ORIGINAL ARTICLE
Absorption, distribution, metabolism, and excretion of [14C]-dasotraline in humans Yu-Luan Chen1, Estela Skende1, Jing Lin1, Yijun Yi2, Peter L. Wang2, Sarah Wills3, H. Scott Wilkinson1, Kenneth S. Koblan1 & Seth C. Hopkins1 1
Sunovion Pharmaceuticals, Inc., Marlborough, Massachusetts XenoBiotic Laboratories, Inc., Plainsboro, New Jersey 3 Covance Laboratories, Inc., Madison, Wisconsin 2
Keywords Absorption, dasotraline, distribution, excretion, glucuronidation, mass spectrometry, metabolite identification, pharmacokinetics, phase II metabolism, sulfate conjugation Correspondence Yu-Luan Chen, Sunovion Pharmaceuticals, Inc., 84 Waterford Drive, Marlborough, MA 01752. Tel: +1 508 787 4286; Fax: +1 508 357 7859; E-mail: [email protected] Funding Information This work was funded by Sunovion Pharmaceuticals, Inc. Received: 10 August 2016; Revised: 3 November 2016; Accepted: 5 November 2016 Pharma Res Per, 5(1), 2017, e00281, doi: 10.1002/prp2.281 doi: 10.1002/prp2.281
Abstract Dasotraline is a dopamine and norepinephrine reuptake inhibitor, and the early clinical trials show a slow absorption and long elimination half-life. To investigate the absorption, distribution, metabolism, and excretion of dasotraline in humans, a single dose of [14C]-dasotraline was administered to eight healthy male adult volunteers. At 35 days, 90.7% of the dosed radioactivity was recovered in the urine (68.3%) and feces (22.4%). The major metabolic pathways involved were: (1) amine oxidation to form oxime M41 and sequential sulfation to form M42 or glucuronidation to form M43; (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) monooxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)-acetamide M102. A total of 8 metabolites were detected and structurally elucidated with 4 in plasma (M41, M42, M43, and M35), 7 in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), and 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide). The 2 most abundant circulating metabolites were sulfate (M42) and glucuronide (M43) conjugates of the oxime of dasotraline, accounting for 60.1% and 15.0% of the total plasma radioactivity, respectively; unchanged dasotraline accounted for 8.59%. The oxime M41 accounted for only 0.62% of the total plasma radioactivity and was detected only at early time points. M35 was a minor glucuronide metabolite, undetectable by radioactivity but identified by mass spectrometry. The results demonstrate that dasotraline was slowly absorbed, and extensively metabolized by oxidation and subsequent phase II conjugations. The findings from this study also demonstrated that metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern. Abbreviations ADHD, attention deficit/hyperactivity disorder; AUC, area under the concentrationtime curve; Cmax, maximum concentration; DAT, dopamine transporters; ESI+/−, electrospray ionization positive/negative mode; FDA, Food and Drug Administration; F, feces; FT-MS/MS, Fourier Transform tandem mass spectrometry; HPLC, highperformance liquid chromatography; IC50, 50% inhibitory concentration; ICH, International Conference on Harmonization; ICRP, International Commission on Radiological Protection; IS, internal standard; LC, liquid chromatography; LLQ, lower limit of quantitation; LSC, liquid scintillation counting; mrem, unit of absorbed radiation dose; MS, mass spectrometry; NET, norepinephrine transporters; NME, new molecular entity; PK, pharmacokinetic; P, plasma; SERT, serotonin transporters; SLE, supported liquid extraction; tmax, time to maximum concentration; TRA, total radioactivity; U, urine.
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2017 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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ADME of Dasotraline in Humans
Introduction Dasotraline (SEP-225289; (1R,4S)-4-(3,4-dichlorophenyl)1,2,3,4-tetrahydronaphthalen-1-6 amine) is a diastereomer of the desmethyl metabolite of sertraline, but is not a metabolite of sertraline, nor is it converted to the demethylated metabolite of sertraline in vivo. In contrast to sertraline, dasotraline preferentially occupies dopamine transporters (DAT) relative to serotonin transporters (SERT) (DeLorenzo et al. 2011). Dasotraline is a potent inhibitor of human DAT (dopamine uptake 50% inhibitory concentration [IC50] 3 nmol/L) and norepinephrine transporters (NET; norepinephrine uptake IC50 4 nmol/L), and a weaker inhibitor of human SERT (serotonin uptake IC50 15 nmol/L) (Hopkins et al. 2016). Dasotraline is slowly absorbed by humans (time to maximum concentration [tmax] of 10–12 h) and is characterized by slow elimination (t1/2 of 47–77 h) with nearly dose-proportional maximum concentration (Cmax) and area under the concentration– time curve (AUC) (Hopkins et al. 2016). A double-blind placebo-controlled proof-of-concept study demonstrated the potential for dasotraline to be efficacious for the treatment of subjects with attention deficit/hyperactivity disorder (ADHD) (Koblan et al. 2015). An understanding of the disposition and metabolism of a new molecular entity (NME) in humans is an integral part of its comprehensive safety evaluation. For the vast majority of NMEs, the information on the drug disposition and metabolic fate and exposure of metabolites is required for regulatory approval (Prakash et al. 2012). To examine the absorption, metabolism, and excretion of dasotraline in humans, a single oral dose of radiolabeled [14C]-dasotraline was administered to 8 healthy male volunteers to follow the recovery of radioactivity over time (mass balance) in excreted urine and feces; meanwhile, radio profiling and the most advanced mass spectrometry (MS) technology OrbitrapTM MS, which has been widely used for unknown structure identification (Liang et al. 2011; Zhu et al. 2011; Cho et al. 2012), were employed to search for and elucidate the molecular structure of specific metabolites of dasotraline in human plasma, and excreted urine and fecal samples.
and stored at 20°C. Unlabeled dasotraline hydrochloride was synthesized from (S)-tetralone at Siegfried LLC (Pennsville, NJ) and provided by Sunovion Pharmaceuticals, Inc. (Marlborough, MA). The structure of [14C]dasotraline is shown in Figure 1. A complete list of reference standards that were used to assist metabolite searching and structure elucidation are given in Table 1. All of these reference materials were synthesized and purified by in-house chemists at Sunovion Pharmaceuticals, Inc. and were stored at ambient temperature. Other chemical reagents such as formic acid, ammonium formate, acetic acid, and ammonium hydroxide were purchased from J.T. Baker (Center Valley, PA). Acetonitrile and methanol were obtained from EMD Science (Phoenix, AZ). Water used in this study was produced on site with a NANOPure II (Barnstead Co.) water purification system. Liquid scintillation cocktails were obtained from R. J. Harvey Instrument Corporation (Hillsdale, NJ) or MP Biomedical (Solon, OH). Carbo-Sorb and Perma Fluor Model 307 from PerkinElmer (Akron, OH) were used for oxidizing samples. ACS aqueous counting scintillant was purchased from Amersham Biosciences (Piscataway, NJ).
