, seven of the 18 subjects enrolled were intermediate metabolizers of CYP2D6. The geometric mean ratio of AUC 0-inf values of intermediate metabolizers versus extensive metabolizers for the deuHTBZ metabolites ranged from 118% to 206%, indicating a range of sensitivity to CYP extensive versus intermediate metabolizer status (Table 4 and Figure 3). The ratios for the most abundant circulating deuHTBZ metabolites, [−]-α-deuHTBZ and [+]-β-deuHTBZ, were 118%  and 194%, respectively. The geometric mean ratio for [+]-α-HTBZ was 122%.

Discussion
Valbenazine and deutetrabenazine have both been shown in well-controlled trials to be safe and effective for the treatment of TD [31][32][33][34] ; however, the differences in their metabolic and pharmacodynamic profiles may have important clinical implications. 6 Valbenazine is metabolized to a single active HTBZ metabolite, [+]-α-HTBZ, which is a potent and selective inhibitor of VMAT2 with no discernible off-target activity at therapeutic exposures. [7][8][9] In contrast, deutetrabenazine is a deuterated form of racemic tetrabenazine and is metabolized to four deuHTBZ stereoisomers. 8,9 Despite the known potential for individual isomers to have different pharmacokinetic and/or active a Figure 4. Relative exposures and VMAT2 activity of the HTBZ metabolites. The green box indicates primary VMAT2 active metabolite. Based on relative abundance and potency observed in this study, [+]-β-deuHTBZ appears to be the primary contributor to VMAT2 inhibition of deutetrabenazine. In contrast, the pharmacological activity for valbenazine is mediated primarily by [+]-α-HTBZ. AUC 0-inf , area under the plasma-time curve from time 0 to infinity; deuHTBZ, deuterated dihydrotetrabenazine; HTBZ, dihydrotetrabenazine; VMAT2, vesicular monoamine transporter 2. a Based on the assumption that K i < 1000 nM is considered relevant VMAT2 activity. b Based on total AUC 0-inf for all circulating HTBZ metabolites after a single dose of deutetrabenazine (24 mg) or valbenazine (40 mg).
pharmacodynamic activity, [12][13][14] previous studies have only reported the pharmacokinetics of the combined α-deuHTBZ and β-deuHTBZ isomers following deutetrabenazine administration. [15][16][17] Thus, there are no published data on the pharmacokinetics and pharmacology of the individual deuHTBZ metabolites. This study is the first to characterize the metabolic profile and binding affinities of the individual deuHTBZ metabolites of deutetrabenazine in comparison with [+]-α-HTBZ, the single valbenazine HTBZ metabolite. Based on relative abundance and potencies observed in this study, [+]-β-deuHTBZ appears to be the primary contributor to VMAT2 inhibition of deutetrabenazine; however, it accounted for only 29% of total circulating deuHTBZ metabolites after administration of deutetrabenazine (Figure 4). The most abundant overall metabolite, [−]-α-deuHTBZ, accounted for 66% of total circulating deuHTBZ metabolites but had the weakest affinity for VMAT2 and had appreciable affinities for other off-target dopaminergic, serotonergic, and adrenergic targets, increasing the potential for unintended effects. The [+]-α-deuHTBZ and [−]-β-deuHTBZ metabolites were minor metabolites, accounting for only 2% and 3%, respectively, of circulating deutetrabenazine HTBZ metabolites. This metabolic profile is similar to the previously reported profile of the tetrabenazine HTBZ metabolites and indicates that the pharmacologic activities of deutetrabenazine and valbenazine arise from different HTBZ isomers ( Figure 4). 8,9 In contrast to deutetrabenazine, whose action is mediated by a blend of four isomers with varying affinities for VMAT2 and other CNS targets, 100% of the circulating HTBZ metabolite from valbenazine ([+]-α-HTBZ) is active, with high selectivity and specificity for VMAT2 and negligible affinity for off-target receptors ( Figure 4). As such, the pharmacological activity for valbenazine is mediated primarily by [+]-α-HTBZ.
The pharmacokinetic profile of [+]-α-HTBZ observed in this study was similar to previous reports. 10 The half-life of [+]-α-HTBZ was 22.2 hours, which was approximately three times longer than that of [+]-β-deuHTBZ, the predominant active deutetrabenazine metabolite (7.7 hours), and also that of [+]α-deuHTBZ (6.4 hours), which likely reflects the formation rate-limited clearance of [+]-α-HTBZ following valbenazine administration. This longer half-life supports once-daily dosing for valbenazine, compared to twice-daily dosing recommended for deutetrabenazine. Although valbenazine can be administered without regard to food, the dosing in this study was conducted with food, as deutetrabenazine is recommended to be administered with food.
While the effects of coadministration of valbenazine with paroxetine, a potent CYP2D6 inhibitor, on [+]-α-HTBZ exposure have been evaluated, 11 the only study to evaluate these effects for deutetrabenazine reported the effects on exposures for combined α-deuHTBZ and β-deuHTBZ, but not for the four individual deuHTBZ isomers. 17 Considering the results from that study in the context of the findings reported here, the majority of the 1.8-fold effect on α isomers likely reflects an increase in the VMAT2-inactive isomer ([−]-α-deuHTBZ), whereas the 5.6-fold effect on β isomers likely reflects an increase in exposure of the primary active VMAT2 isomer ([+]-β-deuHTBZ). 17 This is consistent with our findings that intrinsic CYP2D6 activity (ie, intermediate versus extensive metabolizer) had a greater effect on the exposure of the primary active metabolite of deutetrabenazine ([+]-β-deuHTBZ) compared to the active metabolite of valbenazine ([+]α-HTBZ). To our knowledge, this is the first report on the effects of intrinsic factors (eg, CYP2D6 genotype) on the pharmacokinetics of the four individual deuHTBZ isomers. Further studies are needed to evaluate the effects of the full range of CYP2D6 activity (including poor and ultrarapid metabolizers) on exposures of the four individual deutetrabenazine HTBZ metabolites.

Conclusions
Given the findings presented here, it is more appropriate to consider relative exposures of each of the four individual deuHTBZ isomers when evaluating the impact of intrinsic and extrinsic variables on the deutetrabenazine safety and efficacy profile, rather than quantifying effects on the combined concentrations of [+] and [−] deuHTBZ isomers. Further evaluation of these differences may aid in optimizing TD treatment for individual patients.