Clinical study and sample collection Eight healthy male adult volunteers were administered a single oral dose of a solution containing 8 mg/150 lCi [14C]-dasotraline (actual dose was found to be close to 6 mg/110 lCi [14C]-dasotraline), in an open-label clinical study. The 8 mg dose was selected as a clinically relevant and well-tolerated dose level with sufficient and lasting circulating drug concentrations to ensure the ability to detect measurable levels of dasotraline and its metabolites. Actual doses, as detailed in Table 2, were below the target dose due to the high levels of residual radioactivity measured in the dose vials. All subjects received similar dose concentrations with a mean dose of 6.08 mg/110 lCi of [14C]-dasotraline. This unintentional negative bias in dose (~25% lower than the target dose) given to study subject should not cause any safety concern or affect the study NH2
Materials, Study Conduct and Methods Chemicals, radiolabeled test article and reference standards The test articles were manufactured according to the guidelines of the Good Manufacturing Practices. [14C]Dasotraline hydrochloride was synthesized in 10 steps from [14C]-succinic anhydride by Selcia Ltd (Essex, UK)
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Cl Cl Figure 1. Chemical structure of [14C]-dasotraline (SEP-225289). *, 14 C-labeled
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Y.-L. Chen et al.
ADME of Dasotraline in Humans
Table 1. Information of reference standards.
Reference Standard Dasotraline (SEP-225289)
Monoisotopic Mass (Da) 35 Cl isotope mass
Structure NH 2
Rt (min)
291.058
29.81
306.021
62.81
347.048
63.01
303.058
27.21
333.069
58.41
290.027
63.11
305.0347
47.52
Cl Cl
SEP-227391
O OH
Cl Cl
(S)-Oxime Acetate
NOAc
Cl Cl
Sertraline Imine
N
Cl Cl
(1R,4S)-Acetamide (M102)
NHAc
Cl Cl
(S)-Tetralone
O
Cl Cl
SEP-0380096 (SEP-225289-oxime, M41)
NOH
Cl Cl
1
Retention time was obtained using HPLC method 1. Retention time was obtained using HPLC method 2.
2
conduct and data interpretation. The methodology for calculation of radiation dosimetry is based on publications from the Oak Ridge National Laboratory (Snyder et al. 1975), the International Commission on Radiological Protection (ICRP Publication 30 (Part 1) 1979; ICRP publication 89 2002), the Society of Nuclear Medicine (Weber et al. 1989), and the US Food and Drug Administration (1999). Selection of the total dose of radioactivity was based on dosimetry calculations using the quantitative whole body autoradiography data obtained from Long Evans (partially pigmented) male rats following a single oral administration of 2 mg/kg [14C]-dasotraline. Based on the data from the rat study, it was calculated that the overall exposure for a 70-kg man, receiving a single oral dose of 150 lCi (5.6 MBq) [14C]-dasotraline was 12.2 mrem (122 lSv), which was just approximately 0.407% of the allowable 3000 mrem exposure limit established by the FDA (U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Biologics Evaluation and Research 2010). This dose level would not be expected to represent a significant radiation exposure risk in man. The study was approved by an institutional review board at the investigational site and was conducted in accordance with the International Conference on Harmonization (ICH) Good Clinical Practices guidelines and with the ethical principles of the Declaration of Helsinki. Prior to study entry, all subjects reviewed and signed an informed consent form explaining study procedures and potential risks. The 8 volunteers enrolled in the study were aged 21–34 years old. One subject was withdrawn after the 168 h sample collection due to a family emergency. This subject’s samples were not used for sample pooling for metabolite profiling, identification, or pharmacokinetic analysis. During the clinical study, serial blood samples for plasma were collected at predose, at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 120, 168, 216, 336, 384, 480 h postdose, and at every 24-h time points until discharge (8 mL of blood per time point; a total quantity of 272 mL blood was collected if a subject was in study for 35 days); serial urine samples were collected at predose (−12–0 h), and for 0–6, 6–12, 12–24 h postdose, then at 24-h intervals until the subject was discharged; fecal samples, including toilet tissue were collected predose and at 24-h intervals postdose until the subject was discharged. The study duration was between 23–35 days. Subjects were confined to the clinic for a minimum of 23 days (Day 1 to Day 22) until the criteria for discharge were met, that is, ≥90% of the radioactive dose was recovered from the sum of urine and feces; and plasma, urine, and fecal radioactivity levels were 90% of the total recovered radioactivity was excreted from humans over the first 16 days (data not shown). A total of 2.51% and 4.33% of unchanged dasotraline were excreted through urine and feces, respectively, indicating that the majority of the dosed dasotraline was excreted through extensive metabolism. The mass balance results also demonstrated that the dasotraline was slowly, but highly, absorbed when it was orally administrated to humans with dasotraline median tmax at 10 h postdose.
Radioactivity distribution in blood, plasma and red blood cells As reported in the results section and in Figure 3, mean maximum radioactivity concentrations in blood, red blood cells, and plasma were 12.4 ng-Eq/mL at 72 h, 4.24 ng-Eq/mL at 10 h, and 20.7 ng-Eq/mL at 48 h, respectively. Both the concentration ratios of blood/plasma and red blood cell/plasma indicate a low association of radioactivity with blood cells
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In the plasma samples, radio-chromatograms obtained with the HPLC method 1 are shown in Figure 5 with 4-h (5A), 24-h (5B), 120-h (5C) time point pools from 7 subjects and the AUC-pool created from one subject’s 0–504 h (Subject 004) plasma (5D). Four peaks were detected from plasma metabolite profiling with respective retention times of ~31, ~41, ~43, and ~63 min. These four peaks were identified as unchanged dasotraline, a sulfate of the oxime of dasotraline, a glucuronide of the oxime of dasotraline, and the oxime of dasotraline, respectively. The observation of sulfonation of monoaryl oximes was reported by Mangold et al. (1986, 1989). The structures of dasotraline and the oxime of dasotraline were identified by comparing these unknown peaks with their reference standards’ retention times, molecular weights, and the MS/MS fragmentation patterns. The structures of the oxime-sulfate and the oxime-glucuronide were identified by interpreting their accurate mass measurements, MS/MS patterns, and comparing with the products obtained from the in vitro incubation (at 37°C for 4 h) of the dasotraline-oxime standard with human liver microsomes to generate the glucuronide (in 0.1 mol/L phosphate buffer pH 7.4, containing 50 lmol/L of the oxime of dasotraline, 0.025 mg/mL alamethicin, 4 mmol/L magnesium chloride, 1 mmol/L EDTA, 1 mg/mL microsome protein, 1.0 mmol/L uridinediphosphate-glucuronic acid, i.e., UDPGA) and human liver cytosol fractions to generate the sulfate (in 0.1 mol/L phosphate buffer pH 7.4 containing 50 lmol/L of the oxime of dasotraline, 4 mmol/L magnesium chloride, 1 mmol/L EDTA, liver cytosol protein (1 mg/mL), adenosine 30 -phosphate 50 -phosphosulfate, 1.0 mmol/L). Accurate mass measurement results of these identified peaks are presented in Table 8. Differences between the measured mass and its theoretical mass were 2.05, 0, 3.39, and 3.11 ppm for dasotraline, M41, M42, and M43, respectively, indicating a perfect match of their assigned structures. The MS/MS spectral data of M41, M42, and M43, as shown in Figure 7–9 fully support their assigned structures. In the pooled urine sample from 0 to 432 h across all 7 subjects, the radio-chromatograms obtained with the HPLC method 1 is given in Figure 6A. Peaks of multiple metabolites were detected including the parent and 3 identified ones in plasma, and 4 other metabolites with respective retention times of ~30, ~34, ~40, and ~63 min. These four newly identified structures were assigned as M31A, M32, M35, and (S)-tetralone, respectively. Here, M31A
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Y.-L. Chen et al.
ADME of Dasotraline in Humans
(A)
(B)
Figure 11. Arithmetic mean plasma concentration profiles of dasotraline (ng/mL) and TRA in plasma (ng-Eq/mL) in linear (A) and semi-logarithmic scale (B).
Table 5. PK parameters for unchanged dasotraline and TRA in plasma, Geometric Mean (CV%).
PK Parameter Cmax3 (ng/mL) tmax3 Median (Min, Max) (h) AUC0–last (ng*h/mL) AUC0–inf (ng*h/mL) t1/2 (h) kZ (h1) CL/F (L/h) Vz/F (L) RCmax drug/rd3
Dasotraline in Plasma1 (N = 7) 2.02 (18.4) 10.0 (8.00, 24.0) 243 248 61.3 0.0113 32.3 2860 NA
(37.6) (36.6) (34.2) (34.2) (36.6) (21.5)
TRA in Plasma2 (N = 7) 20.6 (32.0) 60.0 (24.0, 120) 5220 5840 114 0.00611 NA NA 0.0982
(43.7) (36.6) (32.2) (32.2)
(33.9)
1
Measured by a validated quantitative LC-MS/MS method. Measured by LSC. Units for TRA Cmax and AUC are ng-Eq/mL and ng-Eq*h/mL, respectively. 3 N = 8 for Cmax, tmax, and RCmax drug/rd. only. 2
and M32 were a pair of diastereomers and both were glucuronides of hydroxylated dasotraline. M35 was the glucuronide of N-hydroxylated dasotraline and M35 was also later detected in plasma by MS though was not detectable by radioprofiling. The peak at retention time at ~63– 64 min was identified as (S)-tetralone by comparing with its reference standard. This peak was later confirmed to contain M41 co-elution while the HPLC method 1 conditions were used. As presented in Table 8, mass differences between accurate mass measurements and their assigned
structures were 2.40, 4.00, 2.27, and 1.45 ppm for M31A, M32, M35, and (S)-tetralone, respectively. The MS/MS fragmentation patterns of identified metabolites were in good accordance with their corresponding assigned structures. As M35 was also detected in plasma by MS, its structure assignment was supported by MS/ MS spectral data and proposed fragmentation pathways, shown in Figure 10. A few minor peaks were also detected but structures were not identified. In the pooled fecal sample from 0 to 504 h across all 7 subjects, radio-chromatograms obtained with the HPLC method 1 are given in Figure 6B. Besides the unchanged dasotraline peak (~31 min), a dominant peak at retention time of ~63–64 min was detected. In addition, a couple of minor peaks (~28 min and ~61 min) were also detected in fecal samples. The peak at ~61 min was assigned as M102 and its structure was identified as a form of (1R,4S)-acetamide via comparison with its reference material. The dominant peak fraction from the HPLC method 1 was further investigated with an improved chromatographic gradient procedure. As shown in Figure 6C, with the HPLC method 2, it was able to successfully separate M41 (retention time of 47.5 min) from (S)-tetralone (retention time of 46.5 min). This condition also confirmed both M41 and (S)-tetralone existed in urine samples. In feces, (S)-tetralone was about 80% and M41 about 20% in the co-eluted peak from the HPLC method 1. M41 was unambiguously identified as the oxime of dasotraline by comparing retention time, accurate mass measurement, and MS/MS fragmentation pattern with the reference standard.
Proposed metabolic pathways According to the mass balance data and metabolite profiling and structure elucidation result, dasotraline was extensively metabolized by oxidation and subsequent phase II conjugation. A total of 8 metabolites were structurally identified, 7 of which existed in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide), and 4 were circulating in plasma (M41, M42, M43, and M35; Table 4). The proposed metabolic pathways were described in Figure 4. The major biotransformation pathways of dasotraline involved: (1) amine oxidation to form M41 (dasotraline-oxime) and sequential sulfation to form M42 (a sulfate conjugate) or glucuronidation to form M43 (a glucuronide conjugate); (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) mono-oxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)acetamide (M102).
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
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Table 6. Percent AUC values of dasotraline-derived metabolites relative to TRA AUC in the AUC-pooled plasma samples from individual subjects. Mean %AUC
%AUC in individual subjects Analyte
002
003
004
005
006
007
008
(N = 7)
1 (Dasotraline) 2 (M42) 3 (M43) 4 (M41) AUCTRA (ng-Eq*h/mL)
3.83 69.7 4.88 2.44 5539
6.33 53.6 23.2 ND 10,498
9.20 59.0 14.9 1.92 5201
7.95 54.6 20.8 ND 3871
8.53 59.7 14.2 ND 3087
18.5 57.9 11.6 ND 3846
5.79 65.9 15.2 ND 7248
8.59 60.1 15.0 0.62 5613
Table 7. Exploratory PK parameters of TRA, unchanged dasotraline and major metabolites in plasma based on selected time points pooling across 7 subjects at (predose), 4, 12, 24, 48, 120, 216, and 384 h (1-h and 504-h time points were not used for this measurement).
Analyte
AUC0–384 h (ng-Eq*h/mL)
TRA Dasotraline M42 M43
5460 342 3280 1020
%AUC1
Cmax (ng-Eq/mL)
tmax (h)
t1/2 (h)
100 6.28 60.1 18.6
20.7 2.96 12.1 3.76
48 12 48 120
135 89.6 138 1492
1
Concentration (ng-Eq/mL)
Relative to AUCTRA (assume AUCTRA as 100%). As only limited time points were used, AUC values were just for reference. The PK profiles are presented in Figure 12. 2 The data at tmax was also used for estimation of t½ due to insufficient data points.
25 TRA Dasotraline M42 M43
20 15 10 5 0 0
100
200
300
400
Time postdose (h) Figure 12. Mean plasma (pooled same time points across 7 subjects) concentration (ng-Eq/mL) – time profiles of TRA, dasotraline, sulfate of dasotraline-oxime (M42) and glucuronide of dasotraline-oxime (M43).
Circulating metabolites in plasma and their PK characteristics The exploratory PKs of the circulating metabolites was calculated based on the selected time points pooling across 7 subjects at 4, 12, 24, 48, 120, 216, and 384 h and
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S.D.
4.74 5.84 5.99 1.07 2558
are presented in Table 7. The mean radioactivity Cmax and AUC values showed that 2 phase II conjugated metabolites M42 and M43 were the major circulating components, in which AUC of M42, the sulfate conjugate, is the most abundant one and is about tenfold higher than that of the unchanged dasotraline along with an approximate twofold difference in elimination t1/2. There was no evidence that the back conversion of these highly abundant phase II conjugates would contribute to the long half-life of dasotraline in plasma as the oxime of dasotraline had an extremely low exposure and short half-life in circulation. According to MIST guidance (U.S. Food and Drug Administration 2008), phase II conjugates generally render a compound more water soluble and pharmacologically inactive; thus, further evaluation is not warranted. The only oxidative metabolite detected in plasma was the oxime of dasotraline (M41), accounted for a 0.62% of the plasma TRA and was detected at early time points (~4 h) from only 2 of 7 completed subjects and quickly disappeared likely due to subsequent conjugations. As M41 is a very minor metabolite with a short half-life, it is not expected to accumulate and cause a safety concern. According to ICH guidance (ICH 2010), nonclinical characterization of a human metabolite(s) is only warranted when that metabolite(s) is observed at exposures >10% of total drug-related exposure and at significantly greater levels in humans than the maximum exposure seen in the toxicity studies. Therefore, the further characterization of M41 for safety concern will not be necessary. In conclusion, dasotraline is well but slowly absorbed, and extensively metabolized after a single oral dose of [14C]-dasotraline in healthy male adults. The TRA recovery is >90% in urine and feces over the course of 35 study days and the main excretion is through urine. In human plasma, unchanged dasotraline accounted for 8.59% of the TRA in AUC-pooled plasma samples. The 2 most abundant metabolites were sulfate and glucuronide conjugates of the oxime of dasotraline, accounting for 60.1 and 15.0% of TRA in pooled plasma, respectively.
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Y.-L. Chen et al.
ADME of Dasotraline in Humans
Table 8. Accurate mass measurements of dasotraline and its metabolites.
Compound Code Dasotraline (S)-Tetralone M31A M32 M35 M41 M42 M43 (1R,4S)-Acetamide (M102)
m/z [M+H]+
Elemental Composition
Theoretical mass (based on 35Cl)
Measured Mass
Mass Difference (ppm)
292 291 500 (NH4+- adduct) 500 (NH4+- adduct) 484 306 384 (M-1, negative) 482 344
C16H15Cl2N C16H12Cl2O C22H24Cl2O8N
292.0654 291.0338 500.0874
292.0648 291.0338 500.0862
2.05 0 2.40
C22H24Cl2O8N
500.0874
500.0854
4.00
C22H23Cl2NO7 C16H13Cl2NO C16H12Cl2NO4S
484.0924 306.0447 383.9870
484.0913 306.0447 383.9857
2.27 0 3.39
C22H21Cl2NO7 C18H18Cl2NO
482.0768 344.0760
482.0753 344.0755
3.11 1.45
The oxime of dasotraline was at the minimal level (accounting for 0.62% of TRA in pooled plasma) and only detectable in two subjects’ early time points (4 h) and not detectable afterwards. Those two dominant metabolites, that is, sulfate and glucuronide of dasotraline-oxime are phase II conjugates that are known to be pharmacologically inactive and not toxic. Per MIST guidance, these inactive phase II conjugates do not require for further evaluation. The findings from the current trial such as a consistent high recovery (>90%), major metabolic pathways (oxidation followed by conjugations), main excretion route (through urine), and unambiguous identification of major circulating metabolites (oxime-sulfate and oxime-glucuronide) provided rich knowledge and insight to help understand the mechanism of metabolism, PK, and safety of dasotraline. According to the findings from this study, metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern.
Acknowledgements The authors are grateful to the Covance CRU (Madison, WI, USA) for conducting the clinical aspects of this study, to the metabolism group of Covance Laboratories, Inc. (Madison, WI, USA) for the radioactivity measurements, to the Xenobiotics Laboratories, Inc. (Plainsboro, NJ, USA) for metabolite profiling and structure elucidation, and to the PPD Laboratories (Middleton, WI, USA) for the measurement of unchanged plasma dasotraline levels. We acknowledge Dawn L. Lee of FireKite, an Ashfield company, part of UDG Healthcare plc, for editorial assistance with the final version of the manuscript for submission, which was funded by Sunovion Pharmaceuticals, Inc.
Authorship Contributions Participated in research design: Chen, Lin, Skende, Hopkins, Koblan. Conducted experiments: Wills, Yi, Wang. Contributed new reagents: Wilkinson. Performed data analysis: Chen, Skende, Lin, Wilkinson, Hopkins. Wrote or contributed to the writing of the manuscript: All Authors (Chen, Skende, Lin, Yi, Wang, Wills, Wilkinson, Koblan, and Hopkins).
Disclosures YLC, ES, JL, HSW, KSK, and SCH are employees of Sunovion Pharmaceuticals, Inc. YY and PLW are employees of XenoBiotic Laboratories, Inc. SW is an employee of Covance Laboratories, Inc.
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Liang Y, Wang G, Xie L, Sheng L (2011). Recent development in liquid chromatography/mass spectrometry and emerging technologies for metabolite identification. Curr Drug Metab 12: 329–344. Mangold JB, Mangold BLK, Spina A (1986). Rat liver aryl sulfotransferase-catalyzed sulfation and rearrangement of 9fluorenone oxime. Biochim Biophys Acta 874: 37–43. Mangold JB, Spina A, McCann DJ (1989). Sulfation of monoand diaryl oximes by aryl sulfotransferase isozymes. Biochim Biophys Acta 991: 453–458. Prakash C, Li Z, Orlandi C, Klunk L (2012). Assessment of exposure of metabolites in preclinical species and humans at steady state from the single-dose radiolabeled absorption, distribution, metabolism, and excretion studies: a case study. Drug Metab Dispos 40: 1308–1320. Publication ICRP, 30 (Part 1), (1979). Limits for the Intake of Radionuclides by Workers. Ann ICRP 2(3–4).
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Snyder WS, Ford MR, Warner GG, Watson SB (1975). Absorbed Dose Per Unit Cumulated Activity for Selected Radionuclides and Organs. Nm/mird Pamphlet No. 11, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
U.S. Food and Drug Administration (2008). Guidance for Industry – Safety Testing of Drug Metabolites [MIST]. Available at: http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/guidances/uc m079266.pdf (accessed 1 August 2016). U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Biologics Evaluation and Research (2010). Guidance for industry and researchers: the radioactive drug research committee: human research without an investigational new drug application. Available at: http://www.fda.gov/downloads/Drugs/%e2%80%a6/Guidances/ UCM163892.pdf (accessed 1 August 2016). Weber DA, Eckerman KF, Dillman LT (1989). MIRD “Radionuclide data and decay schemes”. Society of Nuclear Medicine, New York, New York. Zhu M, Zhang H, Humphreys WG (2011). Drug metabolite profiling and identification by high-resolution mass spectrometry. J Biol Chem 286: 25419–25425.
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Absorption, distribution, metabolism, and excretion of [14C]-dasotraline in humans Yu-Luan Chen1, Estela Skende1, Jing Lin1, Yijun Yi2, Peter L. Wang2, Sarah Wills3, H. Scott Wilkinson1, Kenneth S. Koblan1 & Seth C. Hopkins1 1
Sunovion Pharmaceuticals, Inc., Marlborough, Massachusetts XenoBiotic Laboratories, Inc., Plainsboro, New Jersey 3 Covance Laboratories, Inc., Madison, Wisconsin 2
Keywords Absorption, dasotraline, distribution, excretion, glucuronidation, mass spectrometry, metabolite identification, pharmacokinetics, phase II metabolism, sulfate conjugation Correspondence Yu-Luan Chen, Sunovion Pharmaceuticals, Inc., 84 Waterford Drive, Marlborough, MA 01752. Tel: +1 508 787 4286; Fax: +1 508 357 7859; E-mail: [email protected] Funding Information This work was funded by Sunovion Pharmaceuticals, Inc. Received: 10 August 2016; Revised: 3 November 2016; Accepted: 5 November 2016 Pharma Res Per, 5(1), 2017, e00281, doi: 10.1002/prp2.281 doi: 10.1002/prp2.281
Abstract Dasotraline is a dopamine and norepinephrine reuptake inhibitor, and the early clinical trials show a slow absorption and long elimination half-life. To investigate the absorption, distribution, metabolism, and excretion of dasotraline in humans, a single dose of [14C]-dasotraline was administered to eight healthy male adult volunteers. At 35 days, 90.7% of the dosed radioactivity was recovered in the urine (68.3%) and feces (22.4%). The major metabolic pathways involved were: (1) amine oxidation to form oxime M41 and sequential sulfation to form M42 or glucuronidation to form M43; (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) monooxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)-acetamide M102. A total of 8 metabolites were detected and structurally elucidated with 4 in plasma (M41, M42, M43, and M35), 7 in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), and 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide). The 2 most abundant circulating metabolites were sulfate (M42) and glucuronide (M43) conjugates of the oxime of dasotraline, accounting for 60.1% and 15.0% of the total plasma radioactivity, respectively; unchanged dasotraline accounted for 8.59%. The oxime M41 accounted for only 0.62% of the total plasma radioactivity and was detected only at early time points. M35 was a minor glucuronide metabolite, undetectable by radioactivity but identified by mass spectrometry. The results demonstrate that dasotraline was slowly absorbed, and extensively metabolized by oxidation and subsequent phase II conjugations. The findings from this study also demonstrated that metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern. Abbreviations ADHD, attention deficit/hyperactivity disorder; AUC, area under the concentrationtime curve; Cmax, maximum concentration; DAT, dopamine transporters; ESI+/−, electrospray ionization positive/negative mode; FDA, Food and Drug Administration; F, feces; FT-MS/MS, Fourier Transform tandem mass spectrometry; HPLC, highperformance liquid chromatography; IC50, 50% inhibitory concentration; ICH, International Conference on Harmonization; ICRP, International Commission on Radiological Protection; IS, internal standard; LC, liquid chromatography; LLQ, lower limit of quantitation; LSC, liquid scintillation counting; mrem, unit of absorbed radiation dose; MS, mass spectrometry; NET, norepinephrine transporters; NME, new molecular entity; PK, pharmacokinetic; P, plasma; SERT, serotonin transporters; SLE, supported liquid extraction; tmax, time to maximum concentration; TRA, total radioactivity; U, urine.
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, 2017 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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ADME of Dasotraline in Humans
Introduction Dasotraline (SEP-225289; (1R,4S)-4-(3,4-dichlorophenyl)1,2,3,4-tetrahydronaphthalen-1-6 amine) is a diastereomer of the desmethyl metabolite of sertraline, but is not a metabolite of sertraline, nor is it converted to the demethylated metabolite of sertraline in vivo. In contrast to sertraline, dasotraline preferentially occupies dopamine transporters (DAT) relative to serotonin transporters (SERT) (DeLorenzo et al. 2011). Dasotraline is a potent inhibitor of human DAT (dopamine uptake 50% inhibitory concentration [IC50] 3 nmol/L) and norepinephrine transporters (NET; norepinephrine uptake IC50 4 nmol/L), and a weaker inhibitor of human SERT (serotonin uptake IC50 15 nmol/L) (Hopkins et al. 2016). Dasotraline is slowly absorbed by humans (time to maximum concentration [tmax] of 10–12 h) and is characterized by slow elimination (t1/2 of 47–77 h) with nearly dose-proportional maximum concentration (Cmax) and area under the concentration– time curve (AUC) (Hopkins et al. 2016). A double-blind placebo-controlled proof-of-concept study demonstrated the potential for dasotraline to be efficacious for the treatment of subjects with attention deficit/hyperactivity disorder (ADHD) (Koblan et al. 2015). An understanding of the disposition and metabolism of a new molecular entity (NME) in humans is an integral part of its comprehensive safety evaluation. For the vast majority of NMEs, the information on the drug disposition and metabolic fate and exposure of metabolites is required for regulatory approval (Prakash et al. 2012). To examine the absorption, metabolism, and excretion of dasotraline in humans, a single oral dose of radiolabeled [14C]-dasotraline was administered to 8 healthy male volunteers to follow the recovery of radioactivity over time (mass balance) in excreted urine and feces; meanwhile, radio profiling and the most advanced mass spectrometry (MS) technology OrbitrapTM MS, which has been widely used for unknown structure identification (Liang et al. 2011; Zhu et al. 2011; Cho et al. 2012), were employed to search for and elucidate the molecular structure of specific metabolites of dasotraline in human plasma, and excreted urine and fecal samples.
and stored at 20°C. Unlabeled dasotraline hydrochloride was synthesized from (S)-tetralone at Siegfried LLC (Pennsville, NJ) and provided by Sunovion Pharmaceuticals, Inc. (Marlborough, MA). The structure of [14C]dasotraline is shown in Figure 1. A complete list of reference standards that were used to assist metabolite searching and structure elucidation are given in Table 1. All of these reference materials were synthesized and purified by in-house chemists at Sunovion Pharmaceuticals, Inc. and were stored at ambient temperature. Other chemical reagents such as formic acid, ammonium formate, acetic acid, and ammonium hydroxide were purchased from J.T. Baker (Center Valley, PA). Acetonitrile and methanol were obtained from EMD Science (Phoenix, AZ). Water used in this study was produced on site with a NANOPure II (Barnstead Co.) water purification system. Liquid scintillation cocktails were obtained from R. J. Harvey Instrument Corporation (Hillsdale, NJ) or MP Biomedical (Solon, OH). Carbo-Sorb and Perma Fluor Model 307 from PerkinElmer (Akron, OH) were used for oxidizing samples. ACS aqueous counting scintillant was purchased from Amersham Biosciences (Piscataway, NJ).
Clinical study and sample collection Eight healthy male adult volunteers were administered a single oral dose of a solution containing 8 mg/150 lCi [14C]-dasotraline (actual dose was found to be close to 6 mg/110 lCi [14C]-dasotraline), in an open-label clinical study. The 8 mg dose was selected as a clinically relevant and well-tolerated dose level with sufficient and lasting circulating drug concentrations to ensure the ability to detect measurable levels of dasotraline and its metabolites. Actual doses, as detailed in Table 2, were below the target dose due to the high levels of residual radioactivity measured in the dose vials. All subjects received similar dose concentrations with a mean dose of 6.08 mg/110 lCi of [14C]-dasotraline. This unintentional negative bias in dose (~25% lower than the target dose) given to study subject should not cause any safety concern or affect the study NH2
Materials, Study Conduct and Methods Chemicals, radiolabeled test article and reference standards The test articles were manufactured according to the guidelines of the Good Manufacturing Practices. [14C]Dasotraline hydrochloride was synthesized in 10 steps from [14C]-succinic anhydride by Selcia Ltd (Essex, UK)
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Cl Cl Figure 1. Chemical structure of [14C]-dasotraline (SEP-225289). *, 14 C-labeled
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Y.-L. Chen et al.
ADME of Dasotraline in Humans
Table 1. Information of reference standards.
Reference Standard Dasotraline (SEP-225289)
Monoisotopic Mass (Da) 35 Cl isotope mass
Structure NH 2
Rt (min)
291.058
29.81
306.021
62.81
347.048
63.01
303.058
27.21
333.069
58.41
290.027
63.11
305.0347
47.52
Cl Cl
SEP-227391
O OH
Cl Cl
(S)-Oxime Acetate
NOAc
Cl Cl
Sertraline Imine
N
Cl Cl
(1R,4S)-Acetamide (M102)
NHAc
Cl Cl
(S)-Tetralone
O
Cl Cl
SEP-0380096 (SEP-225289-oxime, M41)
NOH
Cl Cl
1
Retention time was obtained using HPLC method 1. Retention time was obtained using HPLC method 2.
2
conduct and data interpretation. The methodology for calculation of radiation dosimetry is based on publications from the Oak Ridge National Laboratory (Snyder et al. 1975), the International Commission on Radiological Protection (ICRP Publication 30 (Part 1) 1979; ICRP publication 89 2002), the Society of Nuclear Medicine (Weber et al. 1989), and the US Food and Drug Administration (1999). Selection of the total dose of radioactivity was based on dosimetry calculations using the quantitative whole body autoradiography data obtained from Long Evans (partially pigmented) male rats following a single oral administration of 2 mg/kg [14C]-dasotraline. Based on the data from the rat study, it was calculated that the overall exposure for a 70-kg man, receiving a single oral dose of 150 lCi (5.6 MBq) [14C]-dasotraline was 12.2 mrem (122 lSv), which was just approximately 0.407% of the allowable 3000 mrem exposure limit established by the FDA (U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Biologics Evaluation and Research 2010). This dose level would not be expected to represent a significant radiation exposure risk in man. The study was approved by an institutional review board at the investigational site and was conducted in accordance with the International Conference on Harmonization (ICH) Good Clinical Practices guidelines and with the ethical principles of the Declaration of Helsinki. Prior to study entry, all subjects reviewed and signed an informed consent form explaining study procedures and potential risks. The 8 volunteers enrolled in the study were aged 21–34 years old. One subject was withdrawn after the 168 h sample collection due to a family emergency. This subject’s samples were not used for sample pooling for metabolite profiling, identification, or pharmacokinetic analysis. During the clinical study, serial blood samples for plasma were collected at predose, at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 120, 168, 216, 336, 384, 480 h postdose, and at every 24-h time points until discharge (8 mL of blood per time point; a total quantity of 272 mL blood was collected if a subject was in study for 35 days); serial urine samples were collected at predose (−12–0 h), and for 0–6, 6–12, 12–24 h postdose, then at 24-h intervals until the subject was discharged; fecal samples, including toilet tissue were collected predose and at 24-h intervals postdose until the subject was discharged. The study duration was between 23–35 days. Subjects were confined to the clinic for a minimum of 23 days (Day 1 to Day 22) until the criteria for discharge were met, that is, ≥90% of the radioactive dose was recovered from the sum of urine and feces; and plasma, urine, and fecal radioactivity levels were 90% of the total recovered radioactivity was excreted from humans over the first 16 days (data not shown). A total of 2.51% and 4.33% of unchanged dasotraline were excreted through urine and feces, respectively, indicating that the majority of the dosed dasotraline was excreted through extensive metabolism. The mass balance results also demonstrated that the dasotraline was slowly, but highly, absorbed when it was orally administrated to humans with dasotraline median tmax at 10 h postdose.
Radioactivity distribution in blood, plasma and red blood cells As reported in the results section and in Figure 3, mean maximum radioactivity concentrations in blood, red blood cells, and plasma were 12.4 ng-Eq/mL at 72 h, 4.24 ng-Eq/mL at 10 h, and 20.7 ng-Eq/mL at 48 h, respectively. Both the concentration ratios of blood/plasma and red blood cell/plasma indicate a low association of radioactivity with blood cells
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In the plasma samples, radio-chromatograms obtained with the HPLC method 1 are shown in Figure 5 with 4-h (5A), 24-h (5B), 120-h (5C) time point pools from 7 subjects and the AUC-pool created from one subject’s 0–504 h (Subject 004) plasma (5D). Four peaks were detected from plasma metabolite profiling with respective retention times of ~31, ~41, ~43, and ~63 min. These four peaks were identified as unchanged dasotraline, a sulfate of the oxime of dasotraline, a glucuronide of the oxime of dasotraline, and the oxime of dasotraline, respectively. The observation of sulfonation of monoaryl oximes was reported by Mangold et al. (1986, 1989). The structures of dasotraline and the oxime of dasotraline were identified by comparing these unknown peaks with their reference standards’ retention times, molecular weights, and the MS/MS fragmentation patterns. The structures of the oxime-sulfate and the oxime-glucuronide were identified by interpreting their accurate mass measurements, MS/MS patterns, and comparing with the products obtained from the in vitro incubation (at 37°C for 4 h) of the dasotraline-oxime standard with human liver microsomes to generate the glucuronide (in 0.1 mol/L phosphate buffer pH 7.4, containing 50 lmol/L of the oxime of dasotraline, 0.025 mg/mL alamethicin, 4 mmol/L magnesium chloride, 1 mmol/L EDTA, 1 mg/mL microsome protein, 1.0 mmol/L uridinediphosphate-glucuronic acid, i.e., UDPGA) and human liver cytosol fractions to generate the sulfate (in 0.1 mol/L phosphate buffer pH 7.4 containing 50 lmol/L of the oxime of dasotraline, 4 mmol/L magnesium chloride, 1 mmol/L EDTA, liver cytosol protein (1 mg/mL), adenosine 30 -phosphate 50 -phosphosulfate, 1.0 mmol/L). Accurate mass measurement results of these identified peaks are presented in Table 8. Differences between the measured mass and its theoretical mass were 2.05, 0, 3.39, and 3.11 ppm for dasotraline, M41, M42, and M43, respectively, indicating a perfect match of their assigned structures. The MS/MS spectral data of M41, M42, and M43, as shown in Figure 7–9 fully support their assigned structures. In the pooled urine sample from 0 to 432 h across all 7 subjects, the radio-chromatograms obtained with the HPLC method 1 is given in Figure 6A. Peaks of multiple metabolites were detected including the parent and 3 identified ones in plasma, and 4 other metabolites with respective retention times of ~30, ~34, ~40, and ~63 min. These four newly identified structures were assigned as M31A, M32, M35, and (S)-tetralone, respectively. Here, M31A
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Y.-L. Chen et al.
ADME of Dasotraline in Humans
(A)
(B)
Figure 11. Arithmetic mean plasma concentration profiles of dasotraline (ng/mL) and TRA in plasma (ng-Eq/mL) in linear (A) and semi-logarithmic scale (B).
Table 5. PK parameters for unchanged dasotraline and TRA in plasma, Geometric Mean (CV%).
PK Parameter Cmax3 (ng/mL) tmax3 Median (Min, Max) (h) AUC0–last (ng*h/mL) AUC0–inf (ng*h/mL) t1/2 (h) kZ (h1) CL/F (L/h) Vz/F (L) RCmax drug/rd3
Dasotraline in Plasma1 (N = 7) 2.02 (18.4) 10.0 (8.00, 24.0) 243 248 61.3 0.0113 32.3 2860 NA
(37.6) (36.6) (34.2) (34.2) (36.6) (21.5)
TRA in Plasma2 (N = 7) 20.6 (32.0) 60.0 (24.0, 120) 5220 5840 114 0.00611 NA NA 0.0982
(43.7) (36.6) (32.2) (32.2)
(33.9)
1
Measured by a validated quantitative LC-MS/MS method. Measured by LSC. Units for TRA Cmax and AUC are ng-Eq/mL and ng-Eq*h/mL, respectively. 3 N = 8 for Cmax, tmax, and RCmax drug/rd. only. 2
and M32 were a pair of diastereomers and both were glucuronides of hydroxylated dasotraline. M35 was the glucuronide of N-hydroxylated dasotraline and M35 was also later detected in plasma by MS though was not detectable by radioprofiling. The peak at retention time at ~63– 64 min was identified as (S)-tetralone by comparing with its reference standard. This peak was later confirmed to contain M41 co-elution while the HPLC method 1 conditions were used. As presented in Table 8, mass differences between accurate mass measurements and their assigned
structures were 2.40, 4.00, 2.27, and 1.45 ppm for M31A, M32, M35, and (S)-tetralone, respectively. The MS/MS fragmentation patterns of identified metabolites were in good accordance with their corresponding assigned structures. As M35 was also detected in plasma by MS, its structure assignment was supported by MS/ MS spectral data and proposed fragmentation pathways, shown in Figure 10. A few minor peaks were also detected but structures were not identified. In the pooled fecal sample from 0 to 504 h across all 7 subjects, radio-chromatograms obtained with the HPLC method 1 are given in Figure 6B. Besides the unchanged dasotraline peak (~31 min), a dominant peak at retention time of ~63–64 min was detected. In addition, a couple of minor peaks (~28 min and ~61 min) were also detected in fecal samples. The peak at ~61 min was assigned as M102 and its structure was identified as a form of (1R,4S)-acetamide via comparison with its reference material. The dominant peak fraction from the HPLC method 1 was further investigated with an improved chromatographic gradient procedure. As shown in Figure 6C, with the HPLC method 2, it was able to successfully separate M41 (retention time of 47.5 min) from (S)-tetralone (retention time of 46.5 min). This condition also confirmed both M41 and (S)-tetralone existed in urine samples. In feces, (S)-tetralone was about 80% and M41 about 20% in the co-eluted peak from the HPLC method 1. M41 was unambiguously identified as the oxime of dasotraline by comparing retention time, accurate mass measurement, and MS/MS fragmentation pattern with the reference standard.
Proposed metabolic pathways According to the mass balance data and metabolite profiling and structure elucidation result, dasotraline was extensively metabolized by oxidation and subsequent phase II conjugation. A total of 8 metabolites were structurally identified, 7 of which existed in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide), and 4 were circulating in plasma (M41, M42, M43, and M35; Table 4). The proposed metabolic pathways were described in Figure 4. The major biotransformation pathways of dasotraline involved: (1) amine oxidation to form M41 (dasotraline-oxime) and sequential sulfation to form M42 (a sulfate conjugate) or glucuronidation to form M43 (a glucuronide conjugate); (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) mono-oxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)acetamide (M102).
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
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ADME of Dasotraline in Humans
Table 6. Percent AUC values of dasotraline-derived metabolites relative to TRA AUC in the AUC-pooled plasma samples from individual subjects. Mean %AUC
%AUC in individual subjects Analyte
002
003
004
005
006
007
008
(N = 7)
1 (Dasotraline) 2 (M42) 3 (M43) 4 (M41) AUCTRA (ng-Eq*h/mL)
3.83 69.7 4.88 2.44 5539
6.33 53.6 23.2 ND 10,498
9.20 59.0 14.9 1.92 5201
7.95 54.6 20.8 ND 3871
8.53 59.7 14.2 ND 3087
18.5 57.9 11.6 ND 3846
5.79 65.9 15.2 ND 7248
8.59 60.1 15.0 0.62 5613
Table 7. Exploratory PK parameters of TRA, unchanged dasotraline and major metabolites in plasma based on selected time points pooling across 7 subjects at (predose), 4, 12, 24, 48, 120, 216, and 384 h (1-h and 504-h time points were not used for this measurement).
Analyte
AUC0–384 h (ng-Eq*h/mL)
TRA Dasotraline M42 M43
5460 342 3280 1020
%AUC1
Cmax (ng-Eq/mL)
tmax (h)
t1/2 (h)
100 6.28 60.1 18.6
20.7 2.96 12.1 3.76
48 12 48 120
135 89.6 138 1492
1
Concentration (ng-Eq/mL)
Relative to AUCTRA (assume AUCTRA as 100%). As only limited time points were used, AUC values were just for reference. The PK profiles are presented in Figure 12. 2 The data at tmax was also used for estimation of t½ due to insufficient data points.
25 TRA Dasotraline M42 M43
20 15 10 5 0 0
100
200
300
400
Time postdose (h) Figure 12. Mean plasma (pooled same time points across 7 subjects) concentration (ng-Eq/mL) – time profiles of TRA, dasotraline, sulfate of dasotraline-oxime (M42) and glucuronide of dasotraline-oxime (M43).
Circulating metabolites in plasma and their PK characteristics The exploratory PKs of the circulating metabolites was calculated based on the selected time points pooling across 7 subjects at 4, 12, 24, 48, 120, 216, and 384 h and
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S.D.
4.74 5.84 5.99 1.07 2558
are presented in Table 7. The mean radioactivity Cmax and AUC values showed that 2 phase II conjugated metabolites M42 and M43 were the major circulating components, in which AUC of M42, the sulfate conjugate, is the most abundant one and is about tenfold higher than that of the unchanged dasotraline along with an approximate twofold difference in elimination t1/2. There was no evidence that the back conversion of these highly abundant phase II conjugates would contribute to the long half-life of dasotraline in plasma as the oxime of dasotraline had an extremely low exposure and short half-life in circulation. According to MIST guidance (U.S. Food and Drug Administration 2008), phase II conjugates generally render a compound more water soluble and pharmacologically inactive; thus, further evaluation is not warranted. The only oxidative metabolite detected in plasma was the oxime of dasotraline (M41), accounted for a 0.62% of the plasma TRA and was detected at early time points (~4 h) from only 2 of 7 completed subjects and quickly disappeared likely due to subsequent conjugations. As M41 is a very minor metabolite with a short half-life, it is not expected to accumulate and cause a safety concern. According to ICH guidance (ICH 2010), nonclinical characterization of a human metabolite(s) is only warranted when that metabolite(s) is observed at exposures >10% of total drug-related exposure and at significantly greater levels in humans than the maximum exposure seen in the toxicity studies. Therefore, the further characterization of M41 for safety concern will not be necessary. In conclusion, dasotraline is well but slowly absorbed, and extensively metabolized after a single oral dose of [14C]-dasotraline in healthy male adults. The TRA recovery is >90% in urine and feces over the course of 35 study days and the main excretion is through urine. In human plasma, unchanged dasotraline accounted for 8.59% of the TRA in AUC-pooled plasma samples. The 2 most abundant metabolites were sulfate and glucuronide conjugates of the oxime of dasotraline, accounting for 60.1 and 15.0% of TRA in pooled plasma, respectively.
ª 2016 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.
Y.-L. Chen et al.
ADME of Dasotraline in Humans
Table 8. Accurate mass measurements of dasotraline and its metabolites.
Compound Code Dasotraline (S)-Tetralone M31A M32 M35 M41 M42 M43 (1R,4S)-Acetamide (M102)
m/z [M+H]+
Elemental Composition
Theoretical mass (based on 35Cl)
Measured Mass
Mass Difference (ppm)
292 291 500 (NH4+- adduct) 500 (NH4+- adduct) 484 306 384 (M-1, negative) 482 344
C16H15Cl2N C16H12Cl2O C22H24Cl2O8N
292.0654 291.0338 500.0874
292.0648 291.0338 500.0862
2.05 0 2.40
C22H24Cl2O8N
500.0874
500.0854
4.00
C22H23Cl2NO7 C16H13Cl2NO C16H12Cl2NO4S
484.0924 306.0447 383.9870
484.0913 306.0447 383.9857
2.27 0 3.39
C22H21Cl2NO7 C18H18Cl2NO
482.0768 344.0760
482.0753 344.0755
3.11 1.45
The oxime of dasotraline was at the minimal level (accounting for 0.62% of TRA in pooled plasma) and only detectable in two subjects’ early time points (4 h) and not detectable afterwards. Those two dominant metabolites, that is, sulfate and glucuronide of dasotraline-oxime are phase II conjugates that are known to be pharmacologically inactive and not toxic. Per MIST guidance, these inactive phase II conjugates do not require for further evaluation. The findings from the current trial such as a consistent high recovery (>90%), major metabolic pathways (oxidation followed by conjugations), main excretion route (through urine), and unambiguous identification of major circulating metabolites (oxime-sulfate and oxime-glucuronide) provided rich knowledge and insight to help understand the mechanism of metabolism, PK, and safety of dasotraline. According to the findings from this study, metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern.
Acknowledgements The authors are grateful to the Covance CRU (Madison, WI, USA) for conducting the clinical aspects of this study, to the metabolism group of Covance Laboratories, Inc. (Madison, WI, USA) for the radioactivity measurements, to the Xenobiotics Laboratories, Inc. (Plainsboro, NJ, USA) for metabolite profiling and structure elucidation, and to the PPD Laboratories (Middleton, WI, USA) for the measurement of unchanged plasma dasotraline levels. We acknowledge Dawn L. Lee of FireKite, an Ashfield company, part of UDG Healthcare plc, for editorial assistance with the final version of the manuscript for submission, which was funded by Sunovion Pharmaceuticals, Inc.
Authorship Contributions Participated in research design: Chen, Lin, Skende, Hopkins, Koblan. Conducted experiments: Wills, Yi, Wang. Contributed new reagents: Wilkinson. Performed data analysis: Chen, Skende, Lin, Wilkinson, Hopkins. Wrote or contributed to the writing of the manuscript: All Authors (Chen, Skende, Lin, Yi, Wang, Wills, Wilkinson, Koblan, and Hopkins).
Disclosures YLC, ES, JL, HSW, KSK, and SCH are employees of Sunovion Pharmaceuticals, Inc. YY and PLW are employees of XenoBiotic Laboratories, Inc. SW is an employee of Covance Laboratories, Inc.
